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mab bin1  (Proteintech)


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    Proteintech mab bin1
    N2a cells stably expressing BIN1iso1-TID and Cyto-TID localize to the cytosol and biotinylate proximal proteins. A , schematic of lentiviral vectors expressing Human BIN1iso1 fused to TurboID via a flexible GS linker and a V5-epitope tag and Cytosolic-TurboID with a NES and a V5-epitope tag. B , schematic of experimental design. Stable pools of neuroblastoma (N2a) cells expressing BIN1iso1-TID were provided exogenous biotin and incubated for the times mentioned below prior to washing and lysis. C , biotin dose-response (0 μM, 25 μM, 50 μM biotin) comparing WT N2a cells, stable Cyto-TID N2a cells, and stable BIN1iso1-TID N2a cells. Exogenous biotin was added to the cells for 10 min of labeling prior to washing and lysis. Western blot shows anti-V5 (680) and biotinylated proteins (streptavidin-800). D , 50 μM of exogenous biotin was added for 10, 20, 30, and 60 min. Western blot is probed with <t>anti-BIN1</t> (680) and anti-V5 (800), revealing endogenous BIN1 and expression of TurboID fusion proteins. E , same lysates as above run on a blot and probed with streptavidin, detecting quantity of biotinylated proteins through 60 min time course. F , immunofluorescence (IF) of N2a stable cells expressing BIN1iso1-TID and Cyto-TID fusion proteins. Anti-V5, anti-BIN1, streptavidin, and Hoechst. Images are deconvolved z-stacks displayed as a sum projection (scale bar 10 μM). The boxed region in the V5 and BIN1 image overlay is shown at a higher magnification on the right .
    Mab Bin1, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/mab+bin1/BIN1+Antibody/pmc12475854-57-21-27
    Average 93 stars, based on 23 article reviews
    mab bin1 - by Bioz Stars, 2026-09
    93/100 stars

    Images

    1) Product Images from "Proteomic Characterization of the Alzheimer’s Disease Risk Factor BIN1 Interactome"

    Article Title: Proteomic Characterization of the Alzheimer’s Disease Risk Factor BIN1 Interactome

    Journal: Molecular & Cellular Proteomics : MCP

    doi: 10.1016/j.mcpro.2025.101055

    N2a cells stably expressing BIN1iso1-TID and Cyto-TID localize to the cytosol and biotinylate proximal proteins. A , schematic of lentiviral vectors expressing Human BIN1iso1 fused to TurboID via a flexible GS linker and a V5-epitope tag and Cytosolic-TurboID with a NES and a V5-epitope tag. B , schematic of experimental design. Stable pools of neuroblastoma (N2a) cells expressing BIN1iso1-TID were provided exogenous biotin and incubated for the times mentioned below prior to washing and lysis. C , biotin dose-response (0 μM, 25 μM, 50 μM biotin) comparing WT N2a cells, stable Cyto-TID N2a cells, and stable BIN1iso1-TID N2a cells. Exogenous biotin was added to the cells for 10 min of labeling prior to washing and lysis. Western blot shows anti-V5 (680) and biotinylated proteins (streptavidin-800). D , 50 μM of exogenous biotin was added for 10, 20, 30, and 60 min. Western blot is probed with anti-BIN1 (680) and anti-V5 (800), revealing endogenous BIN1 and expression of TurboID fusion proteins. E , same lysates as above run on a blot and probed with streptavidin, detecting quantity of biotinylated proteins through 60 min time course. F , immunofluorescence (IF) of N2a stable cells expressing BIN1iso1-TID and Cyto-TID fusion proteins. Anti-V5, anti-BIN1, streptavidin, and Hoechst. Images are deconvolved z-stacks displayed as a sum projection (scale bar 10 μM). The boxed region in the V5 and BIN1 image overlay is shown at a higher magnification on the right .
    Figure Legend Snippet: N2a cells stably expressing BIN1iso1-TID and Cyto-TID localize to the cytosol and biotinylate proximal proteins. A , schematic of lentiviral vectors expressing Human BIN1iso1 fused to TurboID via a flexible GS linker and a V5-epitope tag and Cytosolic-TurboID with a NES and a V5-epitope tag. B , schematic of experimental design. Stable pools of neuroblastoma (N2a) cells expressing BIN1iso1-TID were provided exogenous biotin and incubated for the times mentioned below prior to washing and lysis. C , biotin dose-response (0 μM, 25 μM, 50 μM biotin) comparing WT N2a cells, stable Cyto-TID N2a cells, and stable BIN1iso1-TID N2a cells. Exogenous biotin was added to the cells for 10 min of labeling prior to washing and lysis. Western blot shows anti-V5 (680) and biotinylated proteins (streptavidin-800). D , 50 μM of exogenous biotin was added for 10, 20, 30, and 60 min. Western blot is probed with anti-BIN1 (680) and anti-V5 (800), revealing endogenous BIN1 and expression of TurboID fusion proteins. E , same lysates as above run on a blot and probed with streptavidin, detecting quantity of biotinylated proteins through 60 min time course. F , immunofluorescence (IF) of N2a stable cells expressing BIN1iso1-TID and Cyto-TID fusion proteins. Anti-V5, anti-BIN1, streptavidin, and Hoechst. Images are deconvolved z-stacks displayed as a sum projection (scale bar 10 μM). The boxed region in the V5 and BIN1 image overlay is shown at a higher magnification on the right .

    Techniques Used: Stable Transfection, Expressing, Incubation, Lysis, Labeling, Western Blot, Immunofluorescence

    BIN1iso1-TurboID interactome in N2a cells identifies known and unknown BIN1-proximal or interacting proteins. A , overview schematic of sample processing for mass spectrometry. B , data analysis pipeline for mass spectrometry from database search and quantitation through final cutoffs (CV<30%, z-score >1). C , bar graph displaying unique proteins (detection FDR<0.01) for N2a BIN1iso1-TID and Cyto-TID samples in triplicate (n = 3). D , representation of the N2a BIN1iso1-TID interactome proteins with a z-score >2. Known protein interactions among the BIN1iso1-TID-labeled proteins were identified using the STRING database using experimental data, text mining, and database sources as evidence, applying a high confidence score of >0.7. Four major clusters are annotated and labeled based on representative terms. Note that 69 singlets do not have a known association with the other BIN1-proximal proteins. Singlet nodes in magenta represent proteins involved in cytoskeleton regulation, endocytosis, and vesicle-mediated transport. The size of the nodes corresponds to the -Log (Welch’s t test p -value). E , Volcano plot displaying t test Difference versus -Log10(Welch’s t test p -value) for N2a BIN1iso1-TID interactome (z-score >1). F , on the left, heat map of Log2(intensity) for top 30 proteins, by descending z-score and a horizontal bar graph on the right displaying the average ratio BIN1iso1-TID/Cyto-TID. G , lollipop graph of Gene Ontology (GO) term analysis for biological process. Lines and lollipops are colored according to -Log10(FDR) and the lollipop sizes correspond to the number of genes. H , GO term analysis for cellular components. Lines and lollipops are colored according to -Log10(FDR) and the lollipop sizes correspond to the number of genes.
    Figure Legend Snippet: BIN1iso1-TurboID interactome in N2a cells identifies known and unknown BIN1-proximal or interacting proteins. A , overview schematic of sample processing for mass spectrometry. B , data analysis pipeline for mass spectrometry from database search and quantitation through final cutoffs (CV<30%, z-score >1). C , bar graph displaying unique proteins (detection FDR<0.01) for N2a BIN1iso1-TID and Cyto-TID samples in triplicate (n = 3). D , representation of the N2a BIN1iso1-TID interactome proteins with a z-score >2. Known protein interactions among the BIN1iso1-TID-labeled proteins were identified using the STRING database using experimental data, text mining, and database sources as evidence, applying a high confidence score of >0.7. Four major clusters are annotated and labeled based on representative terms. Note that 69 singlets do not have a known association with the other BIN1-proximal proteins. Singlet nodes in magenta represent proteins involved in cytoskeleton regulation, endocytosis, and vesicle-mediated transport. The size of the nodes corresponds to the -Log (Welch’s t test p -value). E , Volcano plot displaying t test Difference versus -Log10(Welch’s t test p -value) for N2a BIN1iso1-TID interactome (z-score >1). F , on the left, heat map of Log2(intensity) for top 30 proteins, by descending z-score and a horizontal bar graph on the right displaying the average ratio BIN1iso1-TID/Cyto-TID. G , lollipop graph of Gene Ontology (GO) term analysis for biological process. Lines and lollipops are colored according to -Log10(FDR) and the lollipop sizes correspond to the number of genes. H , GO term analysis for cellular components. Lines and lollipops are colored according to -Log10(FDR) and the lollipop sizes correspond to the number of genes.

    Techniques Used: Mass Spectrometry, Quantitation Assay, Labeling

    Retroorbital injection of rAAV human BIN1iso1-TID in mice efficiently transduces neurons and biotinylates proximal proteins in neurons. A , schematic overview of experimental design for in vivo rAAV delivery. The rAAV uses a recently developed AAV serotype capsid AAV-PHP.eB with a human SYN1 promoter to drive expression of BIN1iso1-TID and Cyto-TID in neurons. A flexible GS linker fused BIN1iso1 to TID with a V5 epitope tag. The AAV-TID was injected RO into 2-month-old mice and transduction was allowed for 4 weeks. Following this period, exogenous biotin (0.5 mg/ml) was supplemented in the mouse water for 5 days, followed by brain collection. Brains were split in half for immunofluorescence staining, and the hippocampus and cortex were dissected for quantitative mass spectrometry. Biotinylated proteins were pulled out with streptavidin beads and lysates were processed using S-Trap before LC-MS/MS. B , a Western blot using combined hippocampus and cortex lysates probed with streptavidin (800) to detect biotinylated proteins and anti-V5 to detect TurboID fusion proteins. C , IF detection of biotinylated proteins via streptavidin. Sagittal sections from non-transduced controls, Cyto-TID, and BIN1iso1-TID brains (Scale bar 2 mm). D , IF staining of BIN1-TID and Cyto-TID mouse cortex with anti-V5, streptavidin, and Hoechst. The merged image shows V5/Streptavidin/Hoechst overlap (Scale bar = 500 μm). High-magnification z-maximum projection of boxed Cortex Layer 1 region (Scale bar = 50 μm). E , IF staining of BIN1-TID and Cyto-TID mouse hippocampus with anti-V5, streptavidin, and Hoechst. The merged image shows V5/Streptavidin/Hoechst overlap (Scale bar = 500 μm). High magnification z-maximum projection of boxed Hippocampus CA1 SO region (Scale bar = 50 μm). F , IF staining BIN1-TID mouse brains using anti-synaptophysin to label presynapses and streptavidin to detect biotinylated proteins. Images are deconvolved z-stacks of cortex projected as a sum. Colocalization analysis in Huygens generated the colocalization map (Scale bar 10 μm). G , IF staining BIN1-TID mouse brains using anti-synaptophysin to label presynapses and streptavidin to detect biotinylated proteins. Images are deconvolved z-stacks of hippocampus projected as a sum. Colocalization analysis in Huygens generated the colocalization map (Scale bar 10 μm). H , quantification for the ratio of colocalization map SYP/streptavidin objects divided by the total SYP positive objects in the z-stack for cortex and hippocampus images (Huygens colocalization and object analysis, n = 4 brains, 6 images per mouse per region). I , quantification of Spearman colocalization coefficient for SYP/streptavidin hippocampus and cortex images (Huygens colocalization analysis, n = 4 brains, 6 images per mouse per region). J , IF staining BIN1-TID mouse brains using anti-PSD95 to label postsynapses and streptavidin to detect biotinylated proteins. Images are deconvolved z-stacks of cortex projected as a sum. Colocalization analysis in Huygens generated the colocalization map (Scale bar 10 μm). K , IF staining BIN1-TID mouse brains using anti-PSD95 to label postsynapses and streptavidin to detect biotinylated proteins. Images are deconvolved z-stacks of hippocampus projected as a sum. Colocalization analysis in Huygens generated the colocalization map (Scale bar 10 μm). L , quantification for the ratio of colocalization map PSD95/streptavidin objects divided by the total SYP positive objects in the z-stack for cortex and hippocampus images (Huygens colocalization and object analysis, n = 4 brains, 6 images per mouse per region). M , quantification of Spearman colocalization coefficient for PSD95/streptavidin in hippocampus and cortex images (Huygens colocalization analysis, n = 4 brains, 6 images per mouse per region).
    Figure Legend Snippet: Retroorbital injection of rAAV human BIN1iso1-TID in mice efficiently transduces neurons and biotinylates proximal proteins in neurons. A , schematic overview of experimental design for in vivo rAAV delivery. The rAAV uses a recently developed AAV serotype capsid AAV-PHP.eB with a human SYN1 promoter to drive expression of BIN1iso1-TID and Cyto-TID in neurons. A flexible GS linker fused BIN1iso1 to TID with a V5 epitope tag. The AAV-TID was injected RO into 2-month-old mice and transduction was allowed for 4 weeks. Following this period, exogenous biotin (0.5 mg/ml) was supplemented in the mouse water for 5 days, followed by brain collection. Brains were split in half for immunofluorescence staining, and the hippocampus and cortex were dissected for quantitative mass spectrometry. Biotinylated proteins were pulled out with streptavidin beads and lysates were processed using S-Trap before LC-MS/MS. B , a Western blot using combined hippocampus and cortex lysates probed with streptavidin (800) to detect biotinylated proteins and anti-V5 to detect TurboID fusion proteins. C , IF detection of biotinylated proteins via streptavidin. Sagittal sections from non-transduced controls, Cyto-TID, and BIN1iso1-TID brains (Scale bar 2 mm). D , IF staining of BIN1-TID and Cyto-TID mouse cortex with anti-V5, streptavidin, and Hoechst. The merged image shows V5/Streptavidin/Hoechst overlap (Scale bar = 500 μm). High-magnification z-maximum projection of boxed Cortex Layer 1 region (Scale bar = 50 μm). E , IF staining of BIN1-TID and Cyto-TID mouse hippocampus with anti-V5, streptavidin, and Hoechst. The merged image shows V5/Streptavidin/Hoechst overlap (Scale bar = 500 μm). High magnification z-maximum projection of boxed Hippocampus CA1 SO region (Scale bar = 50 μm). F , IF staining BIN1-TID mouse brains using anti-synaptophysin to label presynapses and streptavidin to detect biotinylated proteins. Images are deconvolved z-stacks of cortex projected as a sum. Colocalization analysis in Huygens generated the colocalization map (Scale bar 10 μm). G , IF staining BIN1-TID mouse brains using anti-synaptophysin to label presynapses and streptavidin to detect biotinylated proteins. Images are deconvolved z-stacks of hippocampus projected as a sum. Colocalization analysis in Huygens generated the colocalization map (Scale bar 10 μm). H , quantification for the ratio of colocalization map SYP/streptavidin objects divided by the total SYP positive objects in the z-stack for cortex and hippocampus images (Huygens colocalization and object analysis, n = 4 brains, 6 images per mouse per region). I , quantification of Spearman colocalization coefficient for SYP/streptavidin hippocampus and cortex images (Huygens colocalization analysis, n = 4 brains, 6 images per mouse per region). J , IF staining BIN1-TID mouse brains using anti-PSD95 to label postsynapses and streptavidin to detect biotinylated proteins. Images are deconvolved z-stacks of cortex projected as a sum. Colocalization analysis in Huygens generated the colocalization map (Scale bar 10 μm). K , IF staining BIN1-TID mouse brains using anti-PSD95 to label postsynapses and streptavidin to detect biotinylated proteins. Images are deconvolved z-stacks of hippocampus projected as a sum. Colocalization analysis in Huygens generated the colocalization map (Scale bar 10 μm). L , quantification for the ratio of colocalization map PSD95/streptavidin objects divided by the total SYP positive objects in the z-stack for cortex and hippocampus images (Huygens colocalization and object analysis, n = 4 brains, 6 images per mouse per region). M , quantification of Spearman colocalization coefficient for PSD95/streptavidin in hippocampus and cortex images (Huygens colocalization analysis, n = 4 brains, 6 images per mouse per region).

    Techniques Used: Injection, In Vivo, Expressing, Transduction, Immunofluorescence, Staining, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Western Blot, Generated

    The BIN1iso1-TID interactome in homeostatic mouse brain neurons. BIN1iso1-TID-proximal proteins enriched in mouse brain neurons with a z-score >2, totaling 238, are shown. Known interactions among these proteins were identified using STRING analysis performed as described above ( D ). The representative terms that define five groups of nodes are listed. The node sizes correspond to the -Log (Welch’s t test p -value). Several BIN1-proximal proteins have one (10 proteins) or no interaction (150 proteins) with others and are color-coded with a gradient that reflects the average ratio of BIN1-TID to Cyto-TID.
    Figure Legend Snippet: The BIN1iso1-TID interactome in homeostatic mouse brain neurons. BIN1iso1-TID-proximal proteins enriched in mouse brain neurons with a z-score >2, totaling 238, are shown. Known interactions among these proteins were identified using STRING analysis performed as described above ( D ). The representative terms that define five groups of nodes are listed. The node sizes correspond to the -Log (Welch’s t test p -value). Several BIN1-proximal proteins have one (10 proteins) or no interaction (150 proteins) with others and are color-coded with a gradient that reflects the average ratio of BIN1-TID to Cyto-TID.

    Techniques Used:

    Analysis of the BIN1iso1-TID interactome reveals novel proximal or interacting proteins and functions for BIN1. A , Volcano plot displaying t test Difference versus -Log10(Welch’s t test p -value) for the mouse brain neuron BIN1iso1-TID interactome (z-score >1) B , on the left , heat map of Log2(intensity) for top 30 proteins, by descending z-score and a horizontal bar graph on the right displaying the average ratio BIN1iso1-TID/Cyto-TID. C , lollipop graph of Gene Ontology (GO) term analysis for biological process. Lines and lollipops are colored according to -Log10(FDR) and the sizes of the lollipops correspond to the number of genes with fold enrichment on the x-axis. D , GO term analysis for cellular component. Lines and lollipops are colored according to -Log10(FDR) and the sizes of the lollipops correspond to the number of genes with fold enrichment on the x-axis. E , GO term analysis for molecular function. Lines and lollipop are colored according to -Log10(FDR) and the sizes of the lollipops correspond to the number of genes with fold enrichment on the x-axis. F , presynaptic SynGO BP and CC terms cartoon for the BIN1iso1-TID interactome. Annotated with abundant terms and proteins from each term detected in interactome z-score >1. G , SynGO biological process heat map color coded according to -log10 (Q-value).
    Figure Legend Snippet: Analysis of the BIN1iso1-TID interactome reveals novel proximal or interacting proteins and functions for BIN1. A , Volcano plot displaying t test Difference versus -Log10(Welch’s t test p -value) for the mouse brain neuron BIN1iso1-TID interactome (z-score >1) B , on the left , heat map of Log2(intensity) for top 30 proteins, by descending z-score and a horizontal bar graph on the right displaying the average ratio BIN1iso1-TID/Cyto-TID. C , lollipop graph of Gene Ontology (GO) term analysis for biological process. Lines and lollipops are colored according to -Log10(FDR) and the sizes of the lollipops correspond to the number of genes with fold enrichment on the x-axis. D , GO term analysis for cellular component. Lines and lollipops are colored according to -Log10(FDR) and the sizes of the lollipops correspond to the number of genes with fold enrichment on the x-axis. E , GO term analysis for molecular function. Lines and lollipop are colored according to -Log10(FDR) and the sizes of the lollipops correspond to the number of genes with fold enrichment on the x-axis. F , presynaptic SynGO BP and CC terms cartoon for the BIN1iso1-TID interactome. Annotated with abundant terms and proteins from each term detected in interactome z-score >1. G , SynGO biological process heat map color coded according to -log10 (Q-value).

    Techniques Used:

    Validation of BIN1iso1 N2a cell and mouse brain neuron interactome top hits. A , a Circos plot showing the N2a cell BIN1 interactome overlap with the BIN1 mouse brain neuron interactome (z-score >1). The inner circle represents protein lists, where hits are arranged along the arc. Proteins that hit multiple lists are colored in dark orange , and genes unique to a list are shown in light orange . Purple curves link identical proteins between the datasets, while the blue curves link proteins that belong to the same enriched ontology term. The numbers indicate unique non-overlapping (715 in neurons; 262 in N2a cells) and overlapping (92) proteins in the BIN1 interactome dataset. Multiple isoforms of a protein were counted as one entry for the combined analysis of the two interactome datasets. B , heatmap of top 20 clusters with their representative enriched terms (one per cluster) across protein lists, colored by p -values. Generated using Metascape pathway and process enrichment analysis. C , ninety-two common proteins between N2a BIN1iso1-TID and BIN1iso1-TID mouse brain neurons with a z-score >1 are shown. A medium confidence edge threshold score >0.40 was applied in STRING to create interaction networks among these proteins. The node sizes represent the BIN1iso1-TID mouse brain interactome, and the nodes are color-coded with a gradient indicating the average ratio of the N2a BIN1-TID interactome. Representative terms that characterize a group of nodes are indicated. D , IF of BIN1iso1-TID brain using anti-SYNJ1 and anti-V5. Overlap of images (scale bar = 25 μm). Higher magnification images are z-stacks projected as a Sum (scale bar = 10 μm). E , PLA using anti-BIN1 and anti-SYNJ1 in brains of neuron-specific Emx-Cre: Bin1 knockout and Emx-Cre control mice. The Emx-Cre: Bin1 knockout mouse lacks BIN1 expression in neurons and oligodendrocytes in the forebrain, thus serving as a negative control for PLA. F , IF of BIN1iso1-TID brain using anti-RANG and anti-V5. Overlap of images (scale bar = 25 μm). Higher magnification images are z-stacks projected as a Sum (scale bar = 10 μm). G , PLA of BIN1iso1-TID using anti-V5 and anti-RANG. Co-stained with streptavidin to display biotinylated proteins. H , IF of BIN1iso1-TID brain using anti-PP2BA and anti-BIN1. Overlap of images (scale bar = 25 μm). Higher magnification images are z-stacks projected as a Sum (scale bar = 10 μm). I , PLA of BIN1iso1-TID using anti-PP2BA and anti-BIN1. Co-stained with streptavidin to display biotinylated proteins for reference.
    Figure Legend Snippet: Validation of BIN1iso1 N2a cell and mouse brain neuron interactome top hits. A , a Circos plot showing the N2a cell BIN1 interactome overlap with the BIN1 mouse brain neuron interactome (z-score >1). The inner circle represents protein lists, where hits are arranged along the arc. Proteins that hit multiple lists are colored in dark orange , and genes unique to a list are shown in light orange . Purple curves link identical proteins between the datasets, while the blue curves link proteins that belong to the same enriched ontology term. The numbers indicate unique non-overlapping (715 in neurons; 262 in N2a cells) and overlapping (92) proteins in the BIN1 interactome dataset. Multiple isoforms of a protein were counted as one entry for the combined analysis of the two interactome datasets. B , heatmap of top 20 clusters with their representative enriched terms (one per cluster) across protein lists, colored by p -values. Generated using Metascape pathway and process enrichment analysis. C , ninety-two common proteins between N2a BIN1iso1-TID and BIN1iso1-TID mouse brain neurons with a z-score >1 are shown. A medium confidence edge threshold score >0.40 was applied in STRING to create interaction networks among these proteins. The node sizes represent the BIN1iso1-TID mouse brain interactome, and the nodes are color-coded with a gradient indicating the average ratio of the N2a BIN1-TID interactome. Representative terms that characterize a group of nodes are indicated. D , IF of BIN1iso1-TID brain using anti-SYNJ1 and anti-V5. Overlap of images (scale bar = 25 μm). Higher magnification images are z-stacks projected as a Sum (scale bar = 10 μm). E , PLA using anti-BIN1 and anti-SYNJ1 in brains of neuron-specific Emx-Cre: Bin1 knockout and Emx-Cre control mice. The Emx-Cre: Bin1 knockout mouse lacks BIN1 expression in neurons and oligodendrocytes in the forebrain, thus serving as a negative control for PLA. F , IF of BIN1iso1-TID brain using anti-RANG and anti-V5. Overlap of images (scale bar = 25 μm). Higher magnification images are z-stacks projected as a Sum (scale bar = 10 μm). G , PLA of BIN1iso1-TID using anti-V5 and anti-RANG. Co-stained with streptavidin to display biotinylated proteins. H , IF of BIN1iso1-TID brain using anti-PP2BA and anti-BIN1. Overlap of images (scale bar = 25 μm). Higher magnification images are z-stacks projected as a Sum (scale bar = 10 μm). I , PLA of BIN1iso1-TID using anti-PP2BA and anti-BIN1. Co-stained with streptavidin to display biotinylated proteins for reference.

    Techniques Used: Biomarker Discovery, Generated, Knock-Out, Control, Expressing, Negative Control, Staining

    Phosphorylation site analysis of BIN1iso1-TID neuronal interactome identifies AAK1 and CDK16. A , proteins identified through the phosphorylation site analysis of BIN1iso1-TID mouse brain neurons with a z-score >2 are shown with the detected phosphorylation sites mapped to them. A medium confidence edge threshold score >0.40 was used in STRING to identify interactions among the proteins. Proteins that localize to the synapse are colored orange, while the group with a green background represents endocytic proteins. Note that about half of the BIN1-proximal phosphoproteins meeting our z-score criteria remain as singlets. B , using PhosophoSitePlus prediction. This displays the probability for a specific kinase to phosphorylate the target protein at each experimentally observed phosphorylated site in the interactome. We set >90% as the threshold for inclusion of a particular kinase in our results. The kinases were tallied for phosphoproteins filtered from the BIN1iso1-TID interactome (z-score >2). C , IF of BIN1iso1-TID brain using anti-AAK1 and anti-V5. Overlap of low mag images (scale bar = 25 μm). Higher magnification images are z-stacks projected as a Sum (scale bar = 10 μm). D , PLA using anti-AAK1 and anti-BIN1 antibodies. Mice are neuron-specific Emx-Cre:BIN1 knockout and Emx-Cre control mice. The Emx-Cre: Bin1 knockout mouse lacks BIN1 expression in neurons and oligodendrocytes in the forebrain, thus serving as a negative control for PLA. E , IF of BIN1iso1-TID brain using anti-CDK16 and anti-V5 antibodies. Overlap of low mag images (scale bar = 25 μm). Higher magnification images are z-stacks projected as a Sum (scale bar = 10 μm). F , PLA of BIN1iso1-TID using anti-CDK16 and anti-V5 antibodies. Co-stained with streptavidin to display biotinylated proteins for reference.
    Figure Legend Snippet: Phosphorylation site analysis of BIN1iso1-TID neuronal interactome identifies AAK1 and CDK16. A , proteins identified through the phosphorylation site analysis of BIN1iso1-TID mouse brain neurons with a z-score >2 are shown with the detected phosphorylation sites mapped to them. A medium confidence edge threshold score >0.40 was used in STRING to identify interactions among the proteins. Proteins that localize to the synapse are colored orange, while the group with a green background represents endocytic proteins. Note that about half of the BIN1-proximal phosphoproteins meeting our z-score criteria remain as singlets. B , using PhosophoSitePlus prediction. This displays the probability for a specific kinase to phosphorylate the target protein at each experimentally observed phosphorylated site in the interactome. We set >90% as the threshold for inclusion of a particular kinase in our results. The kinases were tallied for phosphoproteins filtered from the BIN1iso1-TID interactome (z-score >2). C , IF of BIN1iso1-TID brain using anti-AAK1 and anti-V5. Overlap of low mag images (scale bar = 25 μm). Higher magnification images are z-stacks projected as a Sum (scale bar = 10 μm). D , PLA using anti-AAK1 and anti-BIN1 antibodies. Mice are neuron-specific Emx-Cre:BIN1 knockout and Emx-Cre control mice. The Emx-Cre: Bin1 knockout mouse lacks BIN1 expression in neurons and oligodendrocytes in the forebrain, thus serving as a negative control for PLA. E , IF of BIN1iso1-TID brain using anti-CDK16 and anti-V5 antibodies. Overlap of low mag images (scale bar = 25 μm). Higher magnification images are z-stacks projected as a Sum (scale bar = 10 μm). F , PLA of BIN1iso1-TID using anti-CDK16 and anti-V5 antibodies. Co-stained with streptavidin to display biotinylated proteins for reference.

    Techniques Used: Phospho-proteomics, Knock-Out, Control, Expressing, Negative Control, Staining



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    93
    Proteintech mab bin1
    N2a cells stably expressing BIN1iso1-TID and Cyto-TID localize to the cytosol and biotinylate proximal proteins. A , schematic of lentiviral vectors expressing Human BIN1iso1 fused to TurboID via a flexible GS linker and a V5-epitope tag and Cytosolic-TurboID with a NES and a V5-epitope tag. B , schematic of experimental design. Stable pools of neuroblastoma (N2a) cells expressing BIN1iso1-TID were provided exogenous biotin and incubated for the times mentioned below prior to washing and lysis. C , biotin dose-response (0 μM, 25 μM, 50 μM biotin) comparing WT N2a cells, stable Cyto-TID N2a cells, and stable BIN1iso1-TID N2a cells. Exogenous biotin was added to the cells for 10 min of labeling prior to washing and lysis. Western blot shows anti-V5 (680) and biotinylated proteins (streptavidin-800). D , 50 μM of exogenous biotin was added for 10, 20, 30, and 60 min. Western blot is probed with <t>anti-BIN1</t> (680) and anti-V5 (800), revealing endogenous BIN1 and expression of TurboID fusion proteins. E , same lysates as above run on a blot and probed with streptavidin, detecting quantity of biotinylated proteins through 60 min time course. F , immunofluorescence (IF) of N2a stable cells expressing BIN1iso1-TID and Cyto-TID fusion proteins. Anti-V5, anti-BIN1, streptavidin, and Hoechst. Images are deconvolved z-stacks displayed as a sum projection (scale bar 10 μM). The boxed region in the V5 and BIN1 image overlay is shown at a higher magnification on the right .
    Mab Bin1, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    signaling rb anti bin1 mab  (Cell Signaling Technology Inc)
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    Cell Signaling Technology Inc signaling rb anti bin1 mab
    Comparison of instances of differential alternative splicing at transcriptional and protein level for Atp2a1 and <t>Bin1</t> genes. Panels on the left show LeafCutter AS intron clusters with relative exon usage in the DM1 and WT control groups. Panels in the middle show qRT-PCR results with the splicing-specific probes (see ) covering the corresponding exon inclusion ( gray ) and exon exclusion ( blue ). Panels on the right show the corresponding relative abundances of peptides specific to exon inclusion or exclusion in the same sample groups. See also <xref ref-type=supplemental Fig. S4 for a UCSC genome browser-based display of these LeafCutter AS intron clusters. AS, alternative splicing; DM1, myotonic dystrophy, type 1. " width="250" height="auto" />
    Signaling Rb Anti Bin1 Mab, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    mouse mab anti bin1  (Rockland Immunochemicals)
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    Rockland Immunochemicals mouse mab anti bin1
    RNA expression of T-tubule and SR genes RT-qPCR analysis of LV tissue for mRNA content of ( a ) bridging integrator-1, <t>BIN1;</t> junctophilin 2, Jph2; L-type calcium channel, LTCC; ryanodine 2, RyR2 and ( b ) SR calcium ATPase, SERCa2; phospholamban, PLN; alpha- and beta-myosin heavy chain, αMHC and βMHC. The binary log of ΔΔCt calculations of PCR cycle amplifications was used to determine the fold-change relative to the mean values of the Sham group. Bar graph shows mean ± SD for n = 4–6 hearts/group. Statistical analysis used one-way ANOVA with post-hoc Tukey’s multi-group comparisons: * p < 0.05 vs sham; ** p < 0.001 vs sham; # p < 0.01 vs MI + Veh.
    Mouse Mab Anti Bin1, supplied by Rockland Immunochemicals, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rabbit anti bin1 mab epr13463 25  (Abcam)
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    Abcam rabbit anti bin1 mab epr13463 25
    Single allele deletion of <t>Bin1</t> does not alter APP or BACE localization or Aβ levels. A, forebrain homogenates from 4-month-old Bin1+/− mice and WT littermate controls were analyzed by immunoblotting for BIN1, APP, BACE1, and Amph1 levels. B, quantitative analysis of BIN1:H (top) and BIN1:L (bottom) in forebrain lysates from WT and Bin1+/− mice. C, top panel, immunofluorescent staining for BIN1 (magenta) and BACE1 (green) in the hippocampal CA3 region from WT and Bin1+/− mice. Scale bar, 50 μm. Bottom panel, higher magnification of immunofluorescent staining for APP (red) and BACE1 (green) in CA3 neurons and mossy fibers (MF) of the mouse hippocampus. Scale bar, 20 μm. D, forebrain lysates from WT and Bin1+/− mice were analyzed for steady-state levels of endogenous Aβ40 and Aβ42 using a V-PLEX 4G8 immunoassay (Aβ40, n = 12 per genotype; Aβ42, n = 11 WT, and 12 Bin1+/−).
    Rabbit Anti Bin1 Mab Epr13463 25, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rabbit anti bin1 mab epr13463 25  (Danaher Inc)
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    Danaher Inc rabbit anti bin1 mab epr13463 25
    Single allele deletion of <t>Bin1</t> does not alter APP or BACE localization or Aβ levels. A, forebrain homogenates from 4-month-old Bin1+/− mice and WT littermate controls were analyzed by immunoblotting for BIN1, APP, BACE1, and Amph1 levels. B, quantitative analysis of BIN1:H (top) and BIN1:L (bottom) in forebrain lysates from WT and Bin1+/− mice. C, top panel, immunofluorescent staining for BIN1 (magenta) and BACE1 (green) in the hippocampal CA3 region from WT and Bin1+/− mice. Scale bar, 50 μm. Bottom panel, higher magnification of immunofluorescent staining for APP (red) and BACE1 (green) in CA3 neurons and mossy fibers (MF) of the mouse hippocampus. Scale bar, 20 μm. D, forebrain lysates from WT and Bin1+/− mice were analyzed for steady-state levels of endogenous Aβ40 and Aβ42 using a V-PLEX 4G8 immunoassay (Aβ40, n = 12 per genotype; Aβ42, n = 11 WT, and 12 Bin1+/−).
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    bin1 mab antibody  (Millipore)
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    Millipore bin1 mab antibody
    (A) The top 3 GO biological clusters of 40 genes identified in GWAS studies of LOAD risk. (B) Top 5 individual GO terms within the top GO cluster. The dotted line indicates significance level of P= 0.05. (C) Protein interaction network of 11 LOAD risk genes present in the vesicle-mediated transport/endocytosis GO cluster. Network was generated using the GeneMANIA in Cytoscape. This network has a significant enrichment of proteins present in the PSD (P=0.0002). (D) <t>BIN1</t> protein interaction network reveals a significant enrichment of PSD genes (P= 4.0×10 −7 ), trafficking genes (P= 0.0002), and GTPase-related genes (P= 5.0×10 −5 ). (E) Top: Single plane SIM image of a dendritic region stained for Bin1 and presynaptic marker synapsin1. GFP cell fill is outlined (see ). Scale bar = 5 μm Bottom: Representative SIM images of boxed spines in above image. (F) Top: Single plane SIM image of a dendritic region stained for Bin1 and GluA1. GFP cell fill is outlined (see ). Scale bar = 5 μm Bottom: Representative SIM images of boxed spines above image. (G) Representative 3D reconstruction of a stack of SIM images showing the relative localization of Bin1 and GluA1 within a spine. Scale bar = 50 nm (H) Immuno-electron microscopy for Bin1. Left micrograph is low-magnification view of neuropil. Labeled spines have been colorized green and axon terminals pink; a thin immunopositive dendritic process (left) is colorized blue. (I) Bin1-associated gold particles in immuno-EM images are found in the synaptic membrane/PSD, as well as near the PSD in extrasynaptic membranes.
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    Image Search Results


    N2a cells stably expressing BIN1iso1-TID and Cyto-TID localize to the cytosol and biotinylate proximal proteins. A , schematic of lentiviral vectors expressing Human BIN1iso1 fused to TurboID via a flexible GS linker and a V5-epitope tag and Cytosolic-TurboID with a NES and a V5-epitope tag. B , schematic of experimental design. Stable pools of neuroblastoma (N2a) cells expressing BIN1iso1-TID were provided exogenous biotin and incubated for the times mentioned below prior to washing and lysis. C , biotin dose-response (0 μM, 25 μM, 50 μM biotin) comparing WT N2a cells, stable Cyto-TID N2a cells, and stable BIN1iso1-TID N2a cells. Exogenous biotin was added to the cells for 10 min of labeling prior to washing and lysis. Western blot shows anti-V5 (680) and biotinylated proteins (streptavidin-800). D , 50 μM of exogenous biotin was added for 10, 20, 30, and 60 min. Western blot is probed with anti-BIN1 (680) and anti-V5 (800), revealing endogenous BIN1 and expression of TurboID fusion proteins. E , same lysates as above run on a blot and probed with streptavidin, detecting quantity of biotinylated proteins through 60 min time course. F , immunofluorescence (IF) of N2a stable cells expressing BIN1iso1-TID and Cyto-TID fusion proteins. Anti-V5, anti-BIN1, streptavidin, and Hoechst. Images are deconvolved z-stacks displayed as a sum projection (scale bar 10 μM). The boxed region in the V5 and BIN1 image overlay is shown at a higher magnification on the right .

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Proteomic Characterization of the Alzheimer’s Disease Risk Factor BIN1 Interactome

    doi: 10.1016/j.mcpro.2025.101055

    Figure Lengend Snippet: N2a cells stably expressing BIN1iso1-TID and Cyto-TID localize to the cytosol and biotinylate proximal proteins. A , schematic of lentiviral vectors expressing Human BIN1iso1 fused to TurboID via a flexible GS linker and a V5-epitope tag and Cytosolic-TurboID with a NES and a V5-epitope tag. B , schematic of experimental design. Stable pools of neuroblastoma (N2a) cells expressing BIN1iso1-TID were provided exogenous biotin and incubated for the times mentioned below prior to washing and lysis. C , biotin dose-response (0 μM, 25 μM, 50 μM biotin) comparing WT N2a cells, stable Cyto-TID N2a cells, and stable BIN1iso1-TID N2a cells. Exogenous biotin was added to the cells for 10 min of labeling prior to washing and lysis. Western blot shows anti-V5 (680) and biotinylated proteins (streptavidin-800). D , 50 μM of exogenous biotin was added for 10, 20, 30, and 60 min. Western blot is probed with anti-BIN1 (680) and anti-V5 (800), revealing endogenous BIN1 and expression of TurboID fusion proteins. E , same lysates as above run on a blot and probed with streptavidin, detecting quantity of biotinylated proteins through 60 min time course. F , immunofluorescence (IF) of N2a stable cells expressing BIN1iso1-TID and Cyto-TID fusion proteins. Anti-V5, anti-BIN1, streptavidin, and Hoechst. Images are deconvolved z-stacks displayed as a sum projection (scale bar 10 μM). The boxed region in the V5 and BIN1 image overlay is shown at a higher magnification on the right .

    Article Snippet: Da Vinci Green antibody diluent was used to dilute primary antibodies against V5 (Thermo Fisher # R96025 ), BIN1 (Proteintech #14647-1-AP), mAb BIN1 (clone 19H3) , CDK16 (Proteintech #10102-1-AP), PP2Ba (Santa Cruz # sc-17808), SYNJ1 (Atlas Antibodies #HPS011916), AAK1 (Atlas Antibodies #HPA020289), RANG (Atlas Antibodies #HPA065868), SYP (Sigma #S5768), PSD95 (Sigma # MABN68), and Streptavidin conjugates (Invitrogen #84547).

    Techniques: Stable Transfection, Expressing, Incubation, Lysis, Labeling, Western Blot, Immunofluorescence

    BIN1iso1-TurboID interactome in N2a cells identifies known and unknown BIN1-proximal or interacting proteins. A , overview schematic of sample processing for mass spectrometry. B , data analysis pipeline for mass spectrometry from database search and quantitation through final cutoffs (CV<30%, z-score >1). C , bar graph displaying unique proteins (detection FDR<0.01) for N2a BIN1iso1-TID and Cyto-TID samples in triplicate (n = 3). D , representation of the N2a BIN1iso1-TID interactome proteins with a z-score >2. Known protein interactions among the BIN1iso1-TID-labeled proteins were identified using the STRING database using experimental data, text mining, and database sources as evidence, applying a high confidence score of >0.7. Four major clusters are annotated and labeled based on representative terms. Note that 69 singlets do not have a known association with the other BIN1-proximal proteins. Singlet nodes in magenta represent proteins involved in cytoskeleton regulation, endocytosis, and vesicle-mediated transport. The size of the nodes corresponds to the -Log (Welch’s t test p -value). E , Volcano plot displaying t test Difference versus -Log10(Welch’s t test p -value) for N2a BIN1iso1-TID interactome (z-score >1). F , on the left, heat map of Log2(intensity) for top 30 proteins, by descending z-score and a horizontal bar graph on the right displaying the average ratio BIN1iso1-TID/Cyto-TID. G , lollipop graph of Gene Ontology (GO) term analysis for biological process. Lines and lollipops are colored according to -Log10(FDR) and the lollipop sizes correspond to the number of genes. H , GO term analysis for cellular components. Lines and lollipops are colored according to -Log10(FDR) and the lollipop sizes correspond to the number of genes.

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Proteomic Characterization of the Alzheimer’s Disease Risk Factor BIN1 Interactome

    doi: 10.1016/j.mcpro.2025.101055

    Figure Lengend Snippet: BIN1iso1-TurboID interactome in N2a cells identifies known and unknown BIN1-proximal or interacting proteins. A , overview schematic of sample processing for mass spectrometry. B , data analysis pipeline for mass spectrometry from database search and quantitation through final cutoffs (CV<30%, z-score >1). C , bar graph displaying unique proteins (detection FDR<0.01) for N2a BIN1iso1-TID and Cyto-TID samples in triplicate (n = 3). D , representation of the N2a BIN1iso1-TID interactome proteins with a z-score >2. Known protein interactions among the BIN1iso1-TID-labeled proteins were identified using the STRING database using experimental data, text mining, and database sources as evidence, applying a high confidence score of >0.7. Four major clusters are annotated and labeled based on representative terms. Note that 69 singlets do not have a known association with the other BIN1-proximal proteins. Singlet nodes in magenta represent proteins involved in cytoskeleton regulation, endocytosis, and vesicle-mediated transport. The size of the nodes corresponds to the -Log (Welch’s t test p -value). E , Volcano plot displaying t test Difference versus -Log10(Welch’s t test p -value) for N2a BIN1iso1-TID interactome (z-score >1). F , on the left, heat map of Log2(intensity) for top 30 proteins, by descending z-score and a horizontal bar graph on the right displaying the average ratio BIN1iso1-TID/Cyto-TID. G , lollipop graph of Gene Ontology (GO) term analysis for biological process. Lines and lollipops are colored according to -Log10(FDR) and the lollipop sizes correspond to the number of genes. H , GO term analysis for cellular components. Lines and lollipops are colored according to -Log10(FDR) and the lollipop sizes correspond to the number of genes.

    Article Snippet: Da Vinci Green antibody diluent was used to dilute primary antibodies against V5 (Thermo Fisher # R96025 ), BIN1 (Proteintech #14647-1-AP), mAb BIN1 (clone 19H3) , CDK16 (Proteintech #10102-1-AP), PP2Ba (Santa Cruz # sc-17808), SYNJ1 (Atlas Antibodies #HPS011916), AAK1 (Atlas Antibodies #HPA020289), RANG (Atlas Antibodies #HPA065868), SYP (Sigma #S5768), PSD95 (Sigma # MABN68), and Streptavidin conjugates (Invitrogen #84547).

    Techniques: Mass Spectrometry, Quantitation Assay, Labeling

    Retroorbital injection of rAAV human BIN1iso1-TID in mice efficiently transduces neurons and biotinylates proximal proteins in neurons. A , schematic overview of experimental design for in vivo rAAV delivery. The rAAV uses a recently developed AAV serotype capsid AAV-PHP.eB with a human SYN1 promoter to drive expression of BIN1iso1-TID and Cyto-TID in neurons. A flexible GS linker fused BIN1iso1 to TID with a V5 epitope tag. The AAV-TID was injected RO into 2-month-old mice and transduction was allowed for 4 weeks. Following this period, exogenous biotin (0.5 mg/ml) was supplemented in the mouse water for 5 days, followed by brain collection. Brains were split in half for immunofluorescence staining, and the hippocampus and cortex were dissected for quantitative mass spectrometry. Biotinylated proteins were pulled out with streptavidin beads and lysates were processed using S-Trap before LC-MS/MS. B , a Western blot using combined hippocampus and cortex lysates probed with streptavidin (800) to detect biotinylated proteins and anti-V5 to detect TurboID fusion proteins. C , IF detection of biotinylated proteins via streptavidin. Sagittal sections from non-transduced controls, Cyto-TID, and BIN1iso1-TID brains (Scale bar 2 mm). D , IF staining of BIN1-TID and Cyto-TID mouse cortex with anti-V5, streptavidin, and Hoechst. The merged image shows V5/Streptavidin/Hoechst overlap (Scale bar = 500 μm). High-magnification z-maximum projection of boxed Cortex Layer 1 region (Scale bar = 50 μm). E , IF staining of BIN1-TID and Cyto-TID mouse hippocampus with anti-V5, streptavidin, and Hoechst. The merged image shows V5/Streptavidin/Hoechst overlap (Scale bar = 500 μm). High magnification z-maximum projection of boxed Hippocampus CA1 SO region (Scale bar = 50 μm). F , IF staining BIN1-TID mouse brains using anti-synaptophysin to label presynapses and streptavidin to detect biotinylated proteins. Images are deconvolved z-stacks of cortex projected as a sum. Colocalization analysis in Huygens generated the colocalization map (Scale bar 10 μm). G , IF staining BIN1-TID mouse brains using anti-synaptophysin to label presynapses and streptavidin to detect biotinylated proteins. Images are deconvolved z-stacks of hippocampus projected as a sum. Colocalization analysis in Huygens generated the colocalization map (Scale bar 10 μm). H , quantification for the ratio of colocalization map SYP/streptavidin objects divided by the total SYP positive objects in the z-stack for cortex and hippocampus images (Huygens colocalization and object analysis, n = 4 brains, 6 images per mouse per region). I , quantification of Spearman colocalization coefficient for SYP/streptavidin hippocampus and cortex images (Huygens colocalization analysis, n = 4 brains, 6 images per mouse per region). J , IF staining BIN1-TID mouse brains using anti-PSD95 to label postsynapses and streptavidin to detect biotinylated proteins. Images are deconvolved z-stacks of cortex projected as a sum. Colocalization analysis in Huygens generated the colocalization map (Scale bar 10 μm). K , IF staining BIN1-TID mouse brains using anti-PSD95 to label postsynapses and streptavidin to detect biotinylated proteins. Images are deconvolved z-stacks of hippocampus projected as a sum. Colocalization analysis in Huygens generated the colocalization map (Scale bar 10 μm). L , quantification for the ratio of colocalization map PSD95/streptavidin objects divided by the total SYP positive objects in the z-stack for cortex and hippocampus images (Huygens colocalization and object analysis, n = 4 brains, 6 images per mouse per region). M , quantification of Spearman colocalization coefficient for PSD95/streptavidin in hippocampus and cortex images (Huygens colocalization analysis, n = 4 brains, 6 images per mouse per region).

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Proteomic Characterization of the Alzheimer’s Disease Risk Factor BIN1 Interactome

    doi: 10.1016/j.mcpro.2025.101055

    Figure Lengend Snippet: Retroorbital injection of rAAV human BIN1iso1-TID in mice efficiently transduces neurons and biotinylates proximal proteins in neurons. A , schematic overview of experimental design for in vivo rAAV delivery. The rAAV uses a recently developed AAV serotype capsid AAV-PHP.eB with a human SYN1 promoter to drive expression of BIN1iso1-TID and Cyto-TID in neurons. A flexible GS linker fused BIN1iso1 to TID with a V5 epitope tag. The AAV-TID was injected RO into 2-month-old mice and transduction was allowed for 4 weeks. Following this period, exogenous biotin (0.5 mg/ml) was supplemented in the mouse water for 5 days, followed by brain collection. Brains were split in half for immunofluorescence staining, and the hippocampus and cortex were dissected for quantitative mass spectrometry. Biotinylated proteins were pulled out with streptavidin beads and lysates were processed using S-Trap before LC-MS/MS. B , a Western blot using combined hippocampus and cortex lysates probed with streptavidin (800) to detect biotinylated proteins and anti-V5 to detect TurboID fusion proteins. C , IF detection of biotinylated proteins via streptavidin. Sagittal sections from non-transduced controls, Cyto-TID, and BIN1iso1-TID brains (Scale bar 2 mm). D , IF staining of BIN1-TID and Cyto-TID mouse cortex with anti-V5, streptavidin, and Hoechst. The merged image shows V5/Streptavidin/Hoechst overlap (Scale bar = 500 μm). High-magnification z-maximum projection of boxed Cortex Layer 1 region (Scale bar = 50 μm). E , IF staining of BIN1-TID and Cyto-TID mouse hippocampus with anti-V5, streptavidin, and Hoechst. The merged image shows V5/Streptavidin/Hoechst overlap (Scale bar = 500 μm). High magnification z-maximum projection of boxed Hippocampus CA1 SO region (Scale bar = 50 μm). F , IF staining BIN1-TID mouse brains using anti-synaptophysin to label presynapses and streptavidin to detect biotinylated proteins. Images are deconvolved z-stacks of cortex projected as a sum. Colocalization analysis in Huygens generated the colocalization map (Scale bar 10 μm). G , IF staining BIN1-TID mouse brains using anti-synaptophysin to label presynapses and streptavidin to detect biotinylated proteins. Images are deconvolved z-stacks of hippocampus projected as a sum. Colocalization analysis in Huygens generated the colocalization map (Scale bar 10 μm). H , quantification for the ratio of colocalization map SYP/streptavidin objects divided by the total SYP positive objects in the z-stack for cortex and hippocampus images (Huygens colocalization and object analysis, n = 4 brains, 6 images per mouse per region). I , quantification of Spearman colocalization coefficient for SYP/streptavidin hippocampus and cortex images (Huygens colocalization analysis, n = 4 brains, 6 images per mouse per region). J , IF staining BIN1-TID mouse brains using anti-PSD95 to label postsynapses and streptavidin to detect biotinylated proteins. Images are deconvolved z-stacks of cortex projected as a sum. Colocalization analysis in Huygens generated the colocalization map (Scale bar 10 μm). K , IF staining BIN1-TID mouse brains using anti-PSD95 to label postsynapses and streptavidin to detect biotinylated proteins. Images are deconvolved z-stacks of hippocampus projected as a sum. Colocalization analysis in Huygens generated the colocalization map (Scale bar 10 μm). L , quantification for the ratio of colocalization map PSD95/streptavidin objects divided by the total SYP positive objects in the z-stack for cortex and hippocampus images (Huygens colocalization and object analysis, n = 4 brains, 6 images per mouse per region). M , quantification of Spearman colocalization coefficient for PSD95/streptavidin in hippocampus and cortex images (Huygens colocalization analysis, n = 4 brains, 6 images per mouse per region).

    Article Snippet: Da Vinci Green antibody diluent was used to dilute primary antibodies against V5 (Thermo Fisher # R96025 ), BIN1 (Proteintech #14647-1-AP), mAb BIN1 (clone 19H3) , CDK16 (Proteintech #10102-1-AP), PP2Ba (Santa Cruz # sc-17808), SYNJ1 (Atlas Antibodies #HPS011916), AAK1 (Atlas Antibodies #HPA020289), RANG (Atlas Antibodies #HPA065868), SYP (Sigma #S5768), PSD95 (Sigma # MABN68), and Streptavidin conjugates (Invitrogen #84547).

    Techniques: Injection, In Vivo, Expressing, Transduction, Immunofluorescence, Staining, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Western Blot, Generated

    The BIN1iso1-TID interactome in homeostatic mouse brain neurons. BIN1iso1-TID-proximal proteins enriched in mouse brain neurons with a z-score >2, totaling 238, are shown. Known interactions among these proteins were identified using STRING analysis performed as described above ( D ). The representative terms that define five groups of nodes are listed. The node sizes correspond to the -Log (Welch’s t test p -value). Several BIN1-proximal proteins have one (10 proteins) or no interaction (150 proteins) with others and are color-coded with a gradient that reflects the average ratio of BIN1-TID to Cyto-TID.

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Proteomic Characterization of the Alzheimer’s Disease Risk Factor BIN1 Interactome

    doi: 10.1016/j.mcpro.2025.101055

    Figure Lengend Snippet: The BIN1iso1-TID interactome in homeostatic mouse brain neurons. BIN1iso1-TID-proximal proteins enriched in mouse brain neurons with a z-score >2, totaling 238, are shown. Known interactions among these proteins were identified using STRING analysis performed as described above ( D ). The representative terms that define five groups of nodes are listed. The node sizes correspond to the -Log (Welch’s t test p -value). Several BIN1-proximal proteins have one (10 proteins) or no interaction (150 proteins) with others and are color-coded with a gradient that reflects the average ratio of BIN1-TID to Cyto-TID.

    Article Snippet: Da Vinci Green antibody diluent was used to dilute primary antibodies against V5 (Thermo Fisher # R96025 ), BIN1 (Proteintech #14647-1-AP), mAb BIN1 (clone 19H3) , CDK16 (Proteintech #10102-1-AP), PP2Ba (Santa Cruz # sc-17808), SYNJ1 (Atlas Antibodies #HPS011916), AAK1 (Atlas Antibodies #HPA020289), RANG (Atlas Antibodies #HPA065868), SYP (Sigma #S5768), PSD95 (Sigma # MABN68), and Streptavidin conjugates (Invitrogen #84547).

    Techniques:

    Analysis of the BIN1iso1-TID interactome reveals novel proximal or interacting proteins and functions for BIN1. A , Volcano plot displaying t test Difference versus -Log10(Welch’s t test p -value) for the mouse brain neuron BIN1iso1-TID interactome (z-score >1) B , on the left , heat map of Log2(intensity) for top 30 proteins, by descending z-score and a horizontal bar graph on the right displaying the average ratio BIN1iso1-TID/Cyto-TID. C , lollipop graph of Gene Ontology (GO) term analysis for biological process. Lines and lollipops are colored according to -Log10(FDR) and the sizes of the lollipops correspond to the number of genes with fold enrichment on the x-axis. D , GO term analysis for cellular component. Lines and lollipops are colored according to -Log10(FDR) and the sizes of the lollipops correspond to the number of genes with fold enrichment on the x-axis. E , GO term analysis for molecular function. Lines and lollipop are colored according to -Log10(FDR) and the sizes of the lollipops correspond to the number of genes with fold enrichment on the x-axis. F , presynaptic SynGO BP and CC terms cartoon for the BIN1iso1-TID interactome. Annotated with abundant terms and proteins from each term detected in interactome z-score >1. G , SynGO biological process heat map color coded according to -log10 (Q-value).

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Proteomic Characterization of the Alzheimer’s Disease Risk Factor BIN1 Interactome

    doi: 10.1016/j.mcpro.2025.101055

    Figure Lengend Snippet: Analysis of the BIN1iso1-TID interactome reveals novel proximal or interacting proteins and functions for BIN1. A , Volcano plot displaying t test Difference versus -Log10(Welch’s t test p -value) for the mouse brain neuron BIN1iso1-TID interactome (z-score >1) B , on the left , heat map of Log2(intensity) for top 30 proteins, by descending z-score and a horizontal bar graph on the right displaying the average ratio BIN1iso1-TID/Cyto-TID. C , lollipop graph of Gene Ontology (GO) term analysis for biological process. Lines and lollipops are colored according to -Log10(FDR) and the sizes of the lollipops correspond to the number of genes with fold enrichment on the x-axis. D , GO term analysis for cellular component. Lines and lollipops are colored according to -Log10(FDR) and the sizes of the lollipops correspond to the number of genes with fold enrichment on the x-axis. E , GO term analysis for molecular function. Lines and lollipop are colored according to -Log10(FDR) and the sizes of the lollipops correspond to the number of genes with fold enrichment on the x-axis. F , presynaptic SynGO BP and CC terms cartoon for the BIN1iso1-TID interactome. Annotated with abundant terms and proteins from each term detected in interactome z-score >1. G , SynGO biological process heat map color coded according to -log10 (Q-value).

    Article Snippet: Da Vinci Green antibody diluent was used to dilute primary antibodies against V5 (Thermo Fisher # R96025 ), BIN1 (Proteintech #14647-1-AP), mAb BIN1 (clone 19H3) , CDK16 (Proteintech #10102-1-AP), PP2Ba (Santa Cruz # sc-17808), SYNJ1 (Atlas Antibodies #HPS011916), AAK1 (Atlas Antibodies #HPA020289), RANG (Atlas Antibodies #HPA065868), SYP (Sigma #S5768), PSD95 (Sigma # MABN68), and Streptavidin conjugates (Invitrogen #84547).

    Techniques:

    Validation of BIN1iso1 N2a cell and mouse brain neuron interactome top hits. A , a Circos plot showing the N2a cell BIN1 interactome overlap with the BIN1 mouse brain neuron interactome (z-score >1). The inner circle represents protein lists, where hits are arranged along the arc. Proteins that hit multiple lists are colored in dark orange , and genes unique to a list are shown in light orange . Purple curves link identical proteins between the datasets, while the blue curves link proteins that belong to the same enriched ontology term. The numbers indicate unique non-overlapping (715 in neurons; 262 in N2a cells) and overlapping (92) proteins in the BIN1 interactome dataset. Multiple isoforms of a protein were counted as one entry for the combined analysis of the two interactome datasets. B , heatmap of top 20 clusters with their representative enriched terms (one per cluster) across protein lists, colored by p -values. Generated using Metascape pathway and process enrichment analysis. C , ninety-two common proteins between N2a BIN1iso1-TID and BIN1iso1-TID mouse brain neurons with a z-score >1 are shown. A medium confidence edge threshold score >0.40 was applied in STRING to create interaction networks among these proteins. The node sizes represent the BIN1iso1-TID mouse brain interactome, and the nodes are color-coded with a gradient indicating the average ratio of the N2a BIN1-TID interactome. Representative terms that characterize a group of nodes are indicated. D , IF of BIN1iso1-TID brain using anti-SYNJ1 and anti-V5. Overlap of images (scale bar = 25 μm). Higher magnification images are z-stacks projected as a Sum (scale bar = 10 μm). E , PLA using anti-BIN1 and anti-SYNJ1 in brains of neuron-specific Emx-Cre: Bin1 knockout and Emx-Cre control mice. The Emx-Cre: Bin1 knockout mouse lacks BIN1 expression in neurons and oligodendrocytes in the forebrain, thus serving as a negative control for PLA. F , IF of BIN1iso1-TID brain using anti-RANG and anti-V5. Overlap of images (scale bar = 25 μm). Higher magnification images are z-stacks projected as a Sum (scale bar = 10 μm). G , PLA of BIN1iso1-TID using anti-V5 and anti-RANG. Co-stained with streptavidin to display biotinylated proteins. H , IF of BIN1iso1-TID brain using anti-PP2BA and anti-BIN1. Overlap of images (scale bar = 25 μm). Higher magnification images are z-stacks projected as a Sum (scale bar = 10 μm). I , PLA of BIN1iso1-TID using anti-PP2BA and anti-BIN1. Co-stained with streptavidin to display biotinylated proteins for reference.

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Proteomic Characterization of the Alzheimer’s Disease Risk Factor BIN1 Interactome

    doi: 10.1016/j.mcpro.2025.101055

    Figure Lengend Snippet: Validation of BIN1iso1 N2a cell and mouse brain neuron interactome top hits. A , a Circos plot showing the N2a cell BIN1 interactome overlap with the BIN1 mouse brain neuron interactome (z-score >1). The inner circle represents protein lists, where hits are arranged along the arc. Proteins that hit multiple lists are colored in dark orange , and genes unique to a list are shown in light orange . Purple curves link identical proteins between the datasets, while the blue curves link proteins that belong to the same enriched ontology term. The numbers indicate unique non-overlapping (715 in neurons; 262 in N2a cells) and overlapping (92) proteins in the BIN1 interactome dataset. Multiple isoforms of a protein were counted as one entry for the combined analysis of the two interactome datasets. B , heatmap of top 20 clusters with their representative enriched terms (one per cluster) across protein lists, colored by p -values. Generated using Metascape pathway and process enrichment analysis. C , ninety-two common proteins between N2a BIN1iso1-TID and BIN1iso1-TID mouse brain neurons with a z-score >1 are shown. A medium confidence edge threshold score >0.40 was applied in STRING to create interaction networks among these proteins. The node sizes represent the BIN1iso1-TID mouse brain interactome, and the nodes are color-coded with a gradient indicating the average ratio of the N2a BIN1-TID interactome. Representative terms that characterize a group of nodes are indicated. D , IF of BIN1iso1-TID brain using anti-SYNJ1 and anti-V5. Overlap of images (scale bar = 25 μm). Higher magnification images are z-stacks projected as a Sum (scale bar = 10 μm). E , PLA using anti-BIN1 and anti-SYNJ1 in brains of neuron-specific Emx-Cre: Bin1 knockout and Emx-Cre control mice. The Emx-Cre: Bin1 knockout mouse lacks BIN1 expression in neurons and oligodendrocytes in the forebrain, thus serving as a negative control for PLA. F , IF of BIN1iso1-TID brain using anti-RANG and anti-V5. Overlap of images (scale bar = 25 μm). Higher magnification images are z-stacks projected as a Sum (scale bar = 10 μm). G , PLA of BIN1iso1-TID using anti-V5 and anti-RANG. Co-stained with streptavidin to display biotinylated proteins. H , IF of BIN1iso1-TID brain using anti-PP2BA and anti-BIN1. Overlap of images (scale bar = 25 μm). Higher magnification images are z-stacks projected as a Sum (scale bar = 10 μm). I , PLA of BIN1iso1-TID using anti-PP2BA and anti-BIN1. Co-stained with streptavidin to display biotinylated proteins for reference.

    Article Snippet: Da Vinci Green antibody diluent was used to dilute primary antibodies against V5 (Thermo Fisher # R96025 ), BIN1 (Proteintech #14647-1-AP), mAb BIN1 (clone 19H3) , CDK16 (Proteintech #10102-1-AP), PP2Ba (Santa Cruz # sc-17808), SYNJ1 (Atlas Antibodies #HPS011916), AAK1 (Atlas Antibodies #HPA020289), RANG (Atlas Antibodies #HPA065868), SYP (Sigma #S5768), PSD95 (Sigma # MABN68), and Streptavidin conjugates (Invitrogen #84547).

    Techniques: Biomarker Discovery, Generated, Knock-Out, Control, Expressing, Negative Control, Staining

    Phosphorylation site analysis of BIN1iso1-TID neuronal interactome identifies AAK1 and CDK16. A , proteins identified through the phosphorylation site analysis of BIN1iso1-TID mouse brain neurons with a z-score >2 are shown with the detected phosphorylation sites mapped to them. A medium confidence edge threshold score >0.40 was used in STRING to identify interactions among the proteins. Proteins that localize to the synapse are colored orange, while the group with a green background represents endocytic proteins. Note that about half of the BIN1-proximal phosphoproteins meeting our z-score criteria remain as singlets. B , using PhosophoSitePlus prediction. This displays the probability for a specific kinase to phosphorylate the target protein at each experimentally observed phosphorylated site in the interactome. We set >90% as the threshold for inclusion of a particular kinase in our results. The kinases were tallied for phosphoproteins filtered from the BIN1iso1-TID interactome (z-score >2). C , IF of BIN1iso1-TID brain using anti-AAK1 and anti-V5. Overlap of low mag images (scale bar = 25 μm). Higher magnification images are z-stacks projected as a Sum (scale bar = 10 μm). D , PLA using anti-AAK1 and anti-BIN1 antibodies. Mice are neuron-specific Emx-Cre:BIN1 knockout and Emx-Cre control mice. The Emx-Cre: Bin1 knockout mouse lacks BIN1 expression in neurons and oligodendrocytes in the forebrain, thus serving as a negative control for PLA. E , IF of BIN1iso1-TID brain using anti-CDK16 and anti-V5 antibodies. Overlap of low mag images (scale bar = 25 μm). Higher magnification images are z-stacks projected as a Sum (scale bar = 10 μm). F , PLA of BIN1iso1-TID using anti-CDK16 and anti-V5 antibodies. Co-stained with streptavidin to display biotinylated proteins for reference.

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Proteomic Characterization of the Alzheimer’s Disease Risk Factor BIN1 Interactome

    doi: 10.1016/j.mcpro.2025.101055

    Figure Lengend Snippet: Phosphorylation site analysis of BIN1iso1-TID neuronal interactome identifies AAK1 and CDK16. A , proteins identified through the phosphorylation site analysis of BIN1iso1-TID mouse brain neurons with a z-score >2 are shown with the detected phosphorylation sites mapped to them. A medium confidence edge threshold score >0.40 was used in STRING to identify interactions among the proteins. Proteins that localize to the synapse are colored orange, while the group with a green background represents endocytic proteins. Note that about half of the BIN1-proximal phosphoproteins meeting our z-score criteria remain as singlets. B , using PhosophoSitePlus prediction. This displays the probability for a specific kinase to phosphorylate the target protein at each experimentally observed phosphorylated site in the interactome. We set >90% as the threshold for inclusion of a particular kinase in our results. The kinases were tallied for phosphoproteins filtered from the BIN1iso1-TID interactome (z-score >2). C , IF of BIN1iso1-TID brain using anti-AAK1 and anti-V5. Overlap of low mag images (scale bar = 25 μm). Higher magnification images are z-stacks projected as a Sum (scale bar = 10 μm). D , PLA using anti-AAK1 and anti-BIN1 antibodies. Mice are neuron-specific Emx-Cre:BIN1 knockout and Emx-Cre control mice. The Emx-Cre: Bin1 knockout mouse lacks BIN1 expression in neurons and oligodendrocytes in the forebrain, thus serving as a negative control for PLA. E , IF of BIN1iso1-TID brain using anti-CDK16 and anti-V5 antibodies. Overlap of low mag images (scale bar = 25 μm). Higher magnification images are z-stacks projected as a Sum (scale bar = 10 μm). F , PLA of BIN1iso1-TID using anti-CDK16 and anti-V5 antibodies. Co-stained with streptavidin to display biotinylated proteins for reference.

    Article Snippet: Da Vinci Green antibody diluent was used to dilute primary antibodies against V5 (Thermo Fisher # R96025 ), BIN1 (Proteintech #14647-1-AP), mAb BIN1 (clone 19H3) , CDK16 (Proteintech #10102-1-AP), PP2Ba (Santa Cruz # sc-17808), SYNJ1 (Atlas Antibodies #HPS011916), AAK1 (Atlas Antibodies #HPA020289), RANG (Atlas Antibodies #HPA065868), SYP (Sigma #S5768), PSD95 (Sigma # MABN68), and Streptavidin conjugates (Invitrogen #84547).

    Techniques: Phospho-proteomics, Knock-Out, Control, Expressing, Negative Control, Staining

    Comparison of instances of differential alternative splicing at transcriptional and protein level for Atp2a1 and Bin1 genes. Panels on the left show LeafCutter AS intron clusters with relative exon usage in the DM1 and WT control groups. Panels in the middle show qRT-PCR results with the splicing-specific probes (see ) covering the corresponding exon inclusion ( gray ) and exon exclusion ( blue ). Panels on the right show the corresponding relative abundances of peptides specific to exon inclusion or exclusion in the same sample groups. See also <xref ref-type=supplemental Fig. S4 for a UCSC genome browser-based display of these LeafCutter AS intron clusters. AS, alternative splicing; DM1, myotonic dystrophy, type 1. " width="100%" height="100%">

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Integrative Proteogenomics for Differential Expression and Splicing Variation in a DM1 Mouse Model

    doi: 10.1016/j.mcpro.2023.100683

    Figure Lengend Snippet: Comparison of instances of differential alternative splicing at transcriptional and protein level for Atp2a1 and Bin1 genes. Panels on the left show LeafCutter AS intron clusters with relative exon usage in the DM1 and WT control groups. Panels in the middle show qRT-PCR results with the splicing-specific probes (see ) covering the corresponding exon inclusion ( gray ) and exon exclusion ( blue ). Panels on the right show the corresponding relative abundances of peptides specific to exon inclusion or exclusion in the same sample groups. See also supplemental Fig. S4 for a UCSC genome browser-based display of these LeafCutter AS intron clusters. AS, alternative splicing; DM1, myotonic dystrophy, type 1.

    Article Snippet: Primary antibodies were obtained from Cell Signaling—Rb anti-BIN1 mAB (#51844) and Rb anti-SERCA1 mAb (#12293).

    Techniques: Comparison, Alternative Splicing, Control, Quantitative RT-PCR

    Overview of select genes of known importance to DM1, and summary of their performance in our work, summarized in columns (3) to (6)

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Integrative Proteogenomics for Differential Expression and Splicing Variation in a DM1 Mouse Model

    doi: 10.1016/j.mcpro.2023.100683

    Figure Lengend Snippet: Overview of select genes of known importance to DM1, and summary of their performance in our work, summarized in columns (3) to (6)

    Article Snippet: Primary antibodies were obtained from Cell Signaling—Rb anti-BIN1 mAB (#51844) and Rb anti-SERCA1 mAb (#12293).

    Techniques:

    RNA expression of T-tubule and SR genes RT-qPCR analysis of LV tissue for mRNA content of ( a ) bridging integrator-1, BIN1; junctophilin 2, Jph2; L-type calcium channel, LTCC; ryanodine 2, RyR2 and ( b ) SR calcium ATPase, SERCa2; phospholamban, PLN; alpha- and beta-myosin heavy chain, αMHC and βMHC. The binary log of ΔΔCt calculations of PCR cycle amplifications was used to determine the fold-change relative to the mean values of the Sham group. Bar graph shows mean ± SD for n = 4–6 hearts/group. Statistical analysis used one-way ANOVA with post-hoc Tukey’s multi-group comparisons: * p < 0.05 vs sham; ** p < 0.001 vs sham; # p < 0.01 vs MI + Veh.

    Journal: Molecular Medicine

    Article Title: Adverse transverse-tubule remodeling in a rat model of heart failure is attenuated with low-dose triiodothyronine treatment

    doi: 10.1186/s10020-019-0120-3

    Figure Lengend Snippet: RNA expression of T-tubule and SR genes RT-qPCR analysis of LV tissue for mRNA content of ( a ) bridging integrator-1, BIN1; junctophilin 2, Jph2; L-type calcium channel, LTCC; ryanodine 2, RyR2 and ( b ) SR calcium ATPase, SERCa2; phospholamban, PLN; alpha- and beta-myosin heavy chain, αMHC and βMHC. The binary log of ΔΔCt calculations of PCR cycle amplifications was used to determine the fold-change relative to the mean values of the Sham group. Bar graph shows mean ± SD for n = 4–6 hearts/group. Statistical analysis used one-way ANOVA with post-hoc Tukey’s multi-group comparisons: * p < 0.05 vs sham; ** p < 0.001 vs sham; # p < 0.01 vs MI + Veh.

    Article Snippet: Membranes were blocked with TBST/5% milk and incubated overnight at 4 °C with the following primary antibodies (all at 1:500 dilution): rabbit polyclonal anti-Ca v 1.2 antibody (cat. no. ACC-003; Alomone Labs, Jerusalem, Israel), mouse monoclonal (mAb) anti-RyR antibody (cat. no. MA3–916/C3–33; Invitrogen, ThermoFisher), mouse mAb anti-BIN1 (cat no.200–301-E63; Rockland Inc., Limerick PA), mouse mAb anti-Jph2 (cat no.600–401-CC5; Rockland Inc.).

    Techniques: RNA Expression, Quantitative RT-PCR

    Immunoblot analysis of T-tubule and SR proteins. a Representative immunoblots of microsomal fractions from LV tissues of each study group that were probed with anti-BIN1, Jph2, Ca v 1.2 and RyR2 antibodies. Observed molecular weights (kDa) of each protein are indicated. Ponceau red staining of the immunoblots are shown with molecular weight markers. b , c Bar graphs show the quantitation of the luminescence intensity of the indicated protein bands, normalized to Ponceau red staining and expressed relative to the mean sham values. Values are mean ± SD for n = 4–6 hearts/group. Statistical analysis using one-way ANOVA with post-hoc Tukey’s multiple group comparisons; p- values are indicated

    Journal: Molecular Medicine

    Article Title: Adverse transverse-tubule remodeling in a rat model of heart failure is attenuated with low-dose triiodothyronine treatment

    doi: 10.1186/s10020-019-0120-3

    Figure Lengend Snippet: Immunoblot analysis of T-tubule and SR proteins. a Representative immunoblots of microsomal fractions from LV tissues of each study group that were probed with anti-BIN1, Jph2, Ca v 1.2 and RyR2 antibodies. Observed molecular weights (kDa) of each protein are indicated. Ponceau red staining of the immunoblots are shown with molecular weight markers. b , c Bar graphs show the quantitation of the luminescence intensity of the indicated protein bands, normalized to Ponceau red staining and expressed relative to the mean sham values. Values are mean ± SD for n = 4–6 hearts/group. Statistical analysis using one-way ANOVA with post-hoc Tukey’s multiple group comparisons; p- values are indicated

    Article Snippet: Membranes were blocked with TBST/5% milk and incubated overnight at 4 °C with the following primary antibodies (all at 1:500 dilution): rabbit polyclonal anti-Ca v 1.2 antibody (cat. no. ACC-003; Alomone Labs, Jerusalem, Israel), mouse monoclonal (mAb) anti-RyR antibody (cat. no. MA3–916/C3–33; Invitrogen, ThermoFisher), mouse mAb anti-BIN1 (cat no.200–301-E63; Rockland Inc., Limerick PA), mouse mAb anti-Jph2 (cat no.600–401-CC5; Rockland Inc.).

    Techniques: Western Blot, Staining, Molecular Weight, Quantitation Assay

    Single allele deletion of Bin1 does not alter APP or BACE localization or Aβ levels. A, forebrain homogenates from 4-month-old Bin1+/− mice and WT littermate controls were analyzed by immunoblotting for BIN1, APP, BACE1, and Amph1 levels. B, quantitative analysis of BIN1:H (top) and BIN1:L (bottom) in forebrain lysates from WT and Bin1+/− mice. C, top panel, immunofluorescent staining for BIN1 (magenta) and BACE1 (green) in the hippocampal CA3 region from WT and Bin1+/− mice. Scale bar, 50 μm. Bottom panel, higher magnification of immunofluorescent staining for APP (red) and BACE1 (green) in CA3 neurons and mossy fibers (MF) of the mouse hippocampus. Scale bar, 20 μm. D, forebrain lysates from WT and Bin1+/− mice were analyzed for steady-state levels of endogenous Aβ40 and Aβ42 using a V-PLEX 4G8 immunoassay (Aβ40, n = 12 per genotype; Aβ42, n = 11 WT, and 12 Bin1+/−).

    Journal: The Journal of Biological Chemistry

    Article Title: Reduction of the expression of the late-onset Alzheimer's disease (AD) risk-factor BIN1 does not affect amyloid pathology in an AD mouse model

    doi: 10.1074/jbc.RA118.006379

    Figure Lengend Snippet: Single allele deletion of Bin1 does not alter APP or BACE localization or Aβ levels. A, forebrain homogenates from 4-month-old Bin1+/− mice and WT littermate controls were analyzed by immunoblotting for BIN1, APP, BACE1, and Amph1 levels. B, quantitative analysis of BIN1:H (top) and BIN1:L (bottom) in forebrain lysates from WT and Bin1+/− mice. C, top panel, immunofluorescent staining for BIN1 (magenta) and BACE1 (green) in the hippocampal CA3 region from WT and Bin1+/− mice. Scale bar, 50 μm. Bottom panel, higher magnification of immunofluorescent staining for APP (red) and BACE1 (green) in CA3 neurons and mossy fibers (MF) of the mouse hippocampus. Scale bar, 20 μm. D, forebrain lysates from WT and Bin1+/− mice were analyzed for steady-state levels of endogenous Aβ40 and Aβ42 using a V-PLEX 4G8 immunoassay (Aβ40, n = 12 per genotype; Aβ42, n = 11 WT, and 12 Bin1+/−).

    Article Snippet: The following antibodies were used: mouse anti-Aβ mAb 3D6 (a kind gift from the late Dale Schenk, Elan Corporation PLC, South San Francisco, CA), anti–β-actin mAb (660099-1, Proteintech), anti–amphiphysin 1 mAb 8 (sc-21710, Santa Cruz Biotechnology), rabbit anti-APP C-terminal pAb CTM1 ( 54 ), pAb Y188 (ab32136, Abcam), rabbit anti-BACE1 mAb (EPR3956, Abcam), anti-BACE1 human IgG (a kind gift from Jasvinder Atwal, Genentech, South San Francisco, CA), rabbit anti-BIN1 pAb (14647-1, Proteintech), rabbit anti-BIN1 mAb EPR13463-25 (ab185950, Abcam), mouse anti-GFAP mAb (G3893, Sigma), mouse anti-NeuN mAb (MAB377, Millipore), mouse anti-PSD-95 monoclonal ( {"type":"entrez-protein","attrs":{"text":"P78352","term_id":"71658825","term_text":"P78352"}} P78352 , NeuroMab, UC Davis), and mouse anti-synaptophysin mAb SVP38 (sc-12737, Santa Cruz Biotechnology).

    Techniques: Western Blot, Staining

    Reduction of BIN1 expression does not alter amyloid deposition in female 5XFAD mice. A, representative images of Aβ deposit core staining with thioflavin S and mAb 3D6 immunostaining in 4-month-old female 5XFAD and 5XFADBin1+/− mice at 4 months of age. B, quantification of amyloid burden identified by thioflavin S staining (n = 10 per genotype). C, quantification of amyloid burden identified by mAb 3D6 immunostaining (n = 9 5XFAD and 10 5XFADBin1+/−). Forebrain tissue from 4-month-old female 5XFAD and 5XFADBin1+/− mice was sequentially extracted in TBS and formic acid to generate soluble and insoluble Aβ fractions. D and E, the levels of soluble and insoluble Aβ40 and Aβ42 were measured by V-PLEX 6E10 immunoassay (n = 10 per genotype). F, immunoblot analysis of APP, BACE1, GFAP, and Amph1 levels in 5XFAD and 5XFADBin1+/− mice. G, quantitative analysis of the levels of BACE1, APP, GFAP, and Amph1, normalized to actin (n = 8 per genotype). H, immunoblot analysis of full-length APP (FL-APP) and APP C-terminal fragments (CTF) and quantification of β-CTF normalized to FL-APP (n = 4 per genotype).

    Journal: The Journal of Biological Chemistry

    Article Title: Reduction of the expression of the late-onset Alzheimer's disease (AD) risk-factor BIN1 does not affect amyloid pathology in an AD mouse model

    doi: 10.1074/jbc.RA118.006379

    Figure Lengend Snippet: Reduction of BIN1 expression does not alter amyloid deposition in female 5XFAD mice. A, representative images of Aβ deposit core staining with thioflavin S and mAb 3D6 immunostaining in 4-month-old female 5XFAD and 5XFADBin1+/− mice at 4 months of age. B, quantification of amyloid burden identified by thioflavin S staining (n = 10 per genotype). C, quantification of amyloid burden identified by mAb 3D6 immunostaining (n = 9 5XFAD and 10 5XFADBin1+/−). Forebrain tissue from 4-month-old female 5XFAD and 5XFADBin1+/− mice was sequentially extracted in TBS and formic acid to generate soluble and insoluble Aβ fractions. D and E, the levels of soluble and insoluble Aβ40 and Aβ42 were measured by V-PLEX 6E10 immunoassay (n = 10 per genotype). F, immunoblot analysis of APP, BACE1, GFAP, and Amph1 levels in 5XFAD and 5XFADBin1+/− mice. G, quantitative analysis of the levels of BACE1, APP, GFAP, and Amph1, normalized to actin (n = 8 per genotype). H, immunoblot analysis of full-length APP (FL-APP) and APP C-terminal fragments (CTF) and quantification of β-CTF normalized to FL-APP (n = 4 per genotype).

    Article Snippet: The following antibodies were used: mouse anti-Aβ mAb 3D6 (a kind gift from the late Dale Schenk, Elan Corporation PLC, South San Francisco, CA), anti–β-actin mAb (660099-1, Proteintech), anti–amphiphysin 1 mAb 8 (sc-21710, Santa Cruz Biotechnology), rabbit anti-APP C-terminal pAb CTM1 ( 54 ), pAb Y188 (ab32136, Abcam), rabbit anti-BACE1 mAb (EPR3956, Abcam), anti-BACE1 human IgG (a kind gift from Jasvinder Atwal, Genentech, South San Francisco, CA), rabbit anti-BIN1 pAb (14647-1, Proteintech), rabbit anti-BIN1 mAb EPR13463-25 (ab185950, Abcam), mouse anti-GFAP mAb (G3893, Sigma), mouse anti-NeuN mAb (MAB377, Millipore), mouse anti-PSD-95 monoclonal ( {"type":"entrez-protein","attrs":{"text":"P78352","term_id":"71658825","term_text":"P78352"}} P78352 , NeuroMab, UC Davis), and mouse anti-synaptophysin mAb SVP38 (sc-12737, Santa Cruz Biotechnology).

    Techniques: Expressing, Staining, Immunostaining, Western Blot

    Reduction in BIN1 expression does not alter behavioral deficits in the absence of changes in amyloid burden in female 5XFAD mice. A, the percentage of time in the open arms of the elevated-plus maze was quantified for 4-month-old 5XFAD and 5XFADBin1+/− mice. B, discrimination index score from novel object recognition for 5XFAD and 5XFADBin1+/− mice. Box plots show the median, maximum, and minimum values. C, the number of arm entries over the 8-min Y-maze trial. D, the number of spontaneous alternations in the Y-maze over the 8-min trial period. E, freezing behavior in female 5XFAD and 5XFADBin1+/− mice during day 1 of fear conditioning. Lightning bolts represent delivery of the shock stimulus. F, freezing behavior in female 5XFAD and 5XFADBin1+/− mice on day 2 of fear conditioning, 24 h after shock application (n = 11 for both genotypes in all analyses).

    Journal: The Journal of Biological Chemistry

    Article Title: Reduction of the expression of the late-onset Alzheimer's disease (AD) risk-factor BIN1 does not affect amyloid pathology in an AD mouse model

    doi: 10.1074/jbc.RA118.006379

    Figure Lengend Snippet: Reduction in BIN1 expression does not alter behavioral deficits in the absence of changes in amyloid burden in female 5XFAD mice. A, the percentage of time in the open arms of the elevated-plus maze was quantified for 4-month-old 5XFAD and 5XFADBin1+/− mice. B, discrimination index score from novel object recognition for 5XFAD and 5XFADBin1+/− mice. Box plots show the median, maximum, and minimum values. C, the number of arm entries over the 8-min Y-maze trial. D, the number of spontaneous alternations in the Y-maze over the 8-min trial period. E, freezing behavior in female 5XFAD and 5XFADBin1+/− mice during day 1 of fear conditioning. Lightning bolts represent delivery of the shock stimulus. F, freezing behavior in female 5XFAD and 5XFADBin1+/− mice on day 2 of fear conditioning, 24 h after shock application (n = 11 for both genotypes in all analyses).

    Article Snippet: The following antibodies were used: mouse anti-Aβ mAb 3D6 (a kind gift from the late Dale Schenk, Elan Corporation PLC, South San Francisco, CA), anti–β-actin mAb (660099-1, Proteintech), anti–amphiphysin 1 mAb 8 (sc-21710, Santa Cruz Biotechnology), rabbit anti-APP C-terminal pAb CTM1 ( 54 ), pAb Y188 (ab32136, Abcam), rabbit anti-BACE1 mAb (EPR3956, Abcam), anti-BACE1 human IgG (a kind gift from Jasvinder Atwal, Genentech, South San Francisco, CA), rabbit anti-BIN1 pAb (14647-1, Proteintech), rabbit anti-BIN1 mAb EPR13463-25 (ab185950, Abcam), mouse anti-GFAP mAb (G3893, Sigma), mouse anti-NeuN mAb (MAB377, Millipore), mouse anti-PSD-95 monoclonal ( {"type":"entrez-protein","attrs":{"text":"P78352","term_id":"71658825","term_text":"P78352"}} P78352 , NeuroMab, UC Davis), and mouse anti-synaptophysin mAb SVP38 (sc-12737, Santa Cruz Biotechnology).

    Techniques: Expressing

    Characterization of neuronal conditional Bin1 knockout. A, brain homogenates from the hippocampus and cortex of 4-month-old Bin1Fl/Fl and Bin1Fl/Fl:Syn-Cre were analyzed for BIN1 expression by immunoblotting. B, quantification of BIN1:H and BIN1:L in the cortex and hippocampus (Hipp) (n = 6 Bin1Fl/Fl and 4 Bin1Fl/Fl:Syn-Cre). C, immunohistochemical analysis of BIN1 and NeuN expression in the CA1 (left panel) and CA3 (right panel) of Bin1Fl/Fl and Bin1Fl/Fl:Syn-Cre mice. D, PSD and non-PSD (NP) membrane fractions were analyzed for PSD-95, synaptophysin, BIN1, APP, and BACE1 by immunoblotting. E, quantitative analysis of BIN1:H and BIN1:L in crude pre-and post-synaptic membranes (n = 4). F, non-PSD membranes were analyzed for APP, BACE1, Amph1, and synaptophysin by immunoblotting. G, quantitative analysis of APP and BACE1 levels in pre-synaptic fractions (n = 4). H, forebrain lysates were analyzed for steady-state levels of Aβ40 and Aβ42 using V-PLEX 4G8 immunoassay (Aβ40, n = 10 per genotype; Aβ42, n = 9 Bin1Fl/Fl and 10 Bin1Fl/Fl:Syn-Cre).

    Journal: The Journal of Biological Chemistry

    Article Title: Reduction of the expression of the late-onset Alzheimer's disease (AD) risk-factor BIN1 does not affect amyloid pathology in an AD mouse model

    doi: 10.1074/jbc.RA118.006379

    Figure Lengend Snippet: Characterization of neuronal conditional Bin1 knockout. A, brain homogenates from the hippocampus and cortex of 4-month-old Bin1Fl/Fl and Bin1Fl/Fl:Syn-Cre were analyzed for BIN1 expression by immunoblotting. B, quantification of BIN1:H and BIN1:L in the cortex and hippocampus (Hipp) (n = 6 Bin1Fl/Fl and 4 Bin1Fl/Fl:Syn-Cre). C, immunohistochemical analysis of BIN1 and NeuN expression in the CA1 (left panel) and CA3 (right panel) of Bin1Fl/Fl and Bin1Fl/Fl:Syn-Cre mice. D, PSD and non-PSD (NP) membrane fractions were analyzed for PSD-95, synaptophysin, BIN1, APP, and BACE1 by immunoblotting. E, quantitative analysis of BIN1:H and BIN1:L in crude pre-and post-synaptic membranes (n = 4). F, non-PSD membranes were analyzed for APP, BACE1, Amph1, and synaptophysin by immunoblotting. G, quantitative analysis of APP and BACE1 levels in pre-synaptic fractions (n = 4). H, forebrain lysates were analyzed for steady-state levels of Aβ40 and Aβ42 using V-PLEX 4G8 immunoassay (Aβ40, n = 10 per genotype; Aβ42, n = 9 Bin1Fl/Fl and 10 Bin1Fl/Fl:Syn-Cre).

    Article Snippet: The following antibodies were used: mouse anti-Aβ mAb 3D6 (a kind gift from the late Dale Schenk, Elan Corporation PLC, South San Francisco, CA), anti–β-actin mAb (660099-1, Proteintech), anti–amphiphysin 1 mAb 8 (sc-21710, Santa Cruz Biotechnology), rabbit anti-APP C-terminal pAb CTM1 ( 54 ), pAb Y188 (ab32136, Abcam), rabbit anti-BACE1 mAb (EPR3956, Abcam), anti-BACE1 human IgG (a kind gift from Jasvinder Atwal, Genentech, South San Francisco, CA), rabbit anti-BIN1 pAb (14647-1, Proteintech), rabbit anti-BIN1 mAb EPR13463-25 (ab185950, Abcam), mouse anti-GFAP mAb (G3893, Sigma), mouse anti-NeuN mAb (MAB377, Millipore), mouse anti-PSD-95 monoclonal ( {"type":"entrez-protein","attrs":{"text":"P78352","term_id":"71658825","term_text":"P78352"}} P78352 , NeuroMab, UC Davis), and mouse anti-synaptophysin mAb SVP38 (sc-12737, Santa Cruz Biotechnology).

    Techniques: Knock-Out, Expressing, Western Blot, Immunohistochemical staining

    (A) The top 3 GO biological clusters of 40 genes identified in GWAS studies of LOAD risk. (B) Top 5 individual GO terms within the top GO cluster. The dotted line indicates significance level of P= 0.05. (C) Protein interaction network of 11 LOAD risk genes present in the vesicle-mediated transport/endocytosis GO cluster. Network was generated using the GeneMANIA in Cytoscape. This network has a significant enrichment of proteins present in the PSD (P=0.0002). (D) BIN1 protein interaction network reveals a significant enrichment of PSD genes (P= 4.0×10 −7 ), trafficking genes (P= 0.0002), and GTPase-related genes (P= 5.0×10 −5 ). (E) Top: Single plane SIM image of a dendritic region stained for Bin1 and presynaptic marker synapsin1. GFP cell fill is outlined (see ). Scale bar = 5 μm Bottom: Representative SIM images of boxed spines in above image. (F) Top: Single plane SIM image of a dendritic region stained for Bin1 and GluA1. GFP cell fill is outlined (see ). Scale bar = 5 μm Bottom: Representative SIM images of boxed spines above image. (G) Representative 3D reconstruction of a stack of SIM images showing the relative localization of Bin1 and GluA1 within a spine. Scale bar = 50 nm (H) Immuno-electron microscopy for Bin1. Left micrograph is low-magnification view of neuropil. Labeled spines have been colorized green and axon terminals pink; a thin immunopositive dendritic process (left) is colorized blue. (I) Bin1-associated gold particles in immuno-EM images are found in the synaptic membrane/PSD, as well as near the PSD in extrasynaptic membranes.

    Journal: Molecular psychiatry

    Article Title: A novel role for the late-onset Alzheimer’s disease (LOAD)-associated protein Bin1 in regulating postsynaptic trafficking and glutamatergic signaling

    doi: 10.1038/s41380-019-0407-3

    Figure Lengend Snippet: (A) The top 3 GO biological clusters of 40 genes identified in GWAS studies of LOAD risk. (B) Top 5 individual GO terms within the top GO cluster. The dotted line indicates significance level of P= 0.05. (C) Protein interaction network of 11 LOAD risk genes present in the vesicle-mediated transport/endocytosis GO cluster. Network was generated using the GeneMANIA in Cytoscape. This network has a significant enrichment of proteins present in the PSD (P=0.0002). (D) BIN1 protein interaction network reveals a significant enrichment of PSD genes (P= 4.0×10 −7 ), trafficking genes (P= 0.0002), and GTPase-related genes (P= 5.0×10 −5 ). (E) Top: Single plane SIM image of a dendritic region stained for Bin1 and presynaptic marker synapsin1. GFP cell fill is outlined (see ). Scale bar = 5 μm Bottom: Representative SIM images of boxed spines in above image. (F) Top: Single plane SIM image of a dendritic region stained for Bin1 and GluA1. GFP cell fill is outlined (see ). Scale bar = 5 μm Bottom: Representative SIM images of boxed spines above image. (G) Representative 3D reconstruction of a stack of SIM images showing the relative localization of Bin1 and GluA1 within a spine. Scale bar = 50 nm (H) Immuno-electron microscopy for Bin1. Left micrograph is low-magnification view of neuropil. Labeled spines have been colorized green and axon terminals pink; a thin immunopositive dendritic process (left) is colorized blue. (I) Bin1-associated gold particles in immuno-EM images are found in the synaptic membrane/PSD, as well as near the PSD in extrasynaptic membranes.

    Article Snippet: The following primary antibodies were used (mAb: monoclonal antibody, pAb: polyclonal antibody) : Bin1 mAb (Millipore Cat# 05–449), Map2 mAb (Millipore Cat# MAB3418), GluA1 N-term mAb (Millipore Cat# MAB2263), GluA1 C-term pAb (Millipore Cat# ABN241), Synapsin mAb (CellSignaling Cat# 5297), PSD95 mAb (Neuromab Cat# 75–028), CHC pAb (CellSignaling Cat# 2410), Rab5 pAb (CellSignaling Cat# 2143), EEA1 pAb (CellSignaling Cat# 2411), Rab11 pAb (Sigma Cat# R5903), Arf6 pAb (Abcam Cat# ab77581), Active Arf6 mAb (New East Biosciences, Cat# 26918), CaMKIIα mAb (Millipore Cat# 05–532), DsRed pAb (to identify mCherry expression, Clontech Cat# 632496), GFP chicken pAb GFP (to identify GFP expression, Abcam Cat# ab13970).

    Techniques: Generated, Staining, Marker, Immuno-Electron Microscopy, Labeling, Membrane

    (A) Single plane SIM images of relative localization of Bin1 and major trafficking markers in the dendritic compartment. Regions in boxes are shown below each image set in high magnification where region of co-localization are highlighted. Scale bar = 5 μm. (B) Quantification of Bin1 co-localization with major trafficking markers based on SIM images. One-way ANOVA with Bonferroni post-hoc tests * P<0.05, ** P<0.01 (C) Left: Representative flattened confocal images of Rab11 staining in GFP filled control (scr) and Bin1 knockdown (kd) neurons. The outline indicates the GFP-cell fill . Scale bar = 10 μm. Right: Quantification of area occupied by and number of Rab11 positive puncta in scr and kd neurons (n = 12 cells). Area occupied: Mann-Whitney test; Puncta number: unpaired t-test *** P<0.001, **** P<0.0001 (D) Single plane SIM images of the relative localization of Bin1 and Arf6 in dendrites and spines. Scale bar = 5 μm. (E) Quantification of Bin1 co-localization with trafficking markers in SIM images. One-way ANOVA with Bonferroni post-hoc tests ** P<0.01, *** P<0.001 (F) Representative 3D reconstruction of a stack of SIM images showing the relative localization of Bin1 and Arf6 in spines. Inset enlargement; scale bar = 50 nm. (G) Montage of spine depicted in (F) to illustrate Arf6 and Bin1 contact sites (white arrowheads). (H) Results of co-immunoprecipitation experiment that demonstrates pulldown of Arf6 with Bin1 immunoprecipitation from rat cortex homogenates. (I) Flattened confocal image showing proximity-ligation assay (PLA) reveals an interaction of Bin1 and Arf6 in rat cortical neuron cultures, reflected by green PLA puncta. Red outline represents mCherry cell-filled pyramidal neuron. Inset: Zoomed in view of PLA puncta in dendritic spines and shaft. Scale bar = 20 μm (J) Quantification of PLA puncta normalized to number of cell bodies in the imaging frame (identified by DAPI nuclear staining). Bin1 Ab and Arf6 Ab conditions represent negative controls where each antibody was used singly in the PLA assay. Kruskal-Wallis with Dunn’s post-hoc tests * P<0.05, *** P<0.001

    Journal: Molecular psychiatry

    Article Title: A novel role for the late-onset Alzheimer’s disease (LOAD)-associated protein Bin1 in regulating postsynaptic trafficking and glutamatergic signaling

    doi: 10.1038/s41380-019-0407-3

    Figure Lengend Snippet: (A) Single plane SIM images of relative localization of Bin1 and major trafficking markers in the dendritic compartment. Regions in boxes are shown below each image set in high magnification where region of co-localization are highlighted. Scale bar = 5 μm. (B) Quantification of Bin1 co-localization with major trafficking markers based on SIM images. One-way ANOVA with Bonferroni post-hoc tests * P<0.05, ** P<0.01 (C) Left: Representative flattened confocal images of Rab11 staining in GFP filled control (scr) and Bin1 knockdown (kd) neurons. The outline indicates the GFP-cell fill . Scale bar = 10 μm. Right: Quantification of area occupied by and number of Rab11 positive puncta in scr and kd neurons (n = 12 cells). Area occupied: Mann-Whitney test; Puncta number: unpaired t-test *** P<0.001, **** P<0.0001 (D) Single plane SIM images of the relative localization of Bin1 and Arf6 in dendrites and spines. Scale bar = 5 μm. (E) Quantification of Bin1 co-localization with trafficking markers in SIM images. One-way ANOVA with Bonferroni post-hoc tests ** P<0.01, *** P<0.001 (F) Representative 3D reconstruction of a stack of SIM images showing the relative localization of Bin1 and Arf6 in spines. Inset enlargement; scale bar = 50 nm. (G) Montage of spine depicted in (F) to illustrate Arf6 and Bin1 contact sites (white arrowheads). (H) Results of co-immunoprecipitation experiment that demonstrates pulldown of Arf6 with Bin1 immunoprecipitation from rat cortex homogenates. (I) Flattened confocal image showing proximity-ligation assay (PLA) reveals an interaction of Bin1 and Arf6 in rat cortical neuron cultures, reflected by green PLA puncta. Red outline represents mCherry cell-filled pyramidal neuron. Inset: Zoomed in view of PLA puncta in dendritic spines and shaft. Scale bar = 20 μm (J) Quantification of PLA puncta normalized to number of cell bodies in the imaging frame (identified by DAPI nuclear staining). Bin1 Ab and Arf6 Ab conditions represent negative controls where each antibody was used singly in the PLA assay. Kruskal-Wallis with Dunn’s post-hoc tests * P<0.05, *** P<0.001

    Article Snippet: The following primary antibodies were used (mAb: monoclonal antibody, pAb: polyclonal antibody) : Bin1 mAb (Millipore Cat# 05–449), Map2 mAb (Millipore Cat# MAB3418), GluA1 N-term mAb (Millipore Cat# MAB2263), GluA1 C-term pAb (Millipore Cat# ABN241), Synapsin mAb (CellSignaling Cat# 5297), PSD95 mAb (Neuromab Cat# 75–028), CHC pAb (CellSignaling Cat# 2410), Rab5 pAb (CellSignaling Cat# 2143), EEA1 pAb (CellSignaling Cat# 2411), Rab11 pAb (Sigma Cat# R5903), Arf6 pAb (Abcam Cat# ab77581), Active Arf6 mAb (New East Biosciences, Cat# 26918), CaMKIIα mAb (Millipore Cat# 05–532), DsRed pAb (to identify mCherry expression, Clontech Cat# 632496), GFP chicken pAb GFP (to identify GFP expression, Abcam Cat# ab13970).

    Techniques: Staining, Control, Knockdown, MANN-WHITNEY, Immunoprecipitation, Proximity Ligation Assay, Imaging

    (A) Left: Representative flattened confocal images of Arf6 staining in GFP filled control (scr) and Bin1 knockdown (kd) neurons. The outline indicates the GFP-cell fill (see ). Scale bar = 10 μm Right: Quantification of area occupied by and number of Arf6 positive puncta in scr and kd neurons (scr: n = 19 cells, kd = 17 cells). Area occupied: Mann-Whitney test; Puncta number: Welch’s t-test *** P<0.001 (B) Representative single plane confocal images showing total and active Arf6 staining in N2A cells with and without Bin1-GFP transfection. Scale bar = 5 μm (C) Quantification of active/total Arf6 ratio in N2A cells transfected with GFP (n=16 cells) or Bin1-GFP (n=16 cells). All results were normalized to the average of the value of GFP transfection condition for each experiment. Mann-Whitney test *** P<0.001 (D) Representative flattened confocal images of dendrites in GFP-filled neurons expressing scrambled (scr) or Bin1 knockdown (kd) plasmids. Scale bar = 10 μm (E) Quantification shows that knockdown of Bin1 decreases spine size without altering spine density (n = 26 cells for each condition). Spine size: Mann-Whitney test; Spine density: unpaired t-test **** P<0.0001 (F) Histogram showing frequency distribution of all individual spine areas measured in (E) (scr: n = 1049 spines, kd: n = 1120 spines). (G) Representative flattened confocal images of dendrites in GFP-filled neurons in control (ctrl) and Bin1 overexpressing (ox) neurons. Scale bar = 10 μm (H) Quantification shows that overexpression of Bin1 leads to decreased spine density and increased spine size (ctrl: n = 24 cells, ox: n = 20 cells). Spine size: Mann-Whitney test; Spine density: unpaired t-test * P<0.05, *** P<0.001 (I) Histogram showing frequency distribution of all individual spine areas measured in (H) (ctrl: n = 2407 spines, ox: n = 1740 spines) (J) Representative flattened confocal images of PSD95 in scr and kd GFP-expressing neurons. Scale bar = 10 μm (K) The size of PSD95 puncta is not altered in Bin1 knockdown neurons (scr: n = 10 cells, kd = 12 cells). Welch’s t-test.

    Journal: Molecular psychiatry

    Article Title: A novel role for the late-onset Alzheimer’s disease (LOAD)-associated protein Bin1 in regulating postsynaptic trafficking and glutamatergic signaling

    doi: 10.1038/s41380-019-0407-3

    Figure Lengend Snippet: (A) Left: Representative flattened confocal images of Arf6 staining in GFP filled control (scr) and Bin1 knockdown (kd) neurons. The outline indicates the GFP-cell fill (see ). Scale bar = 10 μm Right: Quantification of area occupied by and number of Arf6 positive puncta in scr and kd neurons (scr: n = 19 cells, kd = 17 cells). Area occupied: Mann-Whitney test; Puncta number: Welch’s t-test *** P<0.001 (B) Representative single plane confocal images showing total and active Arf6 staining in N2A cells with and without Bin1-GFP transfection. Scale bar = 5 μm (C) Quantification of active/total Arf6 ratio in N2A cells transfected with GFP (n=16 cells) or Bin1-GFP (n=16 cells). All results were normalized to the average of the value of GFP transfection condition for each experiment. Mann-Whitney test *** P<0.001 (D) Representative flattened confocal images of dendrites in GFP-filled neurons expressing scrambled (scr) or Bin1 knockdown (kd) plasmids. Scale bar = 10 μm (E) Quantification shows that knockdown of Bin1 decreases spine size without altering spine density (n = 26 cells for each condition). Spine size: Mann-Whitney test; Spine density: unpaired t-test **** P<0.0001 (F) Histogram showing frequency distribution of all individual spine areas measured in (E) (scr: n = 1049 spines, kd: n = 1120 spines). (G) Representative flattened confocal images of dendrites in GFP-filled neurons in control (ctrl) and Bin1 overexpressing (ox) neurons. Scale bar = 10 μm (H) Quantification shows that overexpression of Bin1 leads to decreased spine density and increased spine size (ctrl: n = 24 cells, ox: n = 20 cells). Spine size: Mann-Whitney test; Spine density: unpaired t-test * P<0.05, *** P<0.001 (I) Histogram showing frequency distribution of all individual spine areas measured in (H) (ctrl: n = 2407 spines, ox: n = 1740 spines) (J) Representative flattened confocal images of PSD95 in scr and kd GFP-expressing neurons. Scale bar = 10 μm (K) The size of PSD95 puncta is not altered in Bin1 knockdown neurons (scr: n = 10 cells, kd = 12 cells). Welch’s t-test.

    Article Snippet: The following primary antibodies were used (mAb: monoclonal antibody, pAb: polyclonal antibody) : Bin1 mAb (Millipore Cat# 05–449), Map2 mAb (Millipore Cat# MAB3418), GluA1 N-term mAb (Millipore Cat# MAB2263), GluA1 C-term pAb (Millipore Cat# ABN241), Synapsin mAb (CellSignaling Cat# 5297), PSD95 mAb (Neuromab Cat# 75–028), CHC pAb (CellSignaling Cat# 2410), Rab5 pAb (CellSignaling Cat# 2143), EEA1 pAb (CellSignaling Cat# 2411), Rab11 pAb (Sigma Cat# R5903), Arf6 pAb (Abcam Cat# ab77581), Active Arf6 mAb (New East Biosciences, Cat# 26918), CaMKIIα mAb (Millipore Cat# 05–532), DsRed pAb (to identify mCherry expression, Clontech Cat# 632496), GFP chicken pAb GFP (to identify GFP expression, Abcam Cat# ab13970).

    Techniques: Staining, Control, Knockdown, MANN-WHITNEY, Transfection, Expressing, Over Expression

    (A) Representative traces of currents recorded from scr and kd treated cortical neurons (B) Quantification of AMPA mEPSCs in cortical neurons reveals a significant reduction of mEPSC amplitude upon Bin1 knockdown (kd) (scr: n = 14 cells, kd = 22 cells). Unpaired t-test ** P<0.01 (C) Representative traces of currents recorded from control and Bin1 overexpressing (ox) cortical neurons (D) Quantification of AMPA mEPSCs shows that Bin1 overexpression does not affect average mEPSC amplitude but shifts distribution of values toward more high amplitudes (scr: n = 24 cells, kd: n = 22 cells). Mann-Whitney test. (E) Representative single plane SIM image of individual spines and line scans showing areas of colocalization between Bin1 and GluA1. (F) Representative 3D reconstruction of a stack of SIM images, showing the relative localization of Bin1 and GluA1 in spines. Inset, higher-magnification view; scale bar = 50 nm. (G) Montage of spine depicted in (F) to illustrate GluA1 and Bin1 contact sites (white arrowheads). (H) 3D surface plot showing total area of GluA1 staining (y-axis) as a function of both spine area (x-axis) and total area of Bin1 staining (z-axis). Legend shows color-coding for y-axis values on graph. (I) 3D surface plot showing total area of surface GluA1 (surfGluA1) staining (y-axis) as a function of both total area of Bin1 staining (x-axis) and spine area (z-axis). Legend shows color-coding for y-axis values on graph. (J) Reciprocal coimmunoprecipitation of Bin1 and GluA1 from rat cortex homogenate suggests participation in common complexes. (K) Flattened confocal image showing proximity-ligation assay (PLA) reveals an interaction of Bin1 and GluA1 in rat cortical neuron cultures, reflected by green PLA puncta. Red outline represents mCherry cell-filled pyramidal neuron. White arrowheads denote instances of interaction sites at dendritic spines. Inset: Zoomed in view of PLA puncta in dendritic spines and shaft. Scale bar = 20 μm (L) Quantification of PLA puncta normalized to number of cell bodies in the imaging frame (identified by DAPI nuclear staining). Bin1 Ab and GluA1 Ab conditions represent negative controls where each antibody was used singly in the PLA assay. Kruskal-Wallis with Dunn’s post-hoc tests * P<0.05, ** P<0.01

    Journal: Molecular psychiatry

    Article Title: A novel role for the late-onset Alzheimer’s disease (LOAD)-associated protein Bin1 in regulating postsynaptic trafficking and glutamatergic signaling

    doi: 10.1038/s41380-019-0407-3

    Figure Lengend Snippet: (A) Representative traces of currents recorded from scr and kd treated cortical neurons (B) Quantification of AMPA mEPSCs in cortical neurons reveals a significant reduction of mEPSC amplitude upon Bin1 knockdown (kd) (scr: n = 14 cells, kd = 22 cells). Unpaired t-test ** P<0.01 (C) Representative traces of currents recorded from control and Bin1 overexpressing (ox) cortical neurons (D) Quantification of AMPA mEPSCs shows that Bin1 overexpression does not affect average mEPSC amplitude but shifts distribution of values toward more high amplitudes (scr: n = 24 cells, kd: n = 22 cells). Mann-Whitney test. (E) Representative single plane SIM image of individual spines and line scans showing areas of colocalization between Bin1 and GluA1. (F) Representative 3D reconstruction of a stack of SIM images, showing the relative localization of Bin1 and GluA1 in spines. Inset, higher-magnification view; scale bar = 50 nm. (G) Montage of spine depicted in (F) to illustrate GluA1 and Bin1 contact sites (white arrowheads). (H) 3D surface plot showing total area of GluA1 staining (y-axis) as a function of both spine area (x-axis) and total area of Bin1 staining (z-axis). Legend shows color-coding for y-axis values on graph. (I) 3D surface plot showing total area of surface GluA1 (surfGluA1) staining (y-axis) as a function of both total area of Bin1 staining (x-axis) and spine area (z-axis). Legend shows color-coding for y-axis values on graph. (J) Reciprocal coimmunoprecipitation of Bin1 and GluA1 from rat cortex homogenate suggests participation in common complexes. (K) Flattened confocal image showing proximity-ligation assay (PLA) reveals an interaction of Bin1 and GluA1 in rat cortical neuron cultures, reflected by green PLA puncta. Red outline represents mCherry cell-filled pyramidal neuron. White arrowheads denote instances of interaction sites at dendritic spines. Inset: Zoomed in view of PLA puncta in dendritic spines and shaft. Scale bar = 20 μm (L) Quantification of PLA puncta normalized to number of cell bodies in the imaging frame (identified by DAPI nuclear staining). Bin1 Ab and GluA1 Ab conditions represent negative controls where each antibody was used singly in the PLA assay. Kruskal-Wallis with Dunn’s post-hoc tests * P<0.05, ** P<0.01

    Article Snippet: The following primary antibodies were used (mAb: monoclonal antibody, pAb: polyclonal antibody) : Bin1 mAb (Millipore Cat# 05–449), Map2 mAb (Millipore Cat# MAB3418), GluA1 N-term mAb (Millipore Cat# MAB2263), GluA1 C-term pAb (Millipore Cat# ABN241), Synapsin mAb (CellSignaling Cat# 5297), PSD95 mAb (Neuromab Cat# 75–028), CHC pAb (CellSignaling Cat# 2410), Rab5 pAb (CellSignaling Cat# 2143), EEA1 pAb (CellSignaling Cat# 2411), Rab11 pAb (Sigma Cat# R5903), Arf6 pAb (Abcam Cat# ab77581), Active Arf6 mAb (New East Biosciences, Cat# 26918), CaMKIIα mAb (Millipore Cat# 05–532), DsRed pAb (to identify mCherry expression, Clontech Cat# 632496), GFP chicken pAb GFP (to identify GFP expression, Abcam Cat# ab13970).

    Techniques: Knockdown, Control, Over Expression, MANN-WHITNEY, Staining, Proximity Ligation Assay, Imaging

    (A) Representative flattened heat map confocal images of surface GluA1 (surfGluA1) staining in scr and kd GFP cell-filled (white outline) neurons (see for cell fill). Scale bar = 20 μm Bottom: Zoomed in regions outlined in boxes in above images. (B) Quantification indicates that Bin1 knockdown reduces surface expression of GluA1 in spines and shaft (n = 13 cells for each condition). Welch’s t-test ** P<0.01 (C) Schematic of FRAP experimental paradigm, illustrating fluorescence recovery by exocytosis in pH-sensitive SEP-GluA1 expressing cells. (D) Representative single plane confocal image of the apical dendrite of a control cell demonstrating the FRAP paradigm. A 33 μm wide segment was bleached, while only the middle 2 μm section was quantified to limit the effects of lateral diffusion on fluorescence recovery. Dendrite outlines were created using the co-expressed, cell-filling mCherry. (E) Cells in which Bin1 expression was inhibited presented with significantly slower fluorescence recoveries than scramble control cells (n = 12 cells per condition). Repeated measures ANOVA, * P<.05 (F) Single plane confocal images showing heat map time-lapse examples of a control and knockdown cell. Quantified region outlined in magenta. Scale bar = 2 μm (G) Single plane confocal images of total and surface GluA1 staining in N2A cells with transfection conditions indicated to the left of images. Scale bar = 3 μm (H) Quantification of surface/total GluA1 ratios with indicated transfection conditions. All results were normalized to the average of the value of GFP transfection condition for each experiment (n = 16 cells per condition). One-way ANOVA with Bonferroni post-hoc tests ** P<0.01

    Journal: Molecular psychiatry

    Article Title: A novel role for the late-onset Alzheimer’s disease (LOAD)-associated protein Bin1 in regulating postsynaptic trafficking and glutamatergic signaling

    doi: 10.1038/s41380-019-0407-3

    Figure Lengend Snippet: (A) Representative flattened heat map confocal images of surface GluA1 (surfGluA1) staining in scr and kd GFP cell-filled (white outline) neurons (see for cell fill). Scale bar = 20 μm Bottom: Zoomed in regions outlined in boxes in above images. (B) Quantification indicates that Bin1 knockdown reduces surface expression of GluA1 in spines and shaft (n = 13 cells for each condition). Welch’s t-test ** P<0.01 (C) Schematic of FRAP experimental paradigm, illustrating fluorescence recovery by exocytosis in pH-sensitive SEP-GluA1 expressing cells. (D) Representative single plane confocal image of the apical dendrite of a control cell demonstrating the FRAP paradigm. A 33 μm wide segment was bleached, while only the middle 2 μm section was quantified to limit the effects of lateral diffusion on fluorescence recovery. Dendrite outlines were created using the co-expressed, cell-filling mCherry. (E) Cells in which Bin1 expression was inhibited presented with significantly slower fluorescence recoveries than scramble control cells (n = 12 cells per condition). Repeated measures ANOVA, * P<.05 (F) Single plane confocal images showing heat map time-lapse examples of a control and knockdown cell. Quantified region outlined in magenta. Scale bar = 2 μm (G) Single plane confocal images of total and surface GluA1 staining in N2A cells with transfection conditions indicated to the left of images. Scale bar = 3 μm (H) Quantification of surface/total GluA1 ratios with indicated transfection conditions. All results were normalized to the average of the value of GFP transfection condition for each experiment (n = 16 cells per condition). One-way ANOVA with Bonferroni post-hoc tests ** P<0.01

    Article Snippet: The following primary antibodies were used (mAb: monoclonal antibody, pAb: polyclonal antibody) : Bin1 mAb (Millipore Cat# 05–449), Map2 mAb (Millipore Cat# MAB3418), GluA1 N-term mAb (Millipore Cat# MAB2263), GluA1 C-term pAb (Millipore Cat# ABN241), Synapsin mAb (CellSignaling Cat# 5297), PSD95 mAb (Neuromab Cat# 75–028), CHC pAb (CellSignaling Cat# 2410), Rab5 pAb (CellSignaling Cat# 2143), EEA1 pAb (CellSignaling Cat# 2411), Rab11 pAb (Sigma Cat# R5903), Arf6 pAb (Abcam Cat# ab77581), Active Arf6 mAb (New East Biosciences, Cat# 26918), CaMKIIα mAb (Millipore Cat# 05–532), DsRed pAb (to identify mCherry expression, Clontech Cat# 632496), GFP chicken pAb GFP (to identify GFP expression, Abcam Cat# ab13970).

    Techniques: Staining, Knockdown, Expressing, Fluorescence, Control, Diffusion-based Assay, Transfection