Journal: Molecular Cell

Article Title: Modular antibodies reveal DNA damage-induced mono-ADP-ribosylation as a second wave of PARP1 signaling

doi: 10.1016/j.molcel.2023.03.027

Figure Lengend Snippet: Fluorescence-based sensors reveal DNA damage-induced serine mono-ADPr as second wave of PARP1 signaling (A) Real-time live-cell detection of mono-ADPr by bead-loaded Fab antibodies. (B–E) Recruitment kinetics and representative confocal images of: (B) mono-ADPr Fab probe (fluorophore-coupled AbD33205), scale bars, 10 μm. (C) Genetically encoded poly-ADPr probe (RNF146 WWE domain), scale bars, 10 μm. (D) Genetically encoded mono-ADPr probe (macrodomain of MacroD2), scale bars, 5 μm. (E) Poly- and mono-ADPr probes, scale bars, 10 μm. (F) Left: IF images of WT U2OS cells, treated with H 2 O 2 for the indicated times. Right: quantified mean nuclear intensity from mono- or poly-ADPr antibodies. Scale bar, 10 µm. (G) Immunoblotting of WT U2OS cells treated with H 2 O 2 for the indicated time. See also Figure S2 . Data in (B)–(E) are shown as mean ± SEM from a representative of 3 independent experiments.

Article Snippet: Human: U2OS cells , ATCC , Cat# HTB-96.

Techniques: Fluorescence, Western Blot

Journal: Molecular Cell

Article Title: Modular antibodies reveal DNA damage-induced mono-ADP-ribosylation as a second wave of PARP1 signaling

doi: 10.1016/j.molcel.2023.03.027

Figure Lengend Snippet: Cellular HPF1/PARP1 ratios regulate mono-ADPr levels (A) Immunoblotting of in vitro HPF1/PARP1 ADPr reactions with increasing concentrations of recombinant HPF1. (B) Immunoblotting of WT U2OS cells transfected with mCherry-empty vector (mCh-EV) or mCherry-HPF1-WT (mCh-HPF1-WT) and H 2 O 2 treated. (C) Top: schematics of SILAC-based proteomics of histone mono-ADPr marks on HPF1 overexpression and H 2 O 2 treatment. Bottom: scatterplot showing SILAC quantification. Mono-ADPr peptides (black) and other peptides (gray). (D) Top: mono-ADPr probe recruitment kinetics in WT U2OS cells overexpressing mCherry-HPF1-WT (black) or mCherry-HPF1-E284A (red). Bottom: representative confocal images. (E) Immunoblotting of ARH3-KO U2OS cells transfected with mCherry-EV, mCherry-HPF1-WT, or mCherry-HPF1-E284A. (F) Immunoblotting showing mono-ADPr levels on PARGi and H 2 O 2 time-course treatment. (G) Top: mono-ADPr probe recruitment kinetics in WT U2OS cells treated with DMSO (black) or PARGi (red). Bottom: representative confocal images. (H) Top: poly-ADPr probe recruitment kinetics in WT U2OS cells treated with DMSO (black) or 1 μM PARGi (red). Bottom: representative confocal images. Data in (D), (G), and (H) are shown as mean ± SEM from a representative of 3–4 independent experiments. Scale bars, 5 μm.

Article Snippet: Human: U2OS cells , ATCC , Cat# HTB-96.

Techniques: Western Blot, In Vitro, Recombinant, Transfection, Plasmid Preparation, Multiplex sample analysis, Over Expression

Journal: Molecular Cell

Article Title: Modular antibodies reveal DNA damage-induced mono-ADP-ribosylation as a second wave of PARP1 signaling

doi: 10.1016/j.molcel.2023.03.027

Figure Lengend Snippet: Identification of mono-ADPr readers by chromatin proteomics (A) Quantitative proteomics workflows to identify interactomes of Ser-mono-ADPr peptides (1) and H3S10ADPr nucleosome (2). (B) Scatterplot showing proteins enriched (red) by H3S10 mono-ADPr peptide compared with unmodified peptide. n = 2 biological replicates. (C) Chemoenzymatic generation of site-specific H3S10ADPr nucleosomes. (D) Scatterplot showing proteins enriched (red) by the H3S10ADPr nucleosome compared with unmodified nucleosome. n = 2 biological replicates. (E) Subcellular fractionation proteomics workflows for analysis of the mono-ADPr-dependent chromatin-associated proteome. (F) WT U2OS cells were H 2 O 2 -treated, and the chromatin fraction (as in E) was subjected to LC-MS/MS analysis. n = 3 biological replicates. (G) Top: immunoblotting of HPF1-KO U2OS cells transfected with mCherry-HPF1 WT or mCherry-HPF1-E284A, treated with H 2 O 2 for 20 min. Immunoblotting (top) or LC-MS/MS of chromatin fractions. Bottom: volcano plot showing the log 2 -fold change of identified proteins. n = 4 biological replicates. (H–J) ARH3-KO (H) and WT (I) U2OS cells were treated with DMSO or 1 μM olaparib for 48 h, and the chromatin fraction was subjected to LC-MS/MS. Volcano plot showing the log 2 -fold change of identified proteins. (J) Heatmap showing log 2 -fold change of chromatin-associated proteins in the indicated condition. Data from (H)–(J) come from the same experiment. n = 3 biological replicates. (K) Immunoblotting of WT U2OS cells transfected with GFP-EV or GFP-RNF114, olaparib- and H 2 O 2 -treated then subjected to anti-GFP immunoprecipitation. For (B), (D), and (F)–(I), the red dotted line represents significance with p value = 0.05 (−log 10 (adj. p value) > 1.3) cutoff. Significant proteins are indicated in red or blue. See also Figures S3 – and Table S1 .

Article Snippet: Human: U2OS cells , ATCC , Cat# HTB-96.

Techniques: Quantitative Proteomics, Fractionation, Liquid Chromatography with Mass Spectroscopy, Western Blot, Transfection, Immunoprecipitation

Journal: Molecular Cell

Article Title: Modular antibodies reveal DNA damage-induced mono-ADP-ribosylation as a second wave of PARP1 signaling

doi: 10.1016/j.molcel.2023.03.027

Figure Lengend Snippet: Chromatin mono-ADPr functions as a recruitment signal for RNF114 (A) Chromatin fraction analysis of H 2 O 2 -treated WT U2OS cells. Volcano plots showing the log 2 -fold change of identified proteins. Red dotted lines represent significance with p value = 0.05 (−log 10 (adj. p value) > 1.3) cutoff. Significant proteins are indicated in red. n = 4 biological replicates. (B–D) Recruitment kinetics and representative confocal images for GFP-RNF114-WT in: (B) WT U2OS cell untreated (black) or 30 μm olaparib treated (red); (C) HPF1-KO U2OS cells expressing mCherry-HPF1-WT (black) or mCherry-HPF1-E284A (red); (D) WT (black) or ARH3-KO (red) U2OS cells. Scale bars, 5 μm. (E–H) Recruitment kinetics of: poly-ADPr probe (E), APLF (F), mono-ADPr probe (G), and RNF114 (H) in ARH3-KO U2OS cells. Cells were treated (red) or not (black) with 30 μM olaparib 210 s after laser microirradiation. (I–K) Recruitment kinetics of: poly-ADPr probe (I), mono-ADPr probe (J), and RNF114 (K) in ARH3-KO U2OS cells expressing mCherry-ARH3-WT (red) or mCherry-ARH3-D77/78N (black). (L) Recruitment kinetics of GFP-RNF114 (red) and mCherry-ALC1 (black). (M) Effective diffusion coefficient measured by FCS for GFP-RNF114 (left) and mono-ADPr probe (right). ∗∗∗∗ p value < 0.0001, ∗∗∗ p value < 0.001 (unpaired Student’s t test assuming unequal variances). Data in (B)–(L) are shown as mean ± SEM from a representative of 3 independent experiments. See also Figure S6 and Table S1 .

Article Snippet: Human: U2OS cells , ATCC , Cat# HTB-96.

Techniques: Expressing, Diffusion-based Assay

Journal: Molecular Cell

Article Title: Modular antibodies reveal DNA damage-induced mono-ADP-ribosylation as a second wave of PARP1 signaling

doi: 10.1016/j.molcel.2023.03.027

Figure Lengend Snippet: RNF114 recruitment to DNA lesions is mediated by its zinc-finger domains (A) Dot blots of recombinant full-length RNF114 with indicated peptides or poly-ADP-ribose. Bovine serum albumin (BSA) and anti-mono/poly-ADPr (E6F6A) were used as negative and positive controls of ADPr binding, respectively. (B) Dot blots of equal moles of recombinant APLF, ALC1, and RNF114. (C) Domain architectures of RNF114 and deletion mutants. RING (RING-finger domain), Zn1 (zinc finger 1), Zn2 (zinc finger 2), Zn3 (zinc finger 3), and UIM (ubiquitin-interacting motif). Numbers indicate the motifs amino-acid positions. (D) Top: recruitment kinetics of GFP-RNF114-WT or individual GFP-RNF114 deletion constructs (as in C). Bottom: representative confocal images. Scale bars, 5 μm. (E) Top: recruitment kinetics of GFP-RNF114-WT or GFP-RNF114-C176A (as in C). Bottom: representative confocal images. Scale bars, 5 μm. (F) Dot blot of recombinant RNF114 and deletion constructs. (G) Immunoblotting images of WT U2OS cells transfected with indicated plasmids, H 2 O 2 treated and subjected to anti-GFP immunoprecipitation. Bound proteins were immunoblotted and stained with the indicated antibodies. Data in (D) and (E) are shown as mean ± SEM from a representative of 5 independent experiments.

Article Snippet: Human: U2OS cells , ATCC , Cat# HTB-96.

Techniques: Recombinant, Binding Assay, Ubiquitin Proteomics, Construct, Dot Blot, Western Blot, Transfection, Immunoprecipitation, Staining

Journal: Molecular Cell

Article Title: Modular antibodies reveal DNA damage-induced mono-ADP-ribosylation as a second wave of PARP1 signaling

doi: 10.1016/j.molcel.2023.03.027

Figure Lengend Snippet: RNF114 modulates the alternative lengthening of telomeres pathway and the DNA damage response (A) IF images (left) and quantified ABPs (right) in WT and HPF1-KO U2OS cells transfected with indicated plasmids. (B) Left: representative images of WT, ARH3-KO, and HPF1-KO U2OS cells co-transfected with indicated plasmids. Right: quantification of RNF114 positive telomeres (%). See also Figure S7 . (C) Quantification of APBs in WT U2OS cells transfected with siRNA for control (siControl), HPF1 (siHPF1), RNF114 (siRNF114), or HPF1 + siRNF114. See also Figure S7 E. (D) Quantified APBs in WT and RNF114-KO U2OS cells complemented with GFP-RNF114-WT or GFP-RNF114-C176A. (E) Quantified relative amounts of DNA synthesis occurring at damaged telomeres (%Edu + telomeres). (F) Clonogenic cell survival assay of WT and RNF114-KO U2OS cells. (G) Representative IF images (left) and quantified 53BP1 foci (right) in IR-treated WT or RNF114-KO U2OS cells. (H) IF images (left) and quantified γH2Ax foci (right) in IR-treated WT HeLa cells, transfected with siControl or siRNF114. (I) Representative IF images (left) and quantified 53BP1 foci (right) in IR-treated WT, or RNF114-KO U2OS cells stably complemented with GFP-EV, GFP-RNF114-WT, or GFP-RNF114-C176A. (J) Top: recruitment kinetics of GFP-tagged H2AK13/15Ub probe in WT (black) or ARH3-KO (red) U2OS cells. Bottom: representative confocal images. (K) Representative IF images (left) and quantified RIF1 foci (right) in IR-treated WT or RNF114-KO U2OS cells. See also Figure S7 . (L) Representative IF images (left) and quantification (right) of RNF168 foci in WT, or RNF114-KO U2OS cells stably complemented with GFP-EV, GFP-RNF114-WT, or GFP-RNF114-C176A treated with 5 Gy IR for 1 h, fixed with PFA and stained with the indicated antibody. The mean ± SEM from 100 cells from a representative of 3 independent experiments is shown. (M) Quantified relative NHEJ efficiency. Quantification of GFP-positive U2OS-EJ5 cells (relative NHEJ efficiency) after transfection with I-SceI and the indicated siRNAs. Data were normalized to siControl set to 100%. Data in (A)–(M) are shown as mean ± SEM from 3 to 4 independent experiments. ∗∗∗∗ p value < 0.0001; ∗∗∗ p value < 0.001; ∗∗ p value < 0.01; ∗ p value < 0.05; ns, not significant (two-tailed Student’s t test). (G–L) Scale bars, 5 μm.

Article Snippet: Human: U2OS cells , ATCC , Cat# HTB-96.

Techniques: Transfection, Control, DNA Synthesis, Clonogenic Cell Survival Assay, Stable Transfection, Staining, Two Tailed Test

Journal: Molecular Cell

Article Title: Modular antibodies reveal DNA damage-induced mono-ADP-ribosylation as a second wave of PARP1 signaling

doi: 10.1016/j.molcel.2023.03.027

Figure Lengend Snippet:

Article Snippet: Human: U2OS cells , ATCC , Cat# HTB-96.

Techniques: Transduction, Binding Assay, Recombinant, Staining, Sonication, Mass Spectrometry, Protease Inhibitor, Labeling, Transfection, Polymer, Live Cell Imaging, Protein Purification, CRISPR, Imaging, Plasmid Preparation, Software, Western Blot

Journal: The Science of the Total Environment

Article Title: Next-generation nanophotonic-enabled biosensors for intelligent diagnosis of SARS-CoV-2 variants

doi: 10.1016/j.scitotenv.2023.163333

Figure Lengend Snippet: Portable variants of the SARS-COV-2 virus tested based on the smart phone: A. MiSHERLOCK schematic showing the integration of instrument-free viral RNA extraction and concentration from raw saliva, reactions that identify SARS-CoV-2 and variations, fluorescence output and an auxiliary mobile phone app for automatic result interpretation. Reprinted with permission of , Copyright 2021, Science Advances. B. Smartphone-based imaging device for quantum dot barcodes scan immunoassay. Reprinted with permission from , Copyright 2021, ACS Nano Letters.

Article Snippet: B. Smartphone-based imaging device for quantum dot barcodes scan immunoassay.

Techniques: Virus, RNA Extraction, Concentration Assay, Fluorescence, Imaging

Journal: The EMBO Journal

Article Title: Clustering of Tau fibrils impairs the synaptic composition of α3‐Na + /K + ‐ ATP ase and AMPA receptors

doi: 10.15252/embj.201899871

Figure Lengend Snippet: A–C Time‐ and concentration‐dependent clustering of Fib‐Tau in primary neurons. Representative images are shown for certain conditions to illustrate Fib‐Tau clustering time dependence (A, top row) and concentration dependence (A, bottom row). Quantification of the number of Fib‐Tau clusters per μm 2 (B) or fluorescence intensity of clusters (C, indicating size, refer to ). At low concentrations (up to 0.72 nM), the density of clusters increased with time (between 10 and 60 min) but the increase in intensity was small. At high concentrations of Fib‐Tau (≥ 1.8 nM), both density and size increased with increasing time. Box‐plot represents median, interquartile range, and 10–90% distribution; one‐way ANOVA with Dunnett's post hoc test, number of images analyzed from three cultures (from left to right: 25, 25, 25, 25, 70, 45, 45, 45, 45, 45, 30, 40, 40, 40, 40, and 40 images). D–F Single‐particle tracking using quantum dots (SPT‐QD) of biotin‐tagged Fib‐Tau. Representative single molecule trajectories of Fib‐Tau following 10‐ or 60‐min exposure are shown (D). Note after 60‐min exposure (0.36 nM), single molecules are more confined suggesting they are trapped and clustered. Quantification of diffusion coefficient (E) and explored area (F, extracted from mean squared displacement (MSD), see ) shows that both these parameters decrease after 60‐min exposure to Fib‐Tau. Unpaired t ‐test, n is averaged value per cells imaged in three experiments (10 min: 22, 60 min: 23). C Neurons were exposed for 60 min to Fib‐Tau (0.36 nM) labeled with both biotin and ATTO‐488 (red). Cell surface‐exposed biotin was labeled using streptavidin‐550 (green) followed by live imaging. Note that most of the clusters of ATTO‐488 (red) are co‐labeled with streptavidin‐550 (green) indicating that the clusters are at the cell surface. H–J Clearance of Tau clusters from neurons. Neurons were exposed (0.36 nM) to ATTO‐550‐labeled Fib‐Tau for 10 min, and the unbound fibrils were washed. Cells were fixed immediately (10 min) or allowed to recover in culture medium for 60 min. Two representative images (H) and quantifications (I, J) show that following 60‐min recovery most of the Tau clusters disappear/dissociate as indicated by a decrease in their density. Box‐plot represents median, interquartile range, and 10–90% distribution; unpaired t ‐test, n is number of images analyzed from three cultures (49 images). Data information: * P < 0.05; ** P < 0.01; *** P < 0.001; ns = not significant. Scale bar, 5 μm in (G), 2 μm everywhere else.

Article Snippet: To pull down biotin‐labeled Fib‐Tau‐1N3R and 1N4R together with their partner proteins, 100 μl streptavidin magnetic beads (Pierce, Waltham, MA) were washed three times in binding buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.1% Triton X‐100, complete protease inhibitor cocktail).

Techniques: Concentration Assay, Fluorescence, Single-particle Tracking, Diffusion-based Assay, Labeling, Imaging

Journal: The EMBO Journal

Article Title: Clustering of Tau fibrils impairs the synaptic composition of α3‐Na + /K + ‐ ATP ase and AMPA receptors

doi: 10.15252/embj.201899871

Figure Lengend Snippet: Strategy used to purify and identify neuron intrinsic membrane proteins with extracellular domain that interact specifically with Fib‐Tau‐1N3R. Fib‐Tau was labeled 1 h with 10 molar equivalents of NHS‐S‐S‐Biotin. Mouse cortical neuron cultures were exposed for 10 min to biotinylated Fib‐Tau (14.4 nM). Fresh protein extracts from those neurons were incubated with streptavidin magnetic beads to pull down Fib‐Tau together with their specific protein partners. Unexposed neuron extracts were used as a control. Proteins bound to the streptavidin magnetic beads were eluted with Laemmli buffer and subjected to short migration on a SDS–PAGE gel. After Coomassie blue staining, proteins were subjected to in‐gel digestion using trypsin and subsequently identified by nanoLC‐MS/MS analysis, using a nanoLC‐TripleTOF mass spectrometer. Relative quantification between control and exposed neuron samples was performed using a label‐free MS‐based approach. Six independent replicates were analyzed. Venn diagram of 968 proteins identified in Fib‐Tau pull‐downs only (red), in control pull‐downs only (gray), or in both samples (overlap). Of the 92 proteins identified in both samples, 45 proteins were significantly enriched in Fib‐Tau pull‐downs ( t ‐test with P ‐values < 0.05, Benjamini–Hochberg, fold change > 2). Distribution of the 372 synaptic and membrane protein interactors of Fib‐Tau identified in the pull‐down experiments. Locations of proteins at the levels of subcellular structures were annotated using the Gene Ontology Cell Component annotation tool of AMIGO 2 ( http://amigo.geneontology.org/amigo/landing ). Distribution of Fib‐Tau interactors in the plasma membrane, pre‐synaptic membrane, post‐synaptic membrane, pre‐synapse, and post‐synapse is shown. List of synaptic and plasma membrane proteins with extracellular domains significantly enriched in pull‐downs from neurons exposed to Fib‐Tau. For each identified protein, the name of the protein, the gene name, the P ‐value ( t ‐test with Benjamini–Hochberg correction), and the fold change corresponding to the ratio of spectral counts between exposed neuron and control samples are given. In an independent analysis, after 10‐min exposure of neurons to biotinylated Fib‐Tau, a cross‐linking step was performed during 20 min using 1 mM of DTSSP added in the culture medium, in order to cross‐link the protein complexes formed at the cell surface using a membrane impermeable cross‐linker. After cross‐linking, proteins were analyzed and identified exactly as non‐cross‐linked samples. Proteins identified with at least two peptides are labeled “+”, and the other are labeled “−”. Co‐immunoprecipitation of exogenous biotin‐labeled Fib‐Tau with α3‐NKA, GluA2, and GluN1. α3‐NKA, GluA2, and GluN1 were immunoprecipitated using specific antibodies as described in the section. The presence of Fib‐Tau in the immunoprecipitate was assessed using a slot blot apparatus and nitrocellulose membranes probed with streptavidin‐HRP. A 2.4‐, 2.3‐, and 1.8‐fold enrichment in Tau band intensity is observed in α3‐NKA, GluA2, and GluN1 immunoprecipitates, respectively, compared to controls performed with pre‐immune goat or rabbit IgGs. Co‐immunoprecipitates of exogenous biotin‐labeled Fib‐Tau with anti‐α3‐NKA‐, GluA2‐, and GluN1‐specific antibodies were also subjected to SDS–PAGE and Western blot analysis. The presence of Fib‐Tau in the immunoprecipitates was assessed by probing the nitrocellulose membranes with streptavidin‐HRP. Fib‐Tau co‐immunoprecipitates with α3‐NKA and GluA2‐AMPA receptor but not with GluN1‐NMDA receptor. Network describing the interconnectivity of intrinsic membrane proteins extracellularly exposed (presented in panel D, labeled in yellow) and post‐synaptic proteins (proteins with a post‐synapse or a post‐synaptic membrane annotation, presented in panel C and labeled in blue) that interact with Fib‐Tau‐1N3R. This Fib‐Tau‐1N3R interactome was input in the String database (String v10, https://string-db.org/ ) and exported to Cytoscape (version 3.5.1 at http://www.cytoscape.org/ ) to visualize interactions between the identified proteins. A total of 121 proteins were evaluated. We set parameters to only detect interactions that were validated experimentally or described in databases. The thickness of the line corresponds to the confidence of interaction (thin lines, > 0.4; medium lines, > 0.7; thick lines, > 0.9).

Article Snippet: To pull down biotin‐labeled Fib‐Tau‐1N3R and 1N4R together with their partner proteins, 100 μl streptavidin magnetic beads (Pierce, Waltham, MA) were washed three times in binding buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.1% Triton X‐100, complete protease inhibitor cocktail).

Techniques: Membrane, Labeling, Incubation, Magnetic Beads, Control, Migration, SDS Page, Staining, Tandem Mass Spectroscopy, Mass Spectrometry, Quantitative Proteomics, Clinical Proteomics, Immunoprecipitation, Dot Blot, Western Blot

Journal: The EMBO Journal

Article Title: Clustering of Tau fibrils impairs the synaptic composition of α3‐Na + /K + ‐ ATP ase and AMPA receptors

doi: 10.15252/embj.201899871

Figure Lengend Snippet: Venn diagram of 1,065 proteins identified in Fib‐Tau‐1N4R pull‐downs only (red), in control pull‐downs only (gray), or in both samples (overlap). Of the 88 proteins identified in both samples, 63 proteins were significantly enriched in Fib‐Tau‐1N4R pull‐downs ( t ‐test with P ‐values < 0.05, Benjamini–Hochberg, fold change > 2). Distribution of the 379 synaptic and membrane protein interactors of Fib‐Tau‐1N4R identified in the pull‐down experiments. Locations of proteins at the levels of subcellular structures were annotated using the Gene Ontology Cell Component annotation tool of AMIGO 2 ( http://amigo.geneontology.org/amigo/landing ). Distribution of Fib‐Tau interactors in the plasma membrane, pre‐synaptic membrane, post‐synaptic membrane, pre‐synapse, and post‐synapse is shown. Comparison of synaptic and plasma membrane proteins with extracellular domains significantly enriched in pull‐downs from neurons exposed to Fib‐Tau 1N4R and 1N3R. For each identified 1N4R protein, the name of the protein, the gene name, the P ‐value ( t ‐test with Benjamini–Hochberg correction), and the fold change corresponding to the ratio of spectral counts between exposed neuron and control samples are given. Co‐immunoprecipitation of exogenous biotin‐labeled Fib‐Tau‐1N4R with α3‐NKA, GluA2, and GluN1. α3‐NKA, GluA2, and GluN1 were immunoprecipitated using specific antibodies as described in the section. An 1.8‐, 1.2‐, and 2.5‐fold enrichment in Tau band intensity is observed in α3‐NKA, GluA2, and GluN1 immunoprecipitates, respectively, compared to controls performed with pre‐immune goat or rabbit IgGs. Detection of co‐immunoprecipitation by SDS–PAGE and Western blotting. Co‐immunoprecipitation of exogenous biotin‐labeled Fib‐Tau‐1N4R with anti‐α3‐NKA‐, GluA2‐, and GluN1‐specific antibodies. The presence of Fib‐Tau in the immunoprecipitate was assessed by probing the nitrocellulose membranes with streptavidin‐HRP. Overall, the signal was low with high background. Fib‐Tau (**) co‐immunoprecipitates with α3‐NKA and GluN1‐NMDA receptor but not with GluA2‐AMPA receptor.

Article Snippet: To pull down biotin‐labeled Fib‐Tau‐1N3R and 1N4R together with their partner proteins, 100 μl streptavidin magnetic beads (Pierce, Waltham, MA) were washed three times in binding buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.1% Triton X‐100, complete protease inhibitor cocktail).

Techniques: Control, Membrane, Clinical Proteomics, Comparison, Immunoprecipitation, Labeling, SDS Page, Western Blot

Journal: Communications Biology

Article Title: The intracellular lipid-binding domain of human Na + /H + exchanger 1 forms a lipid-protein co-structure essential for activity

doi: 10.1038/s42003-020-01455-6

Figure Lengend Snippet: a Schematic architecture of human NHE1 indicating the subdomains A-D in the tail and with a zoom on the lipid interaction domain (LID) within subdomain A. b Far-UV CD spectrum of the NHE1-LID 539-593 in H 2 O, pH 6.0. c 15 N, 1 H-HSQC spectrum of NHE1-LID 539-593 in the absence of membrane mimetics (pH 6.4). d Secondary chemical shifts (SCS) of C α and C’ from backbone assignments of NHE1-LID showing two transient and lowly populated helices. e Radius of hydration, R h of NHE1-LID 539-593 from different scaling laws and experimentally determined using diffusion NMR ( R h experimental ).

Article Snippet: Membranes were stained with Ponceau S to confirm equal loading, blocked for 1 h at 37 °C in 120 mM NaCl, 10 mM TrisHCl, 5% nonfat dry milk, and incubated with the primary antibodies against NHE1 (Santa Cruz Biotechnology, sc-136239 (54)) or β-actin (Sigma, A5441) in blocking buffer overnight at 4 °C.

Techniques: Membrane, Diffusion-based Assay

Journal: Communications Biology

Article Title: The intracellular lipid-binding domain of human Na + /H + exchanger 1 forms a lipid-protein co-structure essential for activity

doi: 10.1038/s42003-020-01455-6

Figure Lengend Snippet: a Position of individual lipids on the dot blot membrane. b Lipid binding profile of the CHP1/NHE1 503-595 and CHP1/NHE1 503-698 at pH 7.4 and 8.4. c Variants of NHE1 used to test lipid binding specificity. d Effect of various mutations on CHP1/NHE1 503-595 lipid binding. e Far-UV CD spectra of NHE1-LID 539-593 in DHPC detergent (color) and in DMPC:DHPC bicelles (dashed color). The CD spectrum of NHE1-LID 539-593 in water is shown in black. f Far-UV CD spectra of the NHE1-LID in anionic bicelles consisting of DMPG:DMPC:DHPC (dashed color) and in 2% LPPG (color). The CD spectrum of NHE1-LID 539-593 in water is shown in black. g 15 N, 1 H-HSQC spectrum of NHE1-LID 539-593 in 2% LPPG, 320.15 K. h SCSs of C α of the NHE1-LID in 2% LPPG with two highly populated helices, H1 and H2, indicated below the sequence.

Article Snippet: Membranes were stained with Ponceau S to confirm equal loading, blocked for 1 h at 37 °C in 120 mM NaCl, 10 mM TrisHCl, 5% nonfat dry milk, and incubated with the primary antibodies against NHE1 (Santa Cruz Biotechnology, sc-136239 (54)) or β-actin (Sigma, A5441) in blocking buffer overnight at 4 °C.

Techniques: Dot Blot, Membrane, Binding Assay, Circular Dichroism, Sequencing

Journal: Communications Biology

Article Title: The intracellular lipid-binding domain of human Na + /H + exchanger 1 forms a lipid-protein co-structure essential for activity

doi: 10.1038/s42003-020-01455-6

Figure Lengend Snippet: a Sequence of the NHE1-LID with indicated peptide regions corresponding to nLID 542-569 and cLID 567-592 and with Agadir prediction of helicity and helical wheel representations . The basic PI(4,5)P 2 Site II (blue) and hydrophobic motifs (HM1, HM2) (yellow) indicated above. b Far-UV CD spectra of nLID 542-569 alone and in the presence of various lipids and at two different pH values. c Far-UV CD spectra of cLID 567-592 alone and in the presence of various lipids. d Fluorescence emission spectra of nLID 542-569 alone and upon addition of POPC/POPS and POPC/POPS/PI(4,5)P 2 SUVs. e Center of spectral mass analysis of nLID 542-569 fluorescence emission spectra from a SUV titration series revealed an apparent membrane affinity of 0.8 mM for nLID 542-569 .

Article Snippet: Membranes were stained with Ponceau S to confirm equal loading, blocked for 1 h at 37 °C in 120 mM NaCl, 10 mM TrisHCl, 5% nonfat dry milk, and incubated with the primary antibodies against NHE1 (Santa Cruz Biotechnology, sc-136239 (54)) or β-actin (Sigma, A5441) in blocking buffer overnight at 4 °C.

Techniques: Sequencing, Circular Dichroism, Fluorescence, Titration, Membrane

Journal: Communications Biology

Article Title: The intracellular lipid-binding domain of human Na + /H + exchanger 1 forms a lipid-protein co-structure essential for activity

doi: 10.1038/s42003-020-01455-6

Figure Lengend Snippet: a Three different variants of the NHE1-LID 539-593 were analyzed, NHE1-LID 539-593-2G-1 (I574G, F576G in HM1), NHE1-LID 539-593-2G-2 (I586G, L588G in HM2) and NHE1-LID 539-593-4G (I574G, F576G, I586G, L588G), as indicated. b Far-UV CD spectra of cLID 567-592 peptides with glycine mutations in various lipids. c Far UV CD spectra of NHE1-LID LID 539-593-4G alone (black) and in the presence of 2% LPPG (color) and DMPG:DMPC:DHPC bicelles (dashed color). d 15 N, 1 H-HSQC spectrum of NHE1-LID LID 539-593-4G in H 2 O, pH 6.5. e Differences in SCS between NHE1-LID 539-593 and NHE1-LID 539-593-4G in the absence of membrane mimetics. Top: ΔSCSs of C α , bottom: Differences in amide shifts given by ΔδNHs.

Article Snippet: Membranes were stained with Ponceau S to confirm equal loading, blocked for 1 h at 37 °C in 120 mM NaCl, 10 mM TrisHCl, 5% nonfat dry milk, and incubated with the primary antibodies against NHE1 (Santa Cruz Biotechnology, sc-136239 (54)) or β-actin (Sigma, A5441) in blocking buffer overnight at 4 °C.

Techniques: Circular Dichroism, Membrane

Journal: Communications Biology

Article Title: The intracellular lipid-binding domain of human Na + /H + exchanger 1 forms a lipid-protein co-structure essential for activity

doi: 10.1038/s42003-020-01455-6

Figure Lengend Snippet: a QCM-D, with sensor frequency shift (ΔF) on the left y-axis (top lines) and the dissipation factor (ΔD) on the right y-axis (lower lines), colors represent different sensor harmonics reporting on the adsorption of molecules (i.e., lipids and subsequently NHE1-LID) on the sensor surface. Area I to V correspond to the supported lipid bilayer in contact with the sensor (I), injection of MQ water prior (II), injection of the protein solution and its incubation with the membrane (III), removal of excess protein with MQ water (IV) and re-introduction of the buffer (V). b Reflectivity vs. q measured in buffers of different degree of deuteration (see C). c Scattering length density (SLD) profiles obtained from the NR experiment; the experimental data were collected for the sample in contact with buffer prepared with different D 2 O content (d-buffer = 100% D 2 O, smw-buffer = 38% D 2 O:62% H 2 O, h-buffer = 100% H 2 O). d Per-residue histogram of protein-lipid contacts observed during the MD simulation (blue-gray: POPC, green: POPS). e Temporal evolution of protein-POPC and protein-POPS contacts from the MD simulation. f Snapshots from the MD simulation trajectory depicting the different bound orientations of NHE1-LID on the membrane. NHE1-LID shown in ribbon representation, lipids shown in van der Waals’ representation (hydrogens omitted for clarity), colors as in (D) (H1, blue; H2, red; POPC blue-gray; POPS, green). g Schematic representation of the NR experiment setup. Details on the right side depict the protein-layer thickness measured from NR compared to average thickness of the protein obtained from MD.

Article Snippet: Membranes were stained with Ponceau S to confirm equal loading, blocked for 1 h at 37 °C in 120 mM NaCl, 10 mM TrisHCl, 5% nonfat dry milk, and incubated with the primary antibodies against NHE1 (Santa Cruz Biotechnology, sc-136239 (54)) or β-actin (Sigma, A5441) in blocking buffer overnight at 4 °C.

Techniques: QCM-D, Adsorption, Injection, Incubation, Membrane, Residue

Journal: Communications Biology

Article Title: The intracellular lipid-binding domain of human Na + /H + exchanger 1 forms a lipid-protein co-structure essential for activity

doi: 10.1038/s42003-020-01455-6

Figure Lengend Snippet: a , b Plasma membrane expression of NHE1 in AP-1 cells (untransfected or stably expressing wt or 4G-NHE1 as indicated), assessed by pull-down of the biotinylated membrane fraction followed by immunoblotting for NHE1. a Representative immunoblots. β-actin was used as a loading control (no signal in the pull-down fraction, as expected). Uncropped blots are available in Supplementary Fig. . b Plasma membrane NHE1 expression normalized to that in wt clone 1. Data are quantified from n = 4 biologically independent experiments per clone and shown as mean with S.E.M error bars and all individual data points. The membrane expression of 4G-NHE1 did not differ significantly from that of NHE1 (pooled data from both clones for each variant, two-tailed, non-paired Student’s t -test, p > 0.05). c Localization of wt- and 4G-NHE1 in AP-1 cells. Cells were fixed and stained with antibody against NHE1 (red), rhodamine-conjugated phalloidin to visualize F-actin (green), and DAPI to visualize nuclei (blue). Arrows in the detail images highlight the membrane localization of NHE1. Data shown are representative of n = 3 biologically independent experiments per condition. d – g To measure wt- and 4G-NHE1 activity, cells were loaded with BCECF-AM, and pH i monitored using real time fluorescence imaging. d Steady-state pH i , averaged over the two NHE1 wt ( n = 19 biologically independent experiments) and 4 G ( n = 16 biologically independent experiments) cell clones. Data are shown as mean with S.E.M. error bars and all individual data points. e Representative examples of pH i traces. The black arrow indicates the point of removal of NH 4 Cl. f pH i recovery rates for the NHE1 wt ( n = 20 biologically independent experiments) and 4 G ( n = 15 biologically independent experiments) variant, calculated from the initial linear part of the pH i traces after maximal acidification, as in e . Data are shown as mean with S.E.M. error bars. g From traces as in (E), pH i recovery rates as a function of pH i was calculated by fitting the recovery rates over the entire recovery period. Data are shown as mean with S.E.M. error bars. Data in F and G were corrected for relative cell surface expression (data from Panel B) to ensure that the pH i recovery represents the capacity of the membrane-expressed fraction of NHE1. ** p = 0.0024 (panel D) and 0.0016 (panel F) and compared to wt, two-tailed, non-paired Student’s t -test using GraphPad Prism 8.4.1 software and assuming normal distribution. Source files for Fig. 6b,d–g available as supplementary data.

Article Snippet: Membranes were stained with Ponceau S to confirm equal loading, blocked for 1 h at 37 °C in 120 mM NaCl, 10 mM TrisHCl, 5% nonfat dry milk, and incubated with the primary antibodies against NHE1 (Santa Cruz Biotechnology, sc-136239 (54)) or β-actin (Sigma, A5441) in blocking buffer overnight at 4 °C.

Techniques: Clinical Proteomics, Membrane, Expressing, Stable Transfection, Western Blot, Control, Clone Assay, Variant Assay, Two Tailed Test, Staining, Activity Assay, Fluorescence, Imaging, Software

Journal: PeerJ

Article Title: Exosomes in cancer: small vesicular transporters for cancer progression and metastasis, biomarkers in cancer therapeutics

doi: 10.7717/peerj.4763

Figure Lengend Snippet: Overview of the role of exosomes in multiple kinds of cancer.

Article Snippet: ACTB, TUBA1A, FN1, FNLA, CD61, HLA-A, LGALS3BP, Alix, RAB5B, RAB5C, SDCBP, VPF37B, CLTC, ARF1, ANXA2, ANXA5, HSC70, HSP72, RAC1, STOM, MFGE8, MVP, GNA12, PTGFRN, HBA1, tumor susceptibility gene-101 (TSG-101), and Grp94 , The inhoused established human HCC cell lines HKCI-C3 and HKCI-8 cells The hepatocellular carcinoma (HCC) cell line MHCC97L cells The immortalized hepatocyte cell line MIHA cells , qRT-PCR analysis, Ion Torrent Next-Generation Sequencing, and Western blotting , The internalization of exosomes could activate PI3K/AKT and MAPK signaling pathway, and promote secretion of MMP-2 and MMP-9 that favored cell invasion. Also, by proteome analysis Syndecan–syntenin–ALIX is known to support biogenesis of exosomes and the segregation of signaling cargo to these vesicles. The research also detected the components of endosomal protein sorting complex, such as VPS28 and VPS37, whose functions are required for exosome cargo sorting Process. , .

Techniques: Western Blot, Immunofluorescence, AChE Assay, Immunocytochemistry, Inhibition, Chromatin Immunoprecipitation, Marker, Expressing, Activity Assay, Enzyme-linked Immunosorbent Assay, Derivative Assay, Biomarker Assay, In Vitro, In Silico, Diagnostic Assay, Isolation, Flow Cytometry, Variant Assay, In Vivo, Sampling, Modification, Transgenic Assay, Migration, Luciferase, Plasmid Preparation, In Vivo Imaging, Purification, Ex Vivo, Activation Assay, Injection, Endothelial Tube Formation Assay, Viability Assay, Matrigel Assay, Immunoprecipitation, Microscopy, Functional Assay, Knock-Out, Mass Spectrometry, Dot Blot, Electron Microscopy, Methylation, Chromatography, Over Expression, Mutagenesis, Microarray, Binding Assay, Transferring, Real-time Polymerase Chain Reaction, Bradford Assay, Transformation Assay, Animal Model, MTT Assay, Fluorescence, FACS, Diffusion-based Assay, Next-Generation Sequencing, Transplantation Assay, Sequencing, Histone Deacetylase Assay, Infection, Incubation, Transmission Assay, Blocking Assay, Luminex, Fractionation, Irradiation, Reporter Assay