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Image Search Results
Journal: International Journal of Molecular Sciences
Article Title: Photoreceptor Compartment-Specific TULP1 Interactomes
doi: 10.3390/ijms22158066
Figure Lengend Snippet: IP of retinal lysate. The top panels show Western blot analysis of the MAP1B IP experimental samples probed with Tulp1 antibodies. In the IP product lane, a band corresponding to Tulp1 is detected. A corresponding band is seen in the rat retinal lysate and wt mouse retinal homogenate but not in the tulp1−/− retinal lysate, liver lysate or non-specific IgG IP lanes. The bottom panels show Western blot analysis of the MAP1B IP experimental samples probed with MAP1B antibodies. In the IP product, rat retinal lysate, wt mouse retinal lysate and tulp1−/− retinal lysate lanes, a band corresponding to MAP1B is detected. No bands are seen in the liver or non-specific IgG IP sample lanes.
Article Snippet: Slides were then incubated with blocking solution (1% BSA and 10% Donkey serum in freshly prepared 1X PBS) for 2 h at RT and then subsequently incubated with primary antibodies:
Techniques: Western Blot
Journal: International Journal of Molecular Sciences
Article Title: Photoreceptor Compartment-Specific TULP1 Interactomes
doi: 10.3390/ijms22158066
Figure Lengend Snippet: Immunolocalization of MAP1B and Tulp1 in P17 mouse retinas. ( A ) Wt retinal sections stained with MAP1B (red) and Tulp1 (green). ( B ) Tulp1−/− retinal sections stained with MAP1B (red) and Tulp1 (green). Sections were counterstained with DAPI (blue). Scale bar: 50 µm. INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; IS, inner segment layer; OS, outer segment layer.
Article Snippet: Slides were then incubated with blocking solution (1% BSA and 10% Donkey serum in freshly prepared 1X PBS) for 2 h at RT and then subsequently incubated with primary antibodies:
Techniques: Staining
Journal: Frontiers in Oncology
Article Title: CDK1-driven phosphorylation networks promote glioblastoma progression via MAP1B-mediated microtubule destabilization
doi: 10.3389/fonc.2025.1646698
Figure Lengend Snippet: CDK1-mediated MAP1B phosphorylation is positively correlated with the prognosis of GBM. (A) Overlap analysis of CPTAC phosphoproteome data (blue) with CDK1-regulated phosphosites (red). (B) Heatmap of MAP1B phosphosites in the shCDK1 and shCtrl groups. Red indicates upregulated values relative to the mean, while blue indicates downregulated values relative to the mean. The intensity of the color corresponds to the magnitude of the deviation. (C) Prognostic significance of MAP1B phosphorylation sites. Kaplan-Meier survival analysis of GBM patients stratified by phosphorylation levels at S832, S1260, S1899, S1939, S2209, S2271(log-rank test, p < 0.05). High phosphorylation correlates with poor overall survival (n=99). (D, E) CoIP showing interactions between CDK1 and MAP1B. (F) Detecting pS/T phosphorylation of MAP1B in shCDK1 and shCtrl U251 cells. (G) Detecting pS/T phosphorylation of MAP1B in overexpressed-CDK1(OE-CDK1) and empty vector(Vector) U251 cells.
Article Snippet: Phospho-(Ser/Thr) Phe Antibody (#9631, Cell Signaling Technology),
Techniques: Phospho-proteomics, Plasmid Preparation
Journal: Frontiers in Oncology
Article Title: CDK1-driven phosphorylation networks promote glioblastoma progression via MAP1B-mediated microtubule destabilization
doi: 10.3389/fonc.2025.1646698
Figure Lengend Snippet: CDK1-mediated phosphorylation of MAP1B regulates microtubule stability in U251 cells. (A) Wound healing assays showing delayed gap closure in shMAP1B cells compared to shCtrl cells over 36 hours, ***p < 0.001 (t-test), ****p < 0.0001 (t-test). (B) Transwell migration assays confirmed the impaired migration ability of shMAP1B cells. ***p < 0.001 (t-test). (C) CCK-8 proliferation assays revealed a significant decrease in cell viability in shMAP1B cells ***p < 0.001 (t-test). (D) Wiki-pathways analysis of differently expressed phosphoproteins. (E) Representative photographs of alpha tubulin staining of in U251 cells. Scale bar: 20 μm. Cells were stained with an antibody against acetyl-α-tubulin (green). Nuclei were counterstained with DAPI (blue). Scale bar, 20 μm.
Article Snippet: Phospho-(Ser/Thr) Phe Antibody (#9631, Cell Signaling Technology),
Techniques: Phospho-proteomics, Migration, CCK-8 Assay, Staining
Journal: Gels
Article Title: A 3D Collagen–Alginate Hydrogel Model for Mechanoregulation of Autophagy in Periodontal Ligament Cells
doi: 10.3390/gels12010091
Figure Lengend Snippet: 3D compression promotes autophagy of PDLCs. ( A ) qRT-PCR analysis for quantification of ATG7 expression at the indicated time points. ( B ) qRT-PCR analysis for quantification of Beclin1 expression at the indicated time points. ( C ) Representative immunofluorescence images of LC3 staining at different time points. Scale bar: 100 μm. ( D ) Ratio of LC3-II/LC3-I quantified at different time points. ( E ) Immunoblotting analysis of P62 protein levels over time. ( F ) Densitometric quantification of P62 normalized to GAPDH. For ( A , B , D , F ), **, ***, ****, and ns indicate p < 0.01, p < 0.001, p < 0.0001 and no significant difference by one-way ANOVA, respectively. Shown as the mean ± SD.
Article Snippet: Samples were incubated with
Techniques: Quantitative RT-PCR, Expressing, Immunofluorescence, Staining, Western Blot
Journal: Gels
Article Title: A 3D Collagen–Alginate Hydrogel Model for Mechanoregulation of Autophagy in Periodontal Ligament Cells
doi: 10.3390/gels12010091
Figure Lengend Snippet: AKT-mTOR signaling links compression to autophagy in PDLCs. Compression is transmitted through AKT-mTOR signaling. Modulation of this pathway by the AKT activator SC79 and the mTOR pathway modulator 3BDO alters autophagy-related gene expression. Changes in Beclin1, P62, ATG7, ATG5 and LC3-II reflect the formation of autolysosomes and the overall level of autophagic activity in periodontal ligament cells under compression. The light red arrows indicate the increase in the levels of AKT and mTOR.
Article Snippet: Samples were incubated with
Techniques: Gene Expression, Activity Assay
Journal: Autophagy
Article Title: An optimized protocol for immuno-electron microscopy of endogenous LC3
doi: 10.1080/15548627.2022.2056864
Figure Lengend Snippet: Cosmo Bio anti-LC3B specifically recognizes LC3B in EGFP-LC3B transfected cells. (A) Schematic overview of endo-lysosomal and autophagic compartments, outlining their characteristic morphological features and presence of LC3. (B-C) EGFP-LC3B transfected cells were fixed with 2% PFA+0.2% GA for 3 h and double labeled for LC3B and GFP and protein A gold (PAG). (B) The labeling intensities of anti-GFP (1:400; PAG15) and Cosmo Bio anti-LC3B (1:10; PAG10) correlate in cells with high and low EGFP-LC3B overexpression. (C) Example of double labeling with anti-GFP (1:400; PAG15) and another LC3 antibody (CST, 4108, 1:15; PAG10) showing only PAG15 on EGFP-LC3B overexpressing cells. AL, autolysosome; M, mitochondrion; PM, plasma membrane. Scale bars: 300 nm.
Article Snippet: For the LC3-GFP double labeling, LC3 was first labeled with
Techniques: Transfection, Labeling, Over Expression, Clinical Proteomics, Membrane
Journal: Autophagy
Article Title: An optimized protocol for immuno-electron microscopy of endogenous LC3
doi: 10.1080/15548627.2022.2056864
Figure Lengend Snippet: Performance of commercial LC3 antibodies in IF and immuno-EM.
Article Snippet: For the LC3-GFP double labeling, LC3 was first labeled with
Techniques:
Journal: Autophagy
Article Title: An optimized protocol for immuno-electron microscopy of endogenous LC3
doi: 10.1080/15548627.2022.2056864
Figure Lengend Snippet: Imaging autophagy by conventional EM and immunofluorescence. (A) Conventional EM of control U2OS cells. Endo-lysosomal compartments are identified by morphology. (B) Conventional EM of U2OS cells starved for 2.5 h in the presence of BafA1 showing an accumulation of different types of autophagic compartments. Autophagosomes (AP) are recognized by their double-membrane that encapsulates part of the cytoplasm with the same density as the surrounding cytoplasm and containing multiple membrane structures. Autolysosomes (AL) are vacuoles lined by a single membrane. Their lumen is heterogenous in content (membranes, vesicles, amorphous material) and electron density (from light to dense). Both AP and AL contain recognizable endoplasmic reticulum (ER) cisternae (arrows). Asterisks indicate autophagic content. (C-E) IF of semi-thin cryosections of control and starved, BafA1-treated U2OS cells, fixed either with 4% PFA ON (PFA) or 2% PFA+0.2% GA for 2 h (PFA+GA) and labeled with Cosmo Bio anti-LC3B (1:10) and Alexa Fluor 488-conjugated goat anti-mouse IgG (1:300). Images are recorded with identical settings and represented with equal intensity ranges. PFA and PFA+GA fixed cells contain comparably intense LC3B puncta. AL, autolysosome; AP, autophagosome; E, endosome; Ly, lysosome; M, mitochondrion; PM, plasma membrane. Scale bars: 500 nm (A-B); 20 µm (C-E).
Article Snippet: For the LC3-GFP double labeling, LC3 was first labeled with
Techniques: Imaging, Immunofluorescence, Control, Membrane, Labeling, Clinical Proteomics
Journal: Autophagy
Article Title: An optimized protocol for immuno-electron microscopy of endogenous LC3
doi: 10.1080/15548627.2022.2056864
Figure Lengend Snippet: Correlative light electron microscopy (CLEM) of endogenous LC3B on ultrathin cryosections. (A) Ultrathin (60–70 nm) cryosection of starved, BafA1-treated U2OS cells (fixation 4% PFA ON) labeled for LC3B (1:10), rabbit anti-mouse IgG, Alexa Fluor 488-conjugated donkey anti-rabbit IgG and PAG10, showing IF puncta (green) and nuclei (Hoechst, blue). (B) Low magnification EM picture of same section as in (A). Arrows point to LC3B-immunogold labeled compartments (gold not visible at this magnification). (C) Overlay of the fluorescent and EM images in (A) and (B). Higher magnifications of the boxed areas in (C) are shown in (D-F). (D) A phagophore (PG) is visible as a ring of LC3B-positive vesicles. Arrows indicate visible membrane contours. (E, F) Two examples of autolysosomes (AL). LC3B (PAG10) is predominantly associated with autophagic content located in the AL lumen (indicated by asterisks). Note that the higher magnifications in D-F are rotated relative to B. N, nucleus. Scale bars: 2 µm (A-C), 200 nm (D-F).
Article Snippet: For the LC3-GFP double labeling, LC3 was first labeled with
Techniques: Electron Microscopy, Labeling, Membrane
Journal: Autophagy
Article Title: An optimized protocol for immuno-electron microscopy of endogenous LC3
doi: 10.1080/15548627.2022.2056864
Figure Lengend Snippet: Effect of different fixation and labeling regimes on LC3 immuno-EM labeling efficiency. (A-D) Electron micrographs of U2OS cells starved in the presence of BafA1 for 2.5 h. Cells were fixed and immunolabeled for LC3B (1:10; PAG10) according to the standard or fast labeling protocol as indicated. (A, B) Cells fixed ON with 4% PFA labeled by the standard (A) or fast (B) protocol. Both conditions show abundant LC3B label on autophagic content (asterisks) present in autolysosomes (AL). (C) Cells fixed for 15 min with 4% PFA, followed by 6 days of 0.6% PFA and labeled by the fast protocol. (D) Cells fixed for 3 h with 2% PFA+0.2% GA labeled by the standard protocol. Note a reduction in LC3B label compared to A-C. (E) Quantification of the number of LC3B-representing PAG10 particles per organelle for the conditions shown in A-D. Cells were randomly screened for LC3B-positive organelles. The number of organelles (N) screened was 80, 71, 74 and 25 for conditions indicated in A, B, C and D, respectively. Only the PFA+GA condition significantly differs from the others at p ≤ 0.0001 using Student’s t -test assuming unequal variance (***). (F) Starved, BafA1-treated U2OS cells fixed with 4% PFA for 15 min followed by 6 days with 0.6% PFA. Representative image of an autophagosome (AP) labeled for LC3B (1:6) using a gold enhancement step after PAG5 labeling. AP, autophagosome; ER, endoplasmic reticulum; M, mitochondrion. Scale bars: 200 nm.
Article Snippet: For the LC3-GFP double labeling, LC3 was first labeled with
Techniques: Labeling, Immunolabeling
Journal: Autophagy
Article Title: An optimized protocol for immuno-electron microscopy of endogenous LC3
doi: 10.1080/15548627.2022.2056864
Figure Lengend Snippet: Effect of different fixation and labeling regimes on morphology. U2OS cells starved for 2.5 h in the presence of BafA1, fixed and labeled with Cosmo Bio anti-LC3B and PAG10 according to the indicated fixation and labeling protocols. ( A, E, G ) Typical examples of autophagosomes (AP) using different fixation and labeling regimes. The double membrane is partially extracted resulting in a halo with remnants of inner (open arrowheads) and outer (black arrowheads) membrane. Arrows indicate LC3 PAG10 associated with the outer membrane. (C) Phagophore (PG), recognizable by the edge (shafted arrowhead) of the cup-shaped double membrane. ( B, D, F, H ) Examples of autolysosomes (AL) using the indicated fixation and labeling regimes. Autolysosomes typically contain autophagic content (asterisks) positive for LC3B, are sometimes filled with internal vesicles and display an overall heterogeneous content. The different PFA fixations yield comparable morphologies. The PFA+GA fixation yields an overall better ultrastructure, yet still results in the halo around phagophores and autophagosomes. Dilutions Cosmo Bio LC3B antibody: 1:4 (A, C, D), 1:10 (B, E-H). Asterisk, autophagic content. ER, endoplasmic reticulum; G, Golgi; M, mitochondrion; N, nucleus; PM, plasma membrane. Scale bars: 100 nm (A, C, E, G, H), 200 nm (B, D, F).
Article Snippet: For the LC3-GFP double labeling, LC3 was first labeled with
Techniques: Labeling, Membrane, Clinical Proteomics
Journal: Autophagy
Article Title: An optimized protocol for immuno-electron microscopy of endogenous LC3
doi: 10.1080/15548627.2022.2056864
Figure Lengend Snippet: Immuno-EM of endogenous LC3B in primary cells and tissues. (A-B) Primary mouse macrophages were starved for 30 min without addition of BafA1. LC3B labeling (1:6; PAG10, arrows) is detected on autophagosomes (AP) and autolysosomes (AL). Gold particles are associated with the inner (open arrowheads) and outer (black arrowheads) autophagosome membrane. (A) Cells fixed ON with 4% FPA. (B) Cells fixed with 2% PFA+0.2% GA for 2 h. (C-H) In rat pancreas and liver (1:10; PAG10), in the absence of lysosomal inhibitors, LC3B labeling is detected mainly on autophagosomes (AP) and occasionally on an autolysosome (AL). Gold particles are associated with the inner and outer (arrows) autophagosome membrane. (C-F) Rat exocrine pancreas perfusion fixed with 2% PFA+0.2% GA. (C) Overview. (D, E) Examples of autophagosomes (AP) encapsulating mainly ER membranes. (F) Group of 2 (not-labeled) autolysosomes (AL) and an autophagosome (AP) near the Golgi (G), enlarged from the box in (C). LC3 immunogold label is present on inner and outer (arrows) AP membrane. (G, H) Rat liver perfusion fixed with 4% PFA. (H) LC3 immunogold label on a group of autophagosomes (AP) and an autolysosome (AL) in a hepatocyte, enlarged from the box in (G). Arrows indicate LC3 gold on the outer AP membrane. BC, bile canaliculus; EE, early endosome; ER, endoplasmic reticulum; M, mitochondrion; NE, nuclear envelope; PM, plasma membrane; SG, secretory granule. Scale bars: 100 nm (D), 200 nm (A, B, E, F, H), 1 μm (C), 500 nm (G).
Article Snippet: For the LC3-GFP double labeling, LC3 was first labeled with
Techniques: Labeling, Membrane, Clinical Proteomics
Journal: Autophagy
Article Title: An optimized protocol for immuno-electron microscopy of endogenous LC3
doi: 10.1080/15548627.2022.2056864
Figure Lengend Snippet: LC3B colocalizes with endocytosed BSA 5 , LAMP1 and SQSTM1/p62 in autolysosomes of starved, BafA1-treated U2OS cells. (A) Timeline of experimental setup. (B) Epon section showing BSA 5 -containing autolysosome (AL) with similar morphological features as the AL in (C). (C) LC3B (1:4; PAG10) is present in an autolysosome (AL) also containing BSA 5 (black arrowheads). White arrowheads mark the limiting membrane of the AL, showing that LC3B and BSA 5 colocalize in the AL lumen. (D) Double labeling of LAMP1 (1:60; PAG10) and LC3B (1:10; PAG15). Accumulation of LC3B inside a LAMP1-positive autolysosome (AL) also positive for endocytosed BSA 5 (black arrowheads). (E) Colocalization of LC3B (1:6; PAG15) and SQSTM1/p62 (1:100; PAG10) in a typical autolysosome (AL). SQSTM1/p62 label (arrows) marks only a subset of the LC3B-positive material. Fixation for immuno-EM: (C) and (E) 4% PFA ON; (D) 2% PFA, 0.2% GA, 3 h. Asterisks, autophagic content. PM, plasma membrane. Scale bars: 200 nm.
Article Snippet: For the LC3-GFP double labeling, LC3 was first labeled with
Techniques: Membrane, Labeling, Clinical Proteomics
Journal: Autophagy
Article Title: An optimized protocol for immuno-electron microscopy of endogenous LC3
doi: 10.1080/15548627.2022.2056864
Figure Lengend Snippet: LC3B is incorporated in autolysosomes after extended incubation with BafA1. U2OS cells were treated with BafA1 for the indicated durations and starved in presence of BafA1 using EBSS for 2.5 h before fixation with 4% PFA. Double labeling of LC3B (1:10; PAG15) and LAMP1 (1:100; PAG10) reveals that in all conditions the majority of LC3B label is found in LAMP1-positive autolysosomes (AL). (A) 2.5 h; (B) 5 h; (C) 10 h; (D) 24 h of BafA1 incubation. (E) Quantification of >50 LC3B-positive organelles scored for presence of LAMP1 label per condition. These results indicate that fusion of LC3B-positive autophagosomes with lysosomes proceeds after BafA1 treatment. For more examples of LC3B labeling after BafA1 treatment and the effect on autolysosome morphology, see Fig. S3. Asterisks, autophagic content. M, mitochondrion; N, nucleus. Scale bars: 200 nm.
Article Snippet: For the LC3-GFP double labeling, LC3 was first labeled with
Techniques: Incubation, Labeling
Journal: iScience
Article Title: Single-cell atlas unveils cellular heterogeneity and novel markers in human neonatal and adult intervertebral discs
doi: 10.1016/j.isci.2022.104504
Figure Lengend Snippet:
Article Snippet:
Techniques: Recombinant, Gene Expression, Software
Journal: International Journal of Molecular Sciences
Article Title: Photoreceptor Compartment-Specific TULP1 Interactomes
doi: 10.3390/ijms22158066
Figure Lengend Snippet: IP of retinal lysate. The top panels show Western blot analysis of the MAP1B IP experimental samples probed with Tulp1 antibodies. In the IP product lane, a band corresponding to Tulp1 is detected. A corresponding band is seen in the rat retinal lysate and wt mouse retinal homogenate but not in the tulp1−/− retinal lysate, liver lysate or non-specific IgG IP lanes. The bottom panels show Western blot analysis of the MAP1B IP experimental samples probed with MAP1B antibodies. In the IP product, rat retinal lysate, wt mouse retinal lysate and tulp1−/− retinal lysate lanes, a band corresponding to MAP1B is detected. No bands are seen in the liver or non-specific IgG IP sample lanes.
Article Snippet: Reciprocal co-IPs were performed on whole rat retina lysate to confirm identified Tulp1-binding partners using the following target antibodies:
Techniques: Western Blot
Journal: International Journal of Molecular Sciences
Article Title: Photoreceptor Compartment-Specific TULP1 Interactomes
doi: 10.3390/ijms22158066
Figure Lengend Snippet: Immunolocalization of MAP1B and Tulp1 in P17 mouse retinas. ( A ) Wt retinal sections stained with MAP1B (red) and Tulp1 (green). ( B ) Tulp1−/− retinal sections stained with MAP1B (red) and Tulp1 (green). Sections were counterstained with DAPI (blue). Scale bar: 50 µm. INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; IS, inner segment layer; OS, outer segment layer.
Article Snippet: Reciprocal co-IPs were performed on whole rat retina lysate to confirm identified Tulp1-binding partners using the following target antibodies:
Techniques: Staining