ab 323338  (Bio-Rad)

Journal: eLife

Article Title: Clathrin-independent endocytosis and retrograde transport in cancer cells tune immune synapse organization and CD8 T cell response

doi: 10.7554/eLife.105821

Figure Lengend Snippet: ( A ) Illustration of the SNAP-tag-based BG-labeled antibody uptake assay to study membrane protein endocytosis and retrograde transport. ( B ) Confocal images of GalT-GFP-SNAP (green) and TMR-Star (red) in HeLa cells stably expressing the Golgi-resident GFP-fused SNAP-tag construct (HeLa GalT-GFP-SNAP). Actin (phalloidin, white) and nuclei (DAPI, blue) were also stained. Fluorescence intensity profile was made along the dashed line region in enlarged cropped area and shows the colocalization of both signals. Scale bar: 20 μm. ( C–F ) Retrograde transport of ALCAM and ICAM1. Continuous BG-labeled anti-ALCAM ( C, E ) and anti-ICAM1 ( D, F ) antibody uptake for 4 h at 37°C in HeLa GalT-GFP-SNAP cells. ( C, D ) Western blot analysis of HeLa GalT-GFP-SNAP cells transfected for 72 h with siRNAs: negative control (siCtrl) or against retromer subunits (siVPS35 and siVPS26A). Immunodetection made with anti-SNAP, anti-VPS35, anti-VPS26A, and anti-α-Tubulin (loading control) antibodies. Quantification of the covalent IgG-SNAP-GFP-GalT complex is shown as fractions of siCtrl condition (histogram). Quantification of VPS35 and VPS26A depletion is shown in . ( E, F ) Western blot analysis of HeLa GalT-GFP-SNAP cells transfected for 72 h with siRNAs: negative control (siCtrl) or against EndoA3 (siEndoA3). Immunodetection made with anti-SNAP, anti-EndoA3, and anti-clathrin heavy chain (CHC, loading control) antibodies. Quantification of the covalent IgG-SNAP-GFP-GalT complex (IB:anti-SNAP) is shown as fractions of siCtrl condition (histogram). Quantification of EndoA3 depletion is shown in . Data information: In ( B ), images are from a single experiment. Quantification data ( C–F ) are pooled from three independent experiments. Data are presented as mean ± SEM. *p<0.05, **p<0.01. One-sample t test and Wilcoxon test. Figure 1—source data 1. Original files for western blot analyses displayed in . Figure 1—source data 2. PDF files containing original western blots for .

Article Snippet: First, anti-ALCAM (Bio-Rad, MCA1926) and anti-ICAM1 (Bio-Rad, MCA1615) antibodies were labeled with benzylguanine (BG) by incubating them overnight at 4°C with a threefold molar excess of BG-GLA-NHS reagent (New England Biolabs, S9151S; prepared in anhydrous DMSO).

Techniques: Labeling, Membrane, Stable Transfection, Expressing, Construct, Staining, Fluorescence, Western Blot, Transfection, Negative Control, Immunodetection, Control

Journal: eLife

Article Title: Clathrin-independent endocytosis and retrograde transport in cancer cells tune immune synapse organization and CD8 T cell response

doi: 10.7554/eLife.105821

Figure Lengend Snippet: ( A ) Confocal images of GalT-GFP-SNAP (green) and TMR-Star (red) in LB33-MEL cells stably expressing the Golgi-resident GFP-fused SNAP-tag construct (LB33-MEL GalT-GFP-SNAP). Actin (phalloidin, white) and nuclei (DAPI, blue) were also stained. The fluorescence intensity profile was made along the dashed line region in the enlarged cropped area and shows the colocalization of both signals. Scale bar: 20 μm. ( B ) Confocal images of GalT-GFP-SNAP (green) and TGN46 (red) in LB33-MEL GalT-GFP-SNAP cells. Nuclei (DAPI, blue) were also stained. The fluorescence intensity profile was made along the dashed line region in enlarged cropped area and shows the colocalization of both signals, indicating that GalT-GFP-SNAP is correctly localized at the TGN . Scale bar: 10 μm. ( C ) Quantifications of the immunoblots shown in (IB: anti-VPS35 and anti-VPS26A) confirm depletion efficiency of VPS35 and VPS26A in HeLa GalT-GFP-SNAP cells. ( D ) Retrograde transport of ICAM-1. Continuous BG-labeled anti-ICAM1 antibody uptake for 4 h at 37°C in LB33-MEL GalT-GFP-SNAP cells. Western blot analysis of LB33-MEL GalT-GFP-SNAP cells transfected with siRNAs: negative control (siCtrl) or against retromer subunits (siVPS35 and siVPS26A). Immunodetection with anti-SNAP, anti-VPS35, anti-VPS26A, and anti-α-Tubulin (loading control) antibodies. Quantification of the covalent IgG-SNAP complex is shown as fractions of siCtrl condition (histogram). Quantification of VPS35 and VPS26A depletion is shown in ( E ). ( E ) Quantifications of the immunoblots shown in ( D ) confirm depletion efficiency of VPS35 and VPS26A in LB33-MEL GalT-GFP-SNAP cells. ( F ) Retrograde transport of ALCAM. Continuous BG-labeled anti-ALCAM antibody uptake for 4 h at 37°C in HeLa GalT-GFP-SNAP cells. Western blot analysis of HeLa GalT-GFP-SNAP cells transfected for 72 h with siRNAs: negative control (siCtrl) or against Rab6 (siRab6). Immunodetection made with anti-SNAP, anti-Rab6, and anti-clathrin heavy chain (CHC, loading control) antibodies. Quantification of the covalent IgG-SNAP-GFP-GalT complex is shown as fractions of siCtrl condition (histogram). Quantification of Rab6 depletion is shown in ( G ). ( G ) Quantifications of the immunoblots shown in ( F ) confirm depletion efficiency of Rab6 in HeLa GalT-GFP-SNAP cells. ( H ) Quantifications of the immunoblots shown in (IB: anti-EndoA3) confirm depletion efficiency of EndoA3 in HeLa GalT-GFP-SNAP cells. ( I ) Retrograde transport of ALCAM. Continuous BG-labeled anti-ALCAM antibody uptake for 4 h at 37°C in LB33-MEL GalT-GFP-SNAP cells. Western blot analysis of LB33-MEL GalT-GFP-SNAP cells transfected (or not) with plasmids encoding free GFP (GFP+) or EndoA3-GFP (EndoA3+). Immunodetection with anti-SNAP, anti-EndoA3, and anti-α-Tubulin (loading control) antibodies. Quantification of the covalent IgG-SNAP-GFP-GalT complex is shown as fractions of the GFP +condition (histogram). Data information: In ( A, B ), images are from a single experiment. In ( C ), data are pooled from six independent experiments. In ( D–H ), data are pooled from three independent experiments. In ( I ), data are pooled from two independent experiments. Data are presented as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. One-sample t test and Wilcoxon test. Figure 1—figure supplement 1—source data 1. Original files for western blot analyses displayed in . Figure 1—figure supplement 1—source data 2. PDF files containing original western blots for .

Article Snippet: First, anti-ALCAM (Bio-Rad, MCA1926) and anti-ICAM1 (Bio-Rad, MCA1615) antibodies were labeled with benzylguanine (BG) by incubating them overnight at 4°C with a threefold molar excess of BG-GLA-NHS reagent (New England Biolabs, S9151S; prepared in anhydrous DMSO).

Techniques: Stable Transfection, Expressing, Construct, Staining, Fluorescence, Western Blot, Labeling, Transfection, Negative Control, Immunodetection, Control

Journal: eLife

Article Title: Clathrin-independent endocytosis and retrograde transport in cancer cells tune immune synapse organization and CD8 T cell response

doi: 10.7554/eLife.105821

Figure Lengend Snippet: ( A, B ) Live-cell TIRF images of EndoA3-GFP (stable) and ICAM1-mScarlet (transient) in HeLa ( A ) and LB33-MEL ( B ) cells. Time series show enlarged cropped areas corresponding to region 1 in the full-size images and are extracted from . White arrows indicate dynamic co-distribution of both signals. Kymographs were made along the dashed lines in the enlarged cropped areas corresponding to region 2 ( A, B ; ). Scale bars: 10 μm (full-size image) and 2 μm (enlarged cropped areas). ( C–E ) Continuous uptake of anti-ALCAM antibody for 15 min at 37°C in the following LB33-MEL cell lines: wild-type (WT, D ), stably transfected with empty plasmid (Φ, D ), or stably expressing EndoA3-GFP (LB33-MEL EndoA3+, C–E ). In ( E ), cells were transfected with siRNAs: negative control (siCtrl) or against EndoA3 (siEndoA3). Quantification of EndoA3 depletion by western blots in . ( C ) Airyscan images of Anti-ALCAM (red) and EndoA3-GFP (green). White arrowheads show colocalization between ALCAM and EndoA3. Scale bars: 10 μm (full-size image), 1 μm (enlarged cropped areas). ( D, E ) Quantifications of anti-ALCAM internalization, expressed as fractions of WT condition ( D ) or siCtrl condition ( E ). ( D ) n cells: WT, n=270; Φ, n=279; EndoA3+, n=274. ( E ) n cells: siCtrl, n=350; siEndoA3, n=234. Representative image examples in . Data information: All images ( A–C ) are representative of two independent experiments. In ( D, E ), data are pooled from three independent experiments. Data are presented as median and quartiles. ns, not significant. ****p<0.0001 (D, Kruskal–Wallis test with Dunn’s multiple comparison test; E, Mann–Whitney test).

Article Snippet: First, anti-ALCAM (Bio-Rad, MCA1926) and anti-ICAM1 (Bio-Rad, MCA1615) antibodies were labeled with benzylguanine (BG) by incubating them overnight at 4°C with a threefold molar excess of BG-GLA-NHS reagent (New England Biolabs, S9151S; prepared in anhydrous DMSO).

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Expressing, Negative Control, Western Blot, Comparison, MANN-WHITNEY

Journal: eLife

Article Title: Clathrin-independent endocytosis and retrograde transport in cancer cells tune immune synapse organization and CD8 T cell response

doi: 10.7554/eLife.105821

Figure Lengend Snippet: ( A, B ) Confocal images of anti-ALCAM (white spots) internalization in LB33-MEL cells stained for actin (phalloidin, yellow) and nuclei (DAPI, blue). In ( A ), wild-type (WT), stably transfected with empty plasmid (Φ), or stably expressing EndoA3-GFP (EndoA3+) LB33-MEL cells were used. In ( B ), LB33-MEL EndoA3+ cells were transfected with negative control (siCtrl) siRNA or EndoA3 targeting (siEndoA3) siRNA. Quantification for anti-ALCAM internalization shown in . Scale bars: 20 μm. ( C ) Western blot analysis of LB33-MEL cells stably expressing EndoA3-GFP (LB33 MEL EndoA3+) transfected for 72 h with siRNAs: negative control (siCtrl) or against EndoA3 (siEndoA3). Immunodetection made with anti-EndoA3 and anti-α-Tubulin (loading control) antibodies. Quantification of immunoblots shows depletion efficiency of EndoA3 (histogram). Data information: In ( A, B ), images are representative of three independent experiments. In ( C ), western blot images are representative of four independent experiments, from which quantitative data are pooled. Data are presented as mean ± SEM. ****p<0.0001 (one-sample t test and Wilcoxon test). Figure 2—figure supplement 2—source data 1. Original files for western blot analysis displayed in . Figure 2—figure supplement 2—source data 2. PDF file containing original western blots for .

Article Snippet: First, anti-ALCAM (Bio-Rad, MCA1926) and anti-ICAM1 (Bio-Rad, MCA1615) antibodies were labeled with benzylguanine (BG) by incubating them overnight at 4°C with a threefold molar excess of BG-GLA-NHS reagent (New England Biolabs, S9151S; prepared in anhydrous DMSO).

Techniques: Staining, Stable Transfection, Transfection, Plasmid Preparation, Expressing, Negative Control, Western Blot, Immunodetection, Control