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

Journal: PLOS Genetics

Article Title: Impact of maternal compensation on developmental phenotypes in a zebrafish model of severe congenital muscular dystrophy

doi: 10.1371/journal.pgen.1011987

Figure Lengend Snippet: A : Fluorescent staining of the outer layers of the retina showing comparable zpr1 (Arr3a) staining in the photoreceptor outer segments and pedicles in the outer plexiform layer, but reduced and disrupted synaptophysin (syp) staining, protrusion of photoreceptor cell bodies in the outer plexiform layer (arrowheads), and disorganization of horizontal cells lining the top of the outer nuclear layer (asterisks) suggesting defects to the ribbon synapses (40X magnification, scale bars: 20 µm for main panels, 10 µm insets). B : Quantification of synaptophysin intensity in the outer plexiform layer showing a significant reduction in MZKOs (**p = 0.0069). C : Fluorescent staining of transverse cryosections with zn-8 and DAPI showing an overall disruption in morphology of the retina with evidence of retinal ganglion cell axon defasciculation in the optic nerves at the chiasm (10X magnification, scale bars: 100 µm for main panels, 20 µm for chiasm insets).

Article Snippet: The primary antibodies used in these experiments were 1:200 anti-synaptophysin (Abcam), 1:200 zpr1 (ZIRC), and 1:100 zn-8 (Developmental Studies Hybridoma Bank).

Techniques: Staining, Disruption

Journal: Cancers

Article Title: Wnt5a Regulates Focal Adhesion Formation to Promote Migration in Ewing Sarcoma

doi: 10.3390/cancers17223712

Figure Lengend Snippet: ( A ) Western blot for pFAK (Tyr 397) and pSrc (Tyr 416) in seven Ewing sarcoma cell lines treated with vehicle control (DMSO) versus WNT974, with a decrease in both pFAK and pSrc in the majority of cell lines treated with WNT974. ( B ) Immunofluorescence example for pFAK (Tyr 397) in A4573 cells. Note that pFAK (green) is found in the protrusions of the DMSO-treated A4573 cells but is found in the centralized clumps of actin (pink) in the WNT974-treated cells. With WNT974 treatment, there is a statistically significant decrease in pFAK puncta and the amount of pFAK throughout the cells. Green-fluorescent insert demonstrates pFAK with dots representative of Volocity analysis. ( C ) Boyden chamber assay using two different doses of FAK inhibitor GSK2256098 in A4573 and TC71 cells. Note that migration is significantly impaired in both cell lines upon treatment with the FAK inhibitor, even at nanomolar doses. ( D ) Treatment with Src inhibitor, saracatinib (1 μM), also results in a statistically significant decrease in cell migration, also suggesting that Src is important for Ewing sarcoma cell migration. In addition to catalytic proteins, focal adhesions contain adaptor proteins that mediate interactions between the cytoskeleton and the extracellular matrix . ALCAM is an adaptor protein that contains immunoglobulin-rich domains with the amino-terminal V-type immunoglobulin domain required for cell-to-cell adhesive interactions. ALCAM can form homotypic or heterotypic interactions, and its expression is highest at areas of cell-to-cell contact where it can interact with other cell adhesion molecules [ , ]. ALCAM is expressed in over 70% of pediatric sarcomas, so we started by assessing changes in protein expression with WNT974 treatment . ALCAM protein levels were unchanged with WNT974 treatment, so we next evaluated ALCAM distribution in the cell (Supplementary E). As shown in A, the vehicle control-treated cells maintain a small number, typically 2–3, of well-formed long protrusions in which ALCAM can be found throughout, in addition to its presence in the perinuclear region of the cytoplasm. In contrast, in cells treated with WNT974, there is bright centralized clumping of ALCAM and a notable loss of long protrusions in most of the cells. The highest concentration of ALCAM in these cells appears to overlap the nucleus, in contrast to the perinuclear cytoplasmic distribution in the vehicle control-treated cells. In addition, many of the WNT974-treated cells demonstrate a more circumferential ruffled appearance to the plasma membrane. In addition, there is a decrease in the number of long protrusions from the cells with WNT974 treatment ( A). ALCAM is a member of a family of cell adhesion molecules. Another family member that has been implicated in sarcoma biology is MCAM [ , , ]. The involvement of ALCAM in Wnt5a-mediated cytoskeletal rearrangements is specific, because no such changes were appreciated with MCAM immunofluorescence ( A). * p

Article Snippet: Blocked slides were incubated with primary antibodies including ALCAM (Novus, #NBP2-37358, 1:1000), vinculin (Abcam, #ab129002, 1:1000), and phosphorylated Fak (Tyr397) (Abcam, #ab81298, 1:200) overnight at 4 °C, washed in PBS, incubated with Alexa Fluor 488-conjugated, donkey anti-mouse secondary antibody (1:1000; Life Technologies), Alexa Fluor 555-conjugated goat anti-rabbit secondary, or Alexa Fluor 647-conjugated goat anti-mouse, plus Dapi 1:1000 for 1 h, and then visualized with a Nikon Spinning Disc confocal microscope, including 2 μM Z-stacks.

Techniques: Western Blot, Control, Immunofluorescence, Boyden Chamber Assay, Migration, Adhesive, Expressing, Concentration Assay, Clinical Proteomics, Membrane

Journal: Cancers

Article Title: Wnt5a Regulates Focal Adhesion Formation to Promote Migration in Ewing Sarcoma

doi: 10.3390/cancers17223712

Figure Lengend Snippet: ( A ) Immunofluorescence of ALCAM (green) and vinculin (red) in A4573 cells and TC71 cells. Note the co-localization of both proteins and the centralized clumping with WNT974 treatment in both cell lines. When given recombinant Wnt5a, ALCAM and vinculin both appear less bright versus if the cells are treated with WNT974 and then given that same dose of recombinant Wnt5a. This suggests that the cells respond to the amount of Wnt5a in the environment which affects ALCAM and vinculin. Additional panel demonstrating similar findings in TC71 cells. MCAM immunofluorescence does not demonstrate a redistribution pattern upon WNT974 treatment, in contrast to ALCAM. Quantification of protrusions per cell demonstrating a decrease in protrusions upon WNT974 treatment. ( B ) Phalloidin staining of filamentous actin is statistically significantly decreased in A4573 cells treated with WNT974 compared to vehicle control. ( C ) Immunoprecipitation of vinculin and ALCAM in A4573 cells demonstrating a significant increase in association between the two proteins upon WNT974 treatment. Notably, there is a complete dissociation between the proteins upon FAK inhibition treatment. Both changes are different from vehicle control-treated cells, suggesting that changes to how much these two proteins are in contact affects cell migration. ( D ) ( i ) Using palmostatin B, an inhibitor of acyl-protein thioesterase 1 which depalmitoylates cell surface receptors to allow for lateral migration through the membrane, A4573 cells demonstrate a centralized clumping of ALCAM. ( ii ) Additionally, WNT974 and palmostatin B only inhibit migration in Boyden chamber assays in CLHA10, the metastatic-derived cell line, but not in CHLA9, the localized-derived cell line. Only CHLA10 responds to the lowest amount of recombinant Wnt5a as a chemoattractant by significantly increased cell migration to approximately 400% of vehicle migration, consistent with the hypothesis that Wnt5a may be more active in the metastatic process. Each experiment was repeated a minimum of three times. Error bars represent standard error of the mean of triplicate experiments, and asterisks indicate the degree of statistical difference between indicated conditions ** p

Article Snippet: Blocked slides were incubated with primary antibodies including ALCAM (Novus, #NBP2-37358, 1:1000), vinculin (Abcam, #ab129002, 1:1000), and phosphorylated Fak (Tyr397) (Abcam, #ab81298, 1:200) overnight at 4 °C, washed in PBS, incubated with Alexa Fluor 488-conjugated, donkey anti-mouse secondary antibody (1:1000; Life Technologies), Alexa Fluor 555-conjugated goat anti-rabbit secondary, or Alexa Fluor 647-conjugated goat anti-mouse, plus Dapi 1:1000 for 1 h, and then visualized with a Nikon Spinning Disc confocal microscope, including 2 μM Z-stacks.

Techniques: Immunofluorescence, Recombinant, Staining, Control, Immunoprecipitation, Inhibition, Migration, Membrane, Derivative Assay

Journal: Cancers

Article Title: Wnt5a Regulates Focal Adhesion Formation to Promote Migration in Ewing Sarcoma

doi: 10.3390/cancers17223712

Figure Lengend Snippet: CRISPR-Cas9 gene editing confirms the critical role of Wnt5a in Ewing sarcoma migration. ( A ) Western blotting confirms that the Wnt5a CRISPR-Cas9 clones A9.5 and F10.6 lack Wnt5a protein expression and show a decrease in both vinculin and ALCAM expression as well as a shift in ALCAM banding patterns compared with the parental cells A4573. Beta-2-microglobulin (B2M) is a loading control. ( B ) Photomicrograph of Wnt5a CRISPR-Cas9 clone A9.5 which grows as clumps of cells with bright GFP+ signal (100 μm). ( C ) In Boyden chamber assays, the Wnt5a CRISPR-Cas9 clones migrate statistically significantly less than the scramble control. ( D ) In Boyden chamber assays using FBS as a chemoattractant, the Wnt5a CRISPR-Cas9 clones demonstrate impaired migration in comparison to scramble control. In panels C and D, error bars represent standard error of the mean, and asterisks reflect the degree of statistical significance. Experiments were repeated three times. ( E ) PNGase assay of ALCAM in parental A4573 cells, scramble control, and the two Wnt5a knock-out clones demonstrate a different banding pattern in the Wnt5a knock-out clones when treated with PNGase (denoted as +) compared with parental cells or scramble control. ( F ) Western blotting demonstrates a lack of phospho-FAK in the Wnt5a knock-out clones. ( G ) Immunofluorescence analysis of parental A4573 cells, scramble control cells, and the two Wnt5a knock-out clones shows that the parental cells have an average of 2–3 large protrusions and co-localization of ALCAM (green) and vinculin (red) upon treatment with vehicle control (DMSO) whereas upon WNT974 treatment, the cells appear to have decreased vinculin as well as centralized perinuclear clumping of ALCAM. The same pattern is also seen in the scramble control. In contrast, both the morphology of the Wnt5a knock-out clones A9.5 and F10.6, as well as the distribution of ALCAM and vinculin, is not affected by WNT974. The Wnt5a knock-out clones, A9.5 and F10.6, have a statistically significantly increased colocalization between ALCAM and vinculin when treated with WNT974 (quantified by MFI in the fused channel), a contrast to the decreased co-localization seen in scramble control cells treated with WNT974. To the right is quantification of co-localization. Error bars represent standard deviation. ( H ) Staining of cells with fluorescently tagged phalloidin shows significantly less filamentous actin in Wnt5a knock-out clones compared with parental cells and scramble control (60X). Treatment of parental cells and scramble control with WNT974 decreases phalloidin staining, an effect not seen in the Wnt5a knock-out clones. To the right is quantification of the phalloidin staining. Error bars represent standard deviation, and asterisks indicate the degree of statistical significance. Each experiment was repeated a minimum of three times. * p

Article Snippet: Blocked slides were incubated with primary antibodies including ALCAM (Novus, #NBP2-37358, 1:1000), vinculin (Abcam, #ab129002, 1:1000), and phosphorylated Fak (Tyr397) (Abcam, #ab81298, 1:200) overnight at 4 °C, washed in PBS, incubated with Alexa Fluor 488-conjugated, donkey anti-mouse secondary antibody (1:1000; Life Technologies), Alexa Fluor 555-conjugated goat anti-rabbit secondary, or Alexa Fluor 647-conjugated goat anti-mouse, plus Dapi 1:1000 for 1 h, and then visualized with a Nikon Spinning Disc confocal microscope, including 2 μM Z-stacks.

Techniques: CRISPR, Migration, Western Blot, Clone Assay, Expressing, Control, Comparison, Knock-Out, Immunofluorescence, Standard Deviation, Staining

Journal: Cancers

Article Title: Wnt5a Regulates Focal Adhesion Formation to Promote Migration in Ewing Sarcoma

doi: 10.3390/cancers17223712

Figure Lengend Snippet: Working model of the role of Wnt5a in Ewing sarcoma cells. Upon Wnt5a binding with its Fzd receptor(s) and potential co-receptor(s), FAK (Tyr 397) becomes phosphorylated and dimerizes to phosphorylate Src (Tyr 416). Phosphorylated Src then phosphorylates vinculin, allowing it to bind to F-actin bundles and cross-linking it to ALCAM. ALCAM is depalmitolyated by APT1 to allow it to move within the cell membrane, thereby deforming the membrane at the site of proximity to actin bundles.

Article Snippet: Blocked slides were incubated with primary antibodies including ALCAM (Novus, #NBP2-37358, 1:1000), vinculin (Abcam, #ab129002, 1:1000), and phosphorylated Fak (Tyr397) (Abcam, #ab81298, 1:200) overnight at 4 °C, washed in PBS, incubated with Alexa Fluor 488-conjugated, donkey anti-mouse secondary antibody (1:1000; Life Technologies), Alexa Fluor 555-conjugated goat anti-rabbit secondary, or Alexa Fluor 647-conjugated goat anti-mouse, plus Dapi 1:1000 for 1 h, and then visualized with a Nikon Spinning Disc confocal microscope, including 2 μM Z-stacks.

Techniques: Binding Assay, Membrane