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. 2018 Feb 15;29(4):435–451. doi: 10.1091/mbc.E17-06-0356

FIGURE 7:

FIGURE 7:

Hemidesmosome integrin endocytosis requires Arf6. (A, B) Antibody uptake assay after incubating HaCaT cells with the monoclonal antibody (mAb) GoH3 (anti-ITGA6) on ice and shifting cells to 37°C for 1 h. (A) Representative confocal images of ICs +1 µm above HDs in siRNA-transfected cells showing uptaken anti-ITGA6 antibody bound to the extracellular domain of ITGA6 (GoH3 – left panel) and total ITGB4 revealed by immunostaining of the endogenous protein after fixation (right panel). Scale bar = 1 µm. (B) Ratio of internalized GoH3-ITGA6/total ITGB4 measured in several ICs of individual cells for each condition following siRNA transfection from three independent experiments; number of ICs siCTL = 30/262, siArf6 = 27/185, siCav1 = 30/218, and siCavin1 = 25/196 cells/ICs. Student’s t tests: siCTL vs. siArf6, siCav1, or siCavin1. (C) Amount of endocytosed ITGA6 and ITGB4 integrins, following cell surface biotinylation at 4°C followed by internalization at 37°C for 30 min. The amount of biotinylated α6 and β4 integrins was measured by capture ELISA with peroxidase-conjugated streptavidin, corrected for the total amount of ITGA6 or ITGB4 measured by Western blotting; ratios are expressed relative to control cells. (D) ITGA6 and ITGB4 protein levels assessed by Western blotting in HaCaT cells transfected with control or anti-Arf6 or Cav1 siRNAs. (C, D) All data from four independent experiments. Student’s t tests: siCTL vs. siArf6 or siCav1. (E) Quantification of the area covered by HDs at the basal membrane (from ITGA6 staining) of cells treated with vehicle or the dynamin inhibitor Dynasore (Dyna) from three independent experiments; number of cells = 31 and 36. Studen’ts t test: CTL vs. Dyna. (F) Ratio of internalized GoH3-ITGA6/total ITGB4 measured in several ICs of individual cells for each condition following treatment with vehicle or Dynasore from three independent experiments; number of ICs CTL = 18/105, Dyna = 18/131 cells/ICs. Student’s t test: CTL vs. Dyna. (G) FRAP analysis of EGFP-ITGB4 in HDs of cells treated with vehicle or Dynasore from four independent experiments; number of cells = 33 and 33. Mann-Whitney test: CTL vs. Dyna.