Figure 4

Galectin-7 impacts E-cadherin dynamics at the plasma membrane. (a) Total cell surface proteins were purified using labelling with biotin and pull down via avidin affinity. Immunoblots show similar levels of surface E-cadherin in HacaT cells or in shGal7 clones. Transferrin receptor was used as a loading control. (b) Quantification of internalized E-cadherin in HaCaT or shGal7 #2 cells at 15, 30, 60 or 120 min after addition of HECD-1 antibody. (c) Representative images of internalized E-cadherin after 5 min incubation with HECD-1 antibody, directed against E-cadherin ectodomain. Maximum intensity projection of Z-stacks (Z-steps = 0.21 μm) obtained with ImageJ. Scale bar = 10μm (left panels) or 5μm (zoom, right panels). (d,e) Quantification of internalized E-cadherin during 30 min at 37 °C after addition of 0 to 1 μg.ml⁻¹ recombinant galectin-7 (rGal7) in shGal7 #2 clone (d) or control HaCaT (e). (f) Quantification of internalized E-cadherin in HaCaT or shGal7 #2 cells during 30 min at 37 °C after addition of 0 or 0.5 μg.ml⁻¹ recombinant CRD-mutated galectin-7 (R74S). (b,d,e,f) Between 15 and 17 measures per condition were performed in n = 3 independent experiments. Mean values ± s.e.m. are represented. P-values obtained from unpaired t-tests with Welch’s correction are represented. ***p < 0.001; **p < 0.01; *p < 0.05.