Figure 1
From: Annexin A7 is required for ESCRT III-mediated plasma membrane repair
ANXA7 is needed for repair in MCF7-p95ErbB2 cells. (a) Schematic representation of the proteomic setup. MCF7-p95ErbB2 cells were cultured in SILAC medium. Cells were left uninjured (control) or injured by treating with digitonin (20 µg/ml) for 10 min at 37 °C. Cells were disrupted and biotin labelled membrane fragments were affinity purified using streptavidin beads before MS analysis. (b) Plot showing change in cell surface level of proteins presented as the ratio of MCF7-p95ErbB2 cells treated with digitonin over uninjured MCF7-p95ErbB2 as measured by MS analysis (Also see Supplementary Fig. S1A and Table 1). (c) Representative sequential images of HeLa cells showing the translocation of ANXA7-GFP or (d) ANXA7-RFP and ANXA1-GFP (upper panel) or ANXA7-RFP and ANXA5-GFP (lower panel) in MCF7-p95ErbB2 cells in response to focal laser injury (blue arrow indicates injury site). (e) Corresponding translocation kinetic plots. Error bars represent SEM from three independent experiments. (f) Immunoblot showing ANXA7 and S100A11 protein levels in MCF7-p95ErbB2 depleted for ANXA7 or S100A11, as compared to Ctrl siRNA. HSP90 served as loading control (72 h). (g) Plasma membrane repair kinetics upon laser injury measured by membrane impermeable FM1-43 dye influx in MCF7-p95ErbB2 transfected with ANXA7, S100A11 or Ctrl siRNA for 72 h. Error bars represent SEM for at least 10 independent cells per condition. (h) Representative images of MCF7-p95ErbB2 cell transfected with either Ctrl siRNA (upper panel) or ANXA7 siRNA (lower panel) showing the influx of impermeable FM1-43 dye after laser injury (white arrow indicates injury site). The asterisk represent P-values based on Student’s t-test (g): **P ≤ 0.01, as indicated when comparing indicated siRNAs to control siRNA. Full-length blots are presented in Supplementary Fig. S2b.
