Fig. 5.

Mutations at P95 show similar loss of tumor suppressor function and AXL interaction. SKOV3 cells were transduced with an empty vector (CTRL), wild-type (OPCML) or mutant (P95R, P95L, P95S) OPCML and analyzed for protein expression/localization by confocal microscopy (a) and protein expression by western blot (b) with an anti-OPCML antibody. Scale bar = 5 μm. Cells were starved and then stimulated with GAS6 for 30 min, 3 h or 12 h as indicated, and the interaction between OPCML and AXL measured by PLA (c), the signaling to pAKT detected by western blotting (d), the migration tested by gap closure assay (e) and the invasion studied by 3D spheroid invasion assay in Matrigel (f). GAPDH was used as loading control in b, d. The images were taken with a confocal microscope in a or an inverted microscope in e and f. Scale bar = 200 μm (e) and 250 μm (f). All the graphs show the mean ± s.e.m. of three independent experiments. Student t-test compares CTRL and mutants to wild-type OPCML at 24 h of invasion: *p < 0.05, ****p < 0.0001