RAB11 is required for BCAP31-mediated EGFR recycling inhibition and cancer development. (A) BCAP31 interacts with RAB11. The indicated constructs (BCAP31-FLAG and RAB11-HA) were transiently expressed in COS-7 cells, and the whole-cell lysates were immunoprecipitated (IP) with the indicated antibody. (B)
In vitro interaction between BCAP31 and RAB11. Whole-cell lysates from MDA-MB-231 cells were prepared, and IP and IB were performed with antibodies as indicated. (C) MDA-MB-231 cells were fixed for costaining of endogenous BCAP31 (green) and RAB11 (red). (D) Confirmation of RAB11 knockdown using western blotting. (E-H) The effects of KD of BCAP31 and RAB11 on the ligand-independent recycling (E), ligand-independent signalling (F), cell proliferation (G), and colony formation (H) of TNBC cells stably expressing the indicated shRNA vectors. The fold differences represent the mean of the experimental group compared with that of the controls. Error bars indicate the means ± SEM. * p < 0.05 compared with the CTRL. (I) Immunoprecipitation of endogenous EGFR from control or BCAP31-knockdown MDA-MB-231 cells. Bar, 10 μm.