Fig. 4: Rack1 mediates the interaction between P-gp and Src in drug-resistant cells.

a Co-immunoprecipitation assay showed that endogenous P-gp and Src interacted with endogenous Rack1 and Cav1 in drug-resistant breast cancer cells, whereas Rack1 or Cav1 interacted with P-gp and Src, no interaction was detected between Rack1 and Cav1. MCF-7/ADR cells were lysed, immunoprecipitated with anti-P-gp, Src, Rack1, or Cav1 antibodies, and then analyzed by western blotting. b Rack1 knockdown decreased the interaction of Src with P-gp and Cav1 in MCF-7/ADR cells. Control and Rack1-silenced cells were lysed, immunoprecipitated with anti-Src antibody, and then analyzed by western blotting. c Silence of Rack1 expression attenuated ability of P-gp binding to Src, but enhanced the interaction between P-gp and Cav1. Control and Rack1-silenced cells were lysed, immunoprecipitated with anti-P-gp antibody, and then analyzed by western blotting. d Src knockdown has no significant effect on the binding of Rack1 to P-gp. Control and Src-silenced cells were lysed, immunoprecipitated with anti-Rack1 antibody, and then analyzed by western blotting. e Knockdown of Src has no significant effect on the interaction between P-gp and Rack1, whereas enhances the binding ability of Cav1 to P-gp. Control and Src-silenced cells were lysed, immunoprecipitated with anti-P-gp antibody, and then analyzed by western blotting. f Expression of Rack1 or its mutant Rack1^Y246F was effectively rescued in Rack1-depleted MCF-7/ADR cells. Rack1 expression was stably silenced by using an shRNA specifically targeting its noncoding region. Then Rack1-silenced cells were infected with lentivirus expressing Flag-tagged Rack1^WT and Rack1^Y246F mutant, and stable rescued cell lines were selected by using 50 μg/mL of hygromycin B. The cells were lysed and immunoblotted with anti-Flag and Rack1 antibodies. g Re-expression of Rack1^WT, but not Rack1^Y246F, can rescue the binding of Src to Rack1. Rack1^WT- and Rack1^Y246F-rescued cells were lysed, immunoprecipitated with anti-Flag antibody, and then analyzed by western blotting. h Re-expression of Rack1^Y246F decreased the interaction between P-gp and Src, but had no effect on its binding to P-gp. Rack1^WT- and Rack1^Y246F-rescued cells were lysed, immunoprecipitated with anti-Src or P-gp antibodies, and then analyzed by western blotting