Fig. 5

RalA-ANXA2-Src complex is required for activating ERK1/2 signal. A The interaction between RalA and ANXA2 was confirmed by co-immunoprecipitation in MDA231 cells with transient RalA overexpression. B In vitro interactions between RalA and ANXA2 was determined by GST pull-down assay. C Confocal staining presented the co-localization of RalA and ANXA2 in MDA231 cells. Pearson’s correlation (0.7852) of the co-localization between RalA and ANXA2 was analyzed by Las X software. D ZDOCK used to predict the binding sites of RalA and ANXA2. Constructs with deleted mutations Δ80–120 and Δ121–160 of RalA-Flag were used for immunoprecipitation with ANXA2-HA. E WT and RalA KO MDA231 cells were subjected to immunoblotting assays. F Western blot analyses of ERK1/2 and p-ERK1/2 in MDA231 cells transduced with si-ANXA2 or ctrl. GAPDH serves as a loading control. G BC cells with RalA overexpression alone or in combination with si-Src were subjected to transwell migration assays. Scale bar, 100 μm. Error bars, SEM. n = 3. ***p<0.001, ****p < 0.0001 by Student’s t-test. H RalA overexpression MDA231 and MDA468 cells in combination with si-Src were subjected to western blotting assays. I Co-immunoprecipitation assays showed that endogenous Src interacted with endogenous RalA and ANXA2 in BC cells. Endogenous ANXA2 interacted with endogenous RalA and Src in BC cells. J Co-immunoprecipitation assay showed the interaction between ANXA2 and Src in control or RalA overexpression BC cells. K The interaction between ANXA2 and Src in control (DMSO) or RBC8 (Selleck, s7606) treated BC cells were verified by co-immunoprecipitation assays. L FOXD1 overexpression MDA231 and MCF7 cells in combination with RBC8 (0, 5, and 10 μM, 48 h) were subjected to western blotting assays. RalA-GTP levels were analyzed by pulldown assays