Fig. 2. In vivo association of APRO4 with Src. (A-D) NIH3T3 cells were transiently transfected with the described constructs and analysed by co-immunoprecipitation (IP) followed by immunoblotting (IB) with anti-Src, anti-Flag or anti-Xpress (anti-X). X-APRO4N interacted with Src_wt (A) whereas Flag-APRO4C did not (B). (C,D, left panels) Co-immunoprecipitation of Src_wt, K297M, Y529F, K297M Y529F, and W118R R175K with Flag-APRO4 in NIH 3T3 cells co-expressing Src constructs and Flag-APRO4. (C,D, right panels) Co-immunoprecipitation of Src_wt, Src W118R, Src R175K, and Src W118R R175K with Flag-APRO4 or Src_wt with Flag-APRO4-6PA in NIH 3T3 cells co-expressing the described constructs. Note that no IgG band was detected on the Src immunoblot of the anti-Flag immunoprecipitates (C, top right panel) because a polyclonal anti-Src (SRC2) antibody was used. This antibody, unlike the monoclonal anti-Src antibody (MAb 327) that was used in all other figures, does not cross react with the heavy IgG chain. Whole-cell lysates of transfected cells were analysed by western blotting with the appropriate antibody to verify equal expression of all the constructs. Data are representative of three independent experiments.