Figure 6. Effects of tyrosine phosphorylation on the binding of GIT1 to paxillin.

COS7 cells were transfected with GIT1 constructs (A) or cotransfected with the GIT1 constructs together with c-Src (B). The cells cotransfected with c-Src were also treated for 20 min at 37°C with 1 mM pervanadate before lysis (pV). In parallel, a lysate was prepared from cells transfected with orange-paxillin (PXN). After immunoprecipitation of each GFP-GIT1 construct with anti-GFP antibodies (250 μg of protein lysate/immunoprecipitation), the beads were incubated for 2 h at 4°C with aliquots of the lysate containing orange-paxillin (180 μg). Lysates (25 μg) and immunoprecipitates (IP) after pulldown were used for immunoblotting for the indicated antigens.