Complex formation between Grb2 and Gab1 in FGF-stimulated cells.
(A) Quiescent Swiss 3T3 cells were unstimulated or
stimulated with FGF1 and heparin (100 ng/ml and 5 μg/ml,
respectively) for 10 min. Lysates were immunoprecipitated with
anti-Grb2 antibodies and followed by SDS–PAGE and immunoblotting with
anti-pTyr antibodies. (B) Swiss 3T3 cells were
unstimulated or stimulated with FGF1 and heparin (100 ng/ml and 5
μg/ml, respectively) for 10 min. Cell lysates were incubated with
immobilized GST fusion proteins of the N-terminal SH3, SH2, or
C-terminal SH3 domains of Grb2. Bound proteins were eluted and resolved
by SDS–PAGE and then followed by immunoblotting with anti-pTyr
(Upper) or anti-Gab1 antibodies (Lower).
(C) Gab1 binds to the C-terminal SH3 domain of Grb2
through the MBD. 293 cells were transfected with the expression vector
for Flag-tagged MBD of Gab1. The lysates were incubated with
immobilized GST fusion proteins of the SH2, N-SH3, or C-SH3 domains of
Grb2. Bound proteins were eluted and resolved by SDS–PAGE and followed
by immunoblotting with anti-Flag antibodies.