FRS2 and Gab1 potentiate FGF-induced activation of Akt.
(A) Quiescent PC12 or NIH 3T3 cells were unstimulated or
stimulated with FGF1 and heparin (100 ng/ml and 5 μg/ml,
respectively) for 5 min. Equivalent amounts of total cell lysates were
resolved by SDS–PAGE and then followed by immunoblotting with
anti-pS-Akt or anti-Akt antibodies. (B) The 293 cells
were transfected with expression vectors for FGFR1, FRS2α, Gab1 and
Akt as indicated. The expression of FGFR1, FRS2α, and Gab1 was
verified by immunoprecipitation with antibodies specific to these
proteins and followed by SDS–PAGE and immunoblotting with the same
antibodies. Equivalent amounts of total cell lysates were resolved by
SDS–PAGE and immunoblotted with anti-pS-Akt or anti-Akt antibodies.
(C) The MBD of Gab1 inhibits Gab1-potentiated
FGFR-induced activation of Akt. 293 cells were transfected with the
expression vectors for FGFR1, Akt and Gab1-Flag, Gab1(3F)-Flag,
Gab1-MBD-Flag, or Gab1-PH-Flag as indicated. Equivalent amounts of
total cell lysate were resolved by SDS–PAGE and followed by
immunoblotting with anti-FGFR1, anti-Flag, anti-Akt, or anti-pS-Akt
antibodies.