Fig 1.

Identification of Gab2 as a target of Ras/MAPK signaling. (A and C) HEK293 cells were transfected with an empty vector or Myc-tagged Gab1 or Gab2, serum starved overnight, and stimulated for 30 min with PMA (100 ng/ml) or 10 min with EGF (25 ng/ml) or fetal bovine serum (10%). Immunoprecipitated (IP) Myc-Gab1 or Myc-Gab2 was then assayed for phosphorylation with phosphomotif antibodies that recognize the RXXpS/T and RXXpS/TXP consensus motifs. Phosphorylated and total levels of RSK and ERK1/2 were assayed by immunoblotting on total cell lysates. SE, short exposure; LE, long exposure. (B) As for panel A, except that HEK293 cells were cotransfected with Myc-Gab2 and a constitutively active (G12V) or inactive (S17N) form of Ras. (D) RBL-2H3 cells were incubated overnight with anti-2,4-dinitrophenol IgE and stimulated with 2,4-dinitrophenol for 10 min prior to endogenous Gab2 immunoprecipitation. Phosphorylation of endogenous Gab2 was assayed using a phosphomotif antibody that recognizes RXXpS/T sequences.