Fig. 2. hFGFR3 recruits SOCS1 and SOCS3 proteins. (A) HEK-293T cells transiently cotransfected with wild-type hFGFR3 (R3 WT), Myc-tagged SOCS1 (S1), or Myc-tagged SOCS3 (S3), as indicated. The amount of DNA was equalized with empty vector (EV). After 8 hours in serum-free medium, cells were treated with or without 50 ng/ml FGF and 5 µg/ml heparin for 5 minutes. (B) HEK-293 cells were transiently cotransfected with R3 WT or kinase-dead (KD) mutant and with EV, S1 or S3, as indicated. In both experiments, cells were dissolved in lysis buffer and total cell lysates were subjected to 7.5% SDS-PAGE followed by western blotting with anti-hFGFR3 antibody (Total lysates, IB: hFGFR3). Cellular lysate protein (1 mg) was incubated overnight with anti-Myc agarose-conjugated antibody. Immunoprecipitated proteins were separated on 10% SDS-PAGE followed by western blotting with anti-Myc (IP: cMyc, IB: cMyc) or anti-hFGFR3 (IP: cMyc, IB: hFGFR3) antibodies. (C) HEK-293 cells were transiently transfected with R3 WT and five or ten times excess amount of KD mutant, as indicated. The DNA amount was equalized with empty vector. After 8 hours in serum-free medium, cells were stimulated with FGF9 for 5 minutes, lysed and subjected to IP with anti-hFGFR3 antibody. Immunoprecipitated proteins were separated on 10% SDS-PAGE followed by western blotting with anti-phosphotyrosine (IB: P-Tyr) or anti hFGFR3 (IB: hFGFR3) antibodies.