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Fig. 4. A Pyk2-ArgBP2-Cbl complex is recruited to lipid rafts following growth factor stimulation. (A) Serum-starved PC12-Myc-ArgBP2 cells were mock-stimulated or treated for 10 minutes or 2 hours with 50 ng/ml NGF and homogenized lysates were subjected to sucrose gradient centrifugation as described in the Materials and Methods. Immunoprecipitates of Cbl, Pyk2, Myc-ArgBP2, Crk and flotillin-1 from lipid rafts and Triton X-100 soluble fractions were subjected to western blot with the following antibodies: anti-Cbl (RF), anti-Pyk2 (N-19), anti-Myc (9E10), anti-Crk and anti-flotillin-1. (B) HEK 293T cells were transfected with Pyk2 or PKM together with Cbl and FLAG-ArgBP2, serum-starved and stimulated with a mixture of 50 ng/ml EGF and 50 ng/ml PDGF-BB for 20 minutes. Immunoprecipitates of Cbl, Pyk2 and ArgBP2 from lipid rafts and Triton X-100-soluble fractions were analyzed with antibodies against Cbl (RF), Pyk2 (N-19), FLAG(M2). Fyn was detected in total cell lysates. (C) Lysates of HEK 293T cells overexpressing FLAG-CAP, FLAG-ArgBP2 or nArgBP2 were subjected to GST pulldown with GST alone or GST-flotillin-1. Western blotting was done with anti-FLAG and anti-nArgBP2 antibodies. (D) HEK 293T cells overexpressing Pyk2, Cbl and Myc-ArgBP2-SoHo were serum-starved and stimulated with a mixture of 50 ng/ml EGF and 50 ng/ml PDGF-BB for 20 minutes. Immunoprecipitates of Cbl (RF), Pyk2 (600), Myc-ArgBP2-SoHo (9E10) and flotillin-1 from lipid rafts and Triton X-100 soluble fractions are shown. Western blotting was performed with antibodies against Cbl (RF), Pyk2 (N-19), Myc (9E10) and Flotillin-1.

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