Fig. 2. Adaptor protein ArgBP2 links Pyk2 and Cbl in a complex. (A) Pyk2 and Cbl were co-expressed together with FLAG-tagged ArgBP2 (88 kDa), CAP (90 kDa) or CMS (85 kDa) as indicated. Immunoprecipitation (IP) was performed with anti-FLAG (M2) or anti-Cbl (RF) antibodies. Western blotting (WB) was done as indicated with anti-Cbl (TL), anti-Pyk2 (N-19) and anti-FLAG (M5) antibodies. (B) Pyk2 and Cbl were co-expressed in HEK 293T cells with increasing amounts of ArgBP20 Cbl, Pyk2 or FLAG-tagged ArgBP2 were immunoprecipitated (IP) with anti-Cbl (RF), anti-Pyk2 (600) or anti-FLAG (M2) antibodies, respectively. Western blotting (WB) was performed as in A). (C) Glutathione S-transferase (GST) alone or GST fusion proteins of each of the three SH3 domains of ArgBP2 (SH3A, SH3B, SH3C) were incubated with equal amounts of lysates from HEK 293T cells overexpressing Pyk2. GST-ArgBP2-SH3A was also incubated with lysates expressing a kinase inactive mutant of Pyk2 (PKM) or a double mutant of PKM mutated at two prolines in major proline-rich motifs (PKM P717/859A). Western blotting (WB) was performed with anti-Pyk2 (N-19) antibodies. Levels of GST fusion proteins were examined by Ponceau staining. (D) PKM, PKM-P717/859A or Pyk2 were coexpressed with or without Cbl and cell lysates were subjected to immunoprecipitation (IP) with antibodies against Pyk2 (600). Western blotting (WB) was performed with anti-Cbl (RF) and anti-Pyk2 (N-19) antibodies. TCL, total cell lysate.