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Fig. 1. Constitutive association between Pyk2 and Cbl in mammalian cells. (A) PC12-PDGFR cells were starved for 16 hours and were left untreated (–) or stimulated with 100 ng/ml EGF (E) or PDGF (P) for 5 minutes. Following cell lysis, equal amounts of cell lysates were subjected to immunoprecipitation (IP) with antibodies to Pyk2 (600) or Cbl (RF) or pre-immune sera (pre-im) and immunoprecipitates were analyzed by western blotting (WB) with anti-Cbl (TL) and anti-Pyk2 (N-19) antibodies. TCL, total cell lysate. (B) Pyk2 or a kinase inactive mutant of Pyk2 (PKM) were overexpressed in HEK 293T cells alone or together with Cbl, or with Cbl and Src kinase (K+) or Cbl and a kinase inactive mutant of Src (K–). Cbl was immunoprecipitated (IP) with anti-Cbl (RF) antibodies and co-precipitation was monitored by western blotting (WB) with anti-Pyk2 (N-19) antibodies. Levels of proteins in total cell lysates (TCL) were determined with anti-Cbl (TL), anti-Pyk2 (N-19) and anti-Src (Ab1) antibodies. (C) Pyk2 or PKM were overexpressed in HEK 293T cells and equal amounts of cell lysates were subjected to glutathione S-transferase (GST) pulldown with GST fusion proteins of the amino terminus of Cbl spanning the SH2 and RING finger domains (GST-Cbl NT) or the carboxyl terminus of Cbl containing the proline-rich sequences; the acidic box and the leucine zipper domain (GST-Cbl CT) and the western blot (WB) was probed with anti-Pyk2 (600) antibodies.
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