FIG. 1.
Association of p50cdc37, Hsp90, and Raf-1 in vivo and in vitro. (A) Lane 1, anti-Raf-1 IP from [35S]methionine-labeled Cos-1 cells. Lanes 2 to 5, after the primary anti-Raf IP was boiled for 2 min in the presence of 0.5% SDS, a second IP was carried out with anti-Hsp90 or control (c) antibody (lanes 2 and 3) or with polyclonal anti-p50cdc37 or nonimmune rabbit (c) antibody (lanes 4 and 5, respectively). Lanes 6 and 7, anti-p50cdc37 primary IPs and nonimmune rabbit serum IPs, respectively, from [35S]methionine-labeled Cos-1 cells. A second IP with anti-Hsp90 antibody (lane 8) was performed with a fraction of the anti-p50cdc37 primary immunoprecipitate identical to that run in lane 6. The relative migration of molecular weight marker proteins is indicated. (B) Plasmids pMT3-HA-p50cdc37 and pMT3-HA were transiently transfected into Cos-1 cells, and extracts were immunoprecipitated with anti-FLAG antibody (Ab) M5 as a control (lane 1) or anti-HA monoclonal antibody 12CA5 under either denaturing or mild conditions (RIPA or NP-40 LB buffer; lanes 2 and 3, respectively) or, to purify endogenous Raf-1 and p50cdc37 proteins, with anti-Raf-1 (lane 4) and anti-p50cdc37 (lane 5) monoclonal antibodies. Immunoprecipitated proteins were examined by Western blotting (WB) and ECL for the presence of transfected HA-p50cdc37 with anti-HA antibody or for the presence of both transfected and endogenous p50cdc37 with anti-p50cdc37 rabbit antisera. Endogenous Raf-1 and Hsp90 proteins were detected with rabbit-anti-Raf-1 antibodies and rat-anti-Hsp90, respectively (top to bottom panels). IgGH, precipitating IgG antibody heavy chains. (C) FLAG-p50cdc37 (immunoaffinity purified from baculovirus-infected Sf9 cells) and Hsp90 (recombinant E. coli; Stressgen) were assayed in vitro for binding to bacterially produced GST-Raf-1, GST-p50cdc37, or GST alone as indicated by GSH-Sepharose pull-down assays and Western blotting (WB) with the indicated antibodies as described in Materials and Methods. Anti-Hsp90 immunoblotting performed with two distinct Hsp90-specific antibodies (SPA-830 and SPA-771) is shown (bottom two panels). The first two lanes indicate the input amounts of purified proteins added. The arrowhead denotes the position of the full-length GST-Raf-1 above the breakdown products.