Fig. 4. Tyrosine-phosphorylated proteins in osteoclast-like cells. (A) Tyrosine-phosphorylated proteins in lysates of chicken osteoclast precursors cultured for 4 days in the absence or presence of RANKL-ODF were identified on immunoblots with the antibody 4G10 (an identical protein profile was revealed using the anti-phosphotyrosine antibody, mAb PY99 (see Fig. 6C)). Major bands were detected at 60, 70, 85 and 110-130 kDa. (B) Chicken osteoclast-like cells were trypsinized and kept in suspension for 2 hours at 37°C. Subsequently, the cells were seeded onto plastic coated with serum-containing culture medium and lysed after the indicated times. Equal amounts of lysate protein were added to each lane and analyzed by immunoblotting with monoclonal antibody 4G10. (C) Lysates of adherent chicken osteoclast-like cells were immunoprecipitated twice with anti-paxillin antibodies. Lysate supernatants (S) obtained before the first or after the second round of immunoprecipitation as well as the two immunoprecipitates (IP) were analyzed by immunoblotting with antiphosphotyrosine antibody 4G10. (D) Lysates of chicken osteoclast-like cells were subjected to two rounds of immunoprecipitation with antiphosphotyrosine antibody 4G10 and analyzed by western blotting with 4G10 or anti-paxillin antibodies. Lysate supernatants obtained before the first (S1), and before (S2) and after (S3) the second round of immunoprecipitation, as well as the two immunoprecipitates (IP1, IP2) are shown. Note the mobility shift of tyrosine-phosphorylated paxillin in the immunoprecipitates and the disappearance of a slower mobility fraction of paxillin in the supernatants of the immunoprecipitations. However, this fraction represents only a minor subpopulation of total paxillin. (E) Chicken osteoclast-like cells were trypsinized, kept in suspension (Su) for 2 hours or subsequently adhered (Ad) to serum-coated plastic for 40 minutes. Lysates of these cells were immunoprecipitated with anti-Pyk2 antibodies and analyzed by western blotting using 4G10 and anti-Pyk2 antibodies. S, lysate supernatants; IP, immunoprecipitates; asterisks mark the position of immunoglobulin chains in the immunoprecipitates, which were detected by the secondary antibodies.