Fig. 3. Detection of total and phosphorylated MAPKs. (A) CHO_Vß₃ cells and (B) osteoclast cultures were prepared as described in Fig. 2 and plated in LM609-coated wells for 30 minutes. An aliquot of suspended cells was also treated for 5 minutes with TPA (10^-7 M) to show control positive assay for phosphorylated JNK. Total cell lysates were processed by SDS-PAGE and western blot for detection of total and phosphorylated fractions of ERK1/2 (primary antibodies diluted 1:500), p38 (primary antibodies diluted 1:800) and JNK (primary antibodies diluted 1:500). (C) Aliquots of CHO_Vß₃ cells were maintained in suspension or plated in LM609-coated wells for the indicated times, with T0 representing cells cultured in standard conditions, starved overnight in serum-free medium. Cells were processed as described above and total and phosphorylated ERK1/2 were determined. (D) Fractionated CHO_Vß₃ cell and (E) osteoclast lysates, obtained as described in Fig. 2, were probed with anti-total or anti-phosphorylated ERK, and anti-ß₃ integrin antibodies (diluted 1:400) C, cytosolic; M, membrane; I, Triton-X-100-insoluble fractions; Susp, cells in suspension; LM609, adhesion to LM609 for 30 minutes. Similar results were obtained in three independent experiments.