Fig. 7. MEK1/2 activation and effect of MEK1/2 inhibitors. (A) CHO_Vß₃ cells were plated on immobilized LM609 substrate for the indicated times, lysed and total and phosphorylated MEK1/2 was determined. In the same gel, total and phosphorylated ERK1/2 were also detected (shown in Fig. 3C). (B) CHO_Vß₃ cells and (C) osteoclasts were plated on immobilized LM609 substrate. Prior to plating, aliquots of cells were pretreated with the MEK1/2 inhibitors PD98059 (50 µM, 30 minutes) or U0126 (10 µM, 30 minutes). Lysates were subjected to western blot analysis and probed for detection of total and phosphorylated MEK1/2 and ERK1/2. (D)Lysates of (a) CHO_Vß₃ cells and (b) osteoclasts treated as described above were also subjected to cytosol/nuclear fractionation and probed for detection of total and phosphorylated ERK1/2. (E) Osteoclasts cultured in standard conditions were starved in 1% FBS overnight, then stimulated with 20% FBS for 30 minutes. Total cell lysates (left panels) and cytosol/nuclear fractions (right panels) were then probed for detection of total and phosphorylated MEK1/2 and ERK1/2. Similar results were obtained in three independent experiments. Susp, cells in suspension; LM609, adhesion to LM609 for 30 minutes; C, cytosolic fraction; N, nuclear fraction.