Figure 9
Western blot analysis for levels of HA-MEK1 and phosphorylated MAPK and in vitro growth of transfectant clones. For detection of HA-MEK1 and phosphorylated MAPK, mock-transfectant (pUSE-9 and pUSE-12) and activated MEK1-transfectant (H-MEK1-5, H-MEK1-15, H-MEK1-16 and H-MEK1-17) clones were grown and total cell lysate was prepared for Western blot analysis as described under Experimental Procedures. Blots were incubated with mouse anti-α-tubulin (A), rabbit anti-HA-MEK1 (B), mouse anti-MAPK (C), mouse anti-phospho-MAPK (Thr202/Tyr204) antibodies (D). All the antibodies were used at a final concentration of 1 μg per ml. For proliferation study, pUSE-9, pUSE-12, H-MEK1-15, H-MEK1-17 clones were seeded at a density of 2.5 × 104 cells per well in 24-well plates containing MEM supplemented with 10% fetal calf serum. (E) Cell number was counted daily by hemocytometer for 5 days and is plotted against number of days. Means were determined from quadruplicate wells and in no case did standard deviation exceed 15% of the mean value.