Fig. 5. Effect of PD098059 on cellular proliferation and MAP kinase phosphorylation. A, nonmalignant and malignant mammary cell lines were plated at a density of 1 x 104 cells/cm2 in 3.5-cm dishes and incubated in complete growth medium supplemented with PD098059 or with DMSO as vehicle control. Duplicate dishes were counted after 34 days. The number of cells in PD098059-treated dishes was expressed as a percentage of the number of cells in the respective control dishes. Results from at least three separate experiments were used to calculate the mean; bars, SE. The Students t test was used to determine that growth in the first four malignant lines was significantly different when compared with each of the other nonmalignant and malignant cell lines. *, P < 0.05. B, cells plated at equivalent subconfluent densities were treated as in A. After 3 h of treatment, cell lysates were prepared with RIPA buffer, and 10 µg of lysate protein were fractionated by SDS-PAGE and immunoblotted with anti-phosphoERK1/ERK2. The membrane was stripped and reprobed with anti-ERK2 to confirm that equivalent amounts of protein were present.