FIG. 2.

Effect of chemical inhibition of the MEK/ERK pathway on Fra-1 expression. FRTL-5^KRas cells were treated with 10 μM U0126 (Promega) for 4, 8, or 12 h. Total RNA or whole-cell extracts were prepared from untreated FRTL-5 (lane 1) and FRTL-5^KRas (lane 2) cells or from FRTL-5^KRas cells treated with U0126 (lanes 3 to 5). (A) For Northern blotting, 30 μg of total RNA was analyzed by hybridization to the radiolabeled rat fra-1 cDNA probe as indicated in Materials and Methods. The ethidium bromide staining of rRNAs (28S and 18S) was utilized as a control for RNA loading. (B) For immunoblotting analysis, 50-μg samples of cell extracts were processed as described in Materials and Methods. The same membrane was first incubated with the α-Fra-1 antibody and subsequently was stripped and reprobed with α-P-ERK1/2 (New England Biolabs) as a control for the inhibitory activity of the drug. Finally, the blot was incubated with α-ERK1 antibody (New England Biolabs) as a control for equal protein loading. For the in vitro dephosphorylation reaction (B, right-hand panel) the cell extracts from untreated FRTL-5^KRas cells were incubated for 2 h at 37°C with or without 20 U of CIP prior to SDS-10% PAGE and immunoblotting. Northern blotting and immunoblotting data were confirmed by multiple experiments, and similar results were obtained by using the PD 98059 MEK inhibitor.