Overexpression of mutated K-RASV12enhances basal YB-1 phosphorylation. The indicated cell lines were transiently transfected with pEGFP-C1 control vector (con. vector) or pEGFP/K-RASV12 (K-RASV12) as described in Materials and methods. Forty-eight hours after transfection (A) green fluorescent protein (GFP) expression was analyzed by fluorescent microscopy and (B) protein samples were isolated. The expression levels of GFP and K-Ras were assessed by Western blot analysis. P-YB-1 was detected using the same blots. After the membranes were stripped, each blot was incubated with an antibody against total YB-1. Actin was detected as an additional loading control. The function of K-RASV12 on YB-1 phosphorylation was tested in at least three independent experiments, and a representative Western blot is shown. (C) Forty-eight hours after transfecting SKBr3 cells with the pEGFP-C1 control vector or pEGFP/K-RASV12 (K-RAS), cells were mock-irradiated or irradiated with 4 Gy ionizing radiation. Ten minutes after irradiation protein samples were isolated. Following sodium dodecyl sulfate polyacrylamide electrophoresis, the expression levels of GFP and K-Ras, as well as the phosphorylation status of ERK1/2 and YB-1, were assessed by Western blot analysis. The blots were stripped and incubated with YB-1 and ERK1/2 antibodies. A representative Western blot of three independent experiments shown.