Constitutive phosphorylation of YB-1 in MDA-MB-231 cells is K-Ras-dependent. (A) Subconfluent cells were transfected with nontargeting small interfering RNA (siRNA) or siRNA against K-RAS and extracellular signal-regulated kinase 1 (ERK1) as described in the Materials and methods. Protein samples were isolated, and the levels of K-Ras, actin, P-ERK1/2 and P-YB-1 were detected. The blots were stripped and reincubated with ERK1/2 or YB-1. (B) Confluent MDA-MB-231 cells were treated with the MEK inhibitor PD98059 or dimethyl sulfoxide and mock-irradiated or irradiated with 4 Gy ionizing radiation. At the indicated time points after irradiation, protein samples were isolated and P-YB-1 and P-ERK1/2 were detected. The blots were stripped and reincubated with ERK1/2 or YB-1. Representative Western blots of three independent experiments are shown.