Phosphorylation of YB-1 stimulated by ionizing radiation and erbB1 ligands. (A and B) Confluent cells (breast cancer cells MDA-MB-231, MCF-7, HBL100 and SKBr3; normal fibroblasts HSF1, HSF7 and human fetal lung fibroblast (HFL); normal mammary epithelial cells MCF-10A) were cultured in 10% serum. Protein samples were isolated from biologically independent cultures, and a sample of 100 μg of protein from each culture was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). P-YB-1, YB-1, K-Ras and actin were detected by Western blot analysis. (C) From the densitometric values of P-YB-1 and YB-1, the P-YB-1/YB-1 ratios were calculated for tumor cells versus fibroblasts, as well as normal mammary epithelial cells, and graphed. Statistical analyses were performed using Student's t-test. Error bars represent standard deviations (SD). Confluent cells were (D) irradiated with 4 Gy of IR or (E) treated with 100 ng/ml erbB1 ligand. At the indicated time points after stimulation, protein samples were isolated and subjected to SDS-PAGE. The levels of P-YB-1 and YB-1 were assessed by Western blot analysis. The densitometric values represent the P-YB-1/YB-1 ratio normalized to 1 in nonirradiated controls. (D) Phosphorylation of YB-1 after irradiation was tested at least in three independent experiments. (E) ErbB1 ligand-induced YB-1 phosphorylation was tested at least in two independent experiments. EGF, epidermal growth factor; AREG, amphiregulin; TGFα, transforming growth factor α. (F) Cells (confluent status) were kept in serum-free medium or serum containing 10% fetal calf serum medium. Twenty-four hours after serum depletion samples were isolated, and the level of P-YB-1 was assessed by Western blot analysis. Blots were stripped and incubated with antibody against total YB-1.