p90 ribosomal S6 kinase phosphorylates Y-box binding factor-1 at the serine 102 residue. (a) p90 ribosomal S6 kinase RSK1 is detected by immunoblotting following immunoprecipitation (IP) with Y-box binding factor-1 (YB-1) in SUM149 cells. Immunoprecipitation with IgY antibody was used to account for nonspecific binding (left). YB-1 is detected by pulling down and immunoblotting for RSK1. Immunoprecipitations performed with IgG antibody were used to account for nonspecific binding. Secondary detection was performed with horseradish peroxidase protein A (right). WB, western blot. (b) Transfection of SUM149 cells with RSK1, with RSK2 or with RSK1 and RSK2 siRNA for 72 hours reduces P-YB-1S102 while total YB-1 remains unchanged. Actin acts as a loading control (n = 3). (c) HCC1937 cells transfected with RSK1 or activated RSK (Myr-RSK1) express elevated levels of P-YB-1S102 compared with the control vector pKH3 (pRK7 for myr-RSK). A kinase-dead form of RSK (RSK1 KD) failed to induce P-YB-1S102 and was comparable with the control (n = 3). (d) RSK2-/- mouse embryo fibroblasts (MEFs) stimulated with epidermal growth factor (EGF) for a designated amount of time contain less P-YB-1S102 than the wild-type mice. Epidermal growth factor receptor (EGFR) is also reduced, unlike RSK1 that was expressed at a comparable level in both sets of MEFs. The RSK2 immunoblot confirms the genotype of the mice, and actin was used a loading control. The relative expression levels of EGFR in the RSK2-/- MEFs compared with wild-type MEFs are shown under the EGFR blot (n = 2). Ctrl, control.