Rsk1 rsk2 rsk3

DU145 cells were lysed in radioimmunoprecipitation assay buffer (150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM Tris–HCl [pH 7.5], 2 mM ethylenediaminetetraacetic acid) and 15 μg of protein was separated on 6–12% gels by sodium dodecyl sulfate-polyacrylamide gel electrophoresis Proteins were transferred to polyvinylidene difluoride membranes and then membranes were blocked in PBS with 0.1% Tween-20 containing 1% BSA and 1.5% skim milk for 1 h. After washing, the membranes were probed with primary antibody at 4 °C overnight, and then incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. The blot was developed using the EZ-western detection kit (Daeillab Service, Co., Seoul, Korea). Anti-cleaved caspase-8, −cleaved caspase-3, −PARP, −JNK, −p38, −p-ERK1/2, −p-SRC (Tyr-416 and Tyr-527), −SRC, −p-STAT3, −STAT3, −p-PI3K, −PI3K, −p-AKT (Ser-473), −AKT, −Ras, −p-Raf1 (Ser-259 and Ser-338), −p-MEK1/2, −p-p90RSK (Ser-380), and -RSK1/RSK2/RSK3 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-β-Actin, −p-JNK, −p-p38, −ERK2, and -Raf1 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz). The band intensities of specific antibodies were normalized and analyzed by ImageJ (Broken Symmetry Software, version 1.4.3.67).

All reagents were obtained from Sigma-Aldrich, Missouri, USA unless stipulated otherwise. Quetiapine was donated by AstraZeneca, Stockholm, Sweden; aripiprazole by Bristol-Myers Squibb, New Jersey, USA and AG1478 (EGFR inhibitor) purchased from A.G. Scientific, Inc., California, USA. Primary antibodies, including phospho-p44/42 MAPK, p44/42 MAP kinase, phospho-p90RSK, RSK1/RSK2/RSK3 and β-Actin were from Cell Signaling Technology, Massachusetts, USA and c-Fos from Assay Designs, Michigan, USA. Secondary antibodies, including goat anti-mouse and goat anti-rabbit horseradish peroxidase (HRP)-conjugated immunoglobulins (IgGs) were supplied by DAKO, NSW, Australia.