Rabbit anti p mek

Proteins samples were separated by 8% and 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto polyvinylidene difluoride (PVDF) membranes (Millipore, USA). After blocking in 5% non-fat dried milk for 1 h at room temperature, the membranes were incubated with mouse anti-PSPH (1:200) form Santa Cruz Biotechnology, rabbit anti-p-ERK (1:1000), rabbit anti-ERK (1:1000), rabbit anti-p-P38 (1:1000), rabbit anti-P38 (1:1000), rabbit anti-p-MEK (1:1000) and rabbit anti-MEK (1:1000) from Cell Signaling overnight at 4 °C. Horseradish peroxidase (HRP) conjugated anti-mouse IgG1 (1:5000) from Sigma-Aldrich and HRP conjugated anti-rabbit IgG (1:5000) from Sigma-Aldrich were incubated as the secondary antibodies for 2 h at room temperature. Then the membranes were washed in PBS-T for three times between each antibody incubation step. After washing, the bands were detected using LumiBest ECL reagent solution kit (Share-bio, China). Band intensities were quantified SuperSignal West Femto Maximun Sensitivity Substrate (Thermo Fisher Scientific, USA) with β-actin levels used as the loading control. The experiments were repeated twice.