Anti p jnk antibody

For the western blot assays, the spinal cord (C6-C8) was quickly removed and preserved in liquid nitrogen for further analysis. After homogenization in RIPA buffer, the samples were centrifuged at 12,000 g for 30 min at 4°C. The protein concentrations of soluble materials were determined by the Coomassie G250 binding method. The protein samples were separated on 10% polyacrylamide gels containing 0.1% SDS, followed by transfer to polyvinylidene di uoride (PVDF) membranes. The membranes were blocked with 5% skimmed milk for 2 h and then incubated with primary anti-JNK antibody (dilution 1:000,Santa Cruz Biotechnology, Burlingame, CA, USA), anti-p-JNK antibody (dilution 1:000, Cell Signaling Technology, Danvers, MA, USA), anti-c-Jun antibody (dilution 1:000, Cell Signaling Technology, Danvers, MA, USA), anti-p-c-Jun(Ser73) antibody (dilution 1:000, Cell Signaling Technology, Danvers, MA, USA) or β-actin (dilution 1:2000,Santa Cruz Biotechnology, Burlingame, CA, USA) at 4°C overnight. The appropriate secondary antibodies (goat anti-rabbit or -mouse IgG conjugated with horseradish peroxidase) were subsequently used at room temperature for 1 h. Finally, the EC3 Imaging System (UVP Inc., Upland, CA, USA) was used to quantify the p-JNK, JNK, p-c-Jun, c-Jun and β-actin protein bands. Quanti cation was performed using an ImageJ software (NIH, Bethesda, MD, USA) on a computer.