Rabbit anti p jnk

Western blot analysis was performed as described previously [35 (link)], with slight modifications. Briefly, cells were lysed on ice with radioimmunoprecipitation (RIPA) lysis buffer supplemented with protease inhibitor cocktail (Roche, Indianapolis, IN, USA) and sonicated to reduce sample viscosity. Protein concentration was determined using the bicinchoninic acid assay (Pierce, Rockford, IL, USA), equal amounts of protein were separated on 10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). Western blotting was performed using the following antibodies at 4 °C overnight: rabbit anti-β-III-tubulin (1:5000; Covance); rabbit anti-MAP-2 (1:2000; Millipore); rabbit anti-GFAP (1:5000; Abcam); rabbit anti-CNPase, rabbit anti-JNK, rabbit anti-p-JNK, rabbit anti-c-Jun, rabbit anti-p-c-Jun^ser63, and rabbit anti-p-c-Jun^ser73 (all 1:1000; all from Cell Signaling Technology); and mouse anti-β-actin (1:10,000; Sigma-Aldrich). Blots were incubated with horseradish peroxidase-labelled secondary anti-rabbit and anti-mouse antibodies, and immunoreactive bands were visualized on film by enhanced chemiluminescent substrate (Pierce, Rockford, IL, USA) (all 1:10,000, Abcam). Western blots were quantified with ImageJ software from three independent experiments. The intensities of the bands were normalized to β-actin.

After the designed treatment, culture medium was discarded and cells were gently washed with cold PBS, then, lysed in RIPA (P0013B, Beyotime Institute of Biotechnology) buffer. The protein concentrations were detected using the BCA protein assay kit (Shenergy Biocolor Bioscience & Technology Company, Shanghai, China). Equal amounts of proteins were loaded on and seperated by 8%~12% SDS-PAGE electrophoresis, then, transferred to polyvinylidene fluoride membranes (PVDF; Immobilon-P, Cat. No. IPVH00010). Membranes were blocked in TBS containing 0.05% tween20 (TBST) with 5% BSA, and incubated with primary antibodies overnight at 4 °C. After washed with TBST, the membranes were incubated with secondary antibodies and the protein signal was detected using a chemiluminescence solution, ECL kit (Millipore, USA). The intensity of protein bands was quantified using Image J software (Broken Symmetry Software, USA). β-actin was used as loading control. The primary antibodies were used as follows: rabbit anti-p-JNK (9251S, Cell Signaling Technology), rabbit anti-total-JNK (9252S, Cell Signaling Technology), mouse anti-Bcl-2 (2876S, Cell Signaling Technology), rabbit anti-cleaved-Caspase3 (9664S, Cell Signaling Technology), Rabbit anti-NLRX1 (17215-1-AP, Proteintech), rabbit anti-BAX (ab32503, Abcam, Cambridge, UK) and mouse anti-β-actin (TA-09, ZSGB-BIO).