Anti p c jun

Tissue protein was extracted from frozen tissues and cells protein was lased with sodium dodecyl sulfate buffer. Proteins were extracted with RIPA buffer. Proteins with same concentration were separated on a 10% SDS-PAGE and then transferred to PVDF membranes. The membranes were blocked with 3% BSA in Tris-buffered saline with tween (TBST) and incubated with primary antibody DKK4 (1:500, R&D), anti-c-jun (1:400) (Abcam), anti-p-c-jun (1:350) (Santa Cruz), anti-JNK1/2 (1:400) (Abcam), anti-p-JNK (1:500) (Santa Cruz) followed by incubation with secondary antibody. Protein expression was visualized using enhanced chemiluminescence. Comparison between different groups were made by determining the specific protein/β-actin ratio of the immunoreactive area with densitometry. The tests were performed in triplicate.

The proteins in the pre‐treated cells were isolated by using RIPA buffer (Beyotime). The proteins was separated and probed by the primary antibodies, including anti–Bcl‐2 (sc‐509), anti‐Bax (sc‐20067), anti–cleaved Caspase 3 (sc‐373730), anti–cleaved PARP (sc‐56196), anti–MCP‐1 (sc‐130328), anti–IL‐6 (sc‐57315), anti–TNF‐α (sc‐52746), anti‐p38 (sc‐136210), anti–p‐p38 (sc‐7973), anti–c‐Jun (sc‐376488), anti–p‐c‐Jun (sc‐53182), anti‐JNK (sc‐136533), anti–p‐JNK (sc‐293137), anti‐p65 (sc‐514451), anti–p‐p65 (sc‐136548) and anti–β‐actin (sc‐517582, Santa Cruz Biotechnology). Followed by incubation with the secondary antibodies, the positive bands were developed by BeyoECL Star Kit (Beyotime).

The following antibodies were used in this study: anti-pJNK, anti-p-c-jun, anti-Bcl-xL, anti-β-gal, and anti-β-actin (Santa Cruz Biotechnology, Dallas, TX, USA). Anti-C-Caspase-3 was purchased from Cell Signalling Technology (Danvers, MA, USA). The ZAK monoclonal antibody (M02) was purchased from Abnova (Taipei, Taiwan). 3-HF was purchased from Sigma. siZAKβ was kindly provided by Dr. J. J. Yang (Chung Shan Medical University, Taichung, Taiwan).