Phospho jnk antibody

The media and other materials required for culturing cells were purchased from Lonza Inc. (Walkersville, MD, USA). Griess reagent and lipopolysaccharide (LPS: E. coli, serotype O111:B4; L2630) were purchased from Sigma–Aldrich (St. Louis, MO, USA). The WST-1 assay kit was obtained from Daeillab Service Co., Korea. Anti-Toll-like receptor 2 antibody (anti-TLR2), anti-complement receptor 3 antibody (anti-CR3), and anti-Toll-like receptor 4 antibody (anti-TLR4), were obtained from Abcam (Cambridge, MA, USA). Phospho-NF-κB antibody, phospho-p38 (MAPK) antibody, phospho-ERK (MAPK) antibody, and phospho-JNK antibody were purchased from Cell Signalling Technology (Danvers, MA, USA). All chemicals and reagents used in this work were of analytical grade.

Cells were washed with phosphate buffered saline (PBS) three times and then scraped off and lysed with lysis buffer (PBS with 1% Triton X-100 and 1% deoxycholate). The protein concentration of lysates was determined using Bradford reagent (Bio-Rad, Hercules, CA, USA), after which equal amounts of protein were separated electrophoretically using 10% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The gel was then transferred to 0.45 μm nitrocellulose paper and incubated with anti-iNOS, p65, HO-1, Nrf2, phospho-CAMK4, Akt, ERK1/2, JNK, TBP, HDAC antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA), CAMK4, phospho-Akt, phospho-ERK1/2, phospho-JNK antibodies (Cell Signaling Technology, Beverly, MA, USA) or α-tubulin antibody (Bio Genex, Fremont, CA, USA), and secondary antibody and then detected by an enhanced chemiluminescence detection system according to the recommended procedure (GE Healthcare, Piscataway, NJ, USA).

GSH assay and TUNEL staining kits were purchased from the Beyotime Institute of Biotechnology (#S0053, #C1090 and #P0012S, Shanghai, China). Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) kits were purchased from Nanjing Jiancheng Bioengineering Institute (#C009–2 and #C010–2, Nanjing, Jiangsu, China). Elisa kits for ADMA and 4-hydroxynonenal (4-HNE) were purchased from Bio-Techne Co., Ltd. (#NBP2–66728, Minneapolis, MN, USA) and Donggeboye Biological Technology Co., LTD. (#DG30947M, Beijing, China), respectively. The Masson’s trichrome staining kit was obtained from Solarbio Science & Technology Co. LTD (#G1340, Beijing, China). Antibodies against DDAH1, cytochrome P450 2E1 (CYP2E1), glutathione S-transferase A1 (GSTA1) and β-actin were purchased from Signalway Antibody LLC (#37368, #48247, #22536, #21800, Greenbelt, MD, USA). P65, phospho-65, c-jun N-terminal kinase (JNK) and phospho-JNK antibodies were from Cell Signaling Technology (#8242, #3033, #9252, #9251, Danvers, MA, USA). APAP and dihydroethidium (DHE) were purchased from MedChemExpress LLC (#HY-66005, Monmouth Junction, NJ, USA) and Sigma Chemical Co. (#D7008, St. Louis, MO, USA), respectively. All other compounds and chemicals were of analytical purity.