Phosphorylated jnk

C57BL/6 mice (6-8wks old) were purchased from SIPPR-BK Experimental Animal Ltd Co. (Shanghai, China). All experiments and animal care were performed according to protocols approved by the Zhejiang University Institutional Animal Care and Use Committee. TIPE2 small interfering RNA (siRNA) and control siRNA were from GenePharma (Shanghai, China). PMA were from Sigma (Sigma-Aldrich LLC, USA). Abs specific to β-actin, and HRP-coupled secondary Abs were from Santa Cruz Biotechnology (Santa Cruz, CA). Abs specific to ERK, JNK, P38, phosphorylated ERK, phosphorylated JNK, and phosphorylated P38 were from Cell Signaling Technology (Danvers, MA). Abs specific to TIPE2 was from Abcam (Cambridge, MA). The JNK inhibitor SP600125, the P38 inhibitor SB203580 and the ERK inhibitor PD98059 were obtained from Selleck (Shanghai, China).

Cell counting kit-8 (CCK-8) and Annexin V-FITC/PI apoptosis detection kits were purchased from Vazyme Biotechnology Company (Nanjing, China). T-BHP, dimethyl sulfoxide (DMSO), and 2′,7′-dichlorodihydrofluorescein diacetate (DCFHDA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cisplatin and N-acetylcysteine (NAC) were purchased from Macklin Biochemical Corporation (Shanghai, China). CpG ODN1826 was purchased from InvivoGen (San Diego, CA, USA). The JC-1 kit was purchased from Beyotime (Shanghai, China). RAW264.7 and AML12 (alpha mouse liver 12) cells were received from American Type Culture Collection (Manassas, VA, USA). Dulbecco's Modified Eagle Medium (DMEM), Dulbecco's phosphate-buffered saline (DPBS), fetal bovine serum (FBS), and Opti-MEM were purchased from Gibco (Waltham, MA, USA). Antibodies for cleaved-caspase 3, caspase 3, caspase 8, cleaved-caspase 8, caspase 9, cleaved-caspase 9, PARP, cleaved PARP, phosphorylated ERK1/2 (p-ERK1/2), ERK1/2, phosphorylated Akt (p-Akt), phosphorylated p38 MAPK, p38 MAPK, phosphorylated JNK, JNK, anti-rabbit IgG, anti-mouse IgG, and GAPDH were all purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-NOX2 antibody (ab80508) was purchased from Abcam (Cambridge, MA, USA). AST and ALT detection kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

Antibodies against IkB-α (nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor alpha; 1:1,000), JNK (c-Jun N-terminal), phosphorylated JNK (1:1,000), β-actin (1:1,000) and TLR4 (1:1,000) were purchased from Cell Signaling Technology (United States). Total protein of liver and adipose tissue was extracted by adding lytic buffer to each ice-cold sample. Proteins were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane (Millikon). Each membrane was sealed in the presence of Tris buffered saline containing Tween (TBST) containing 5% skimmed milk powder at room temperature for 1 h. Then, a defined concentration of diluted antibody was added as described by the manufacturer. The blot was incubated overnight in a flip shaking bed at 4 °C and washed six times for 5 min each time using TBST. TBST containing 5% skimmed milk powder a 1:8,000 dilution of mouse anti-rabbit antibody labeled with horseradish peroxidase (HRP) and incubated at room temperature for 2 h. The blot was washed six times for 5 min each time. An enhanced chemiluminescence (ECL) detection kit (Applygen Technologies, Beijing, China) was used to display protein bands. The protein bands were analyzed by ImageJ image analysis software (NIH, Bethesda, MD, United States).

Cells were lysed using M-PER Mammalian Protein Extraction Reagent (Thermo Scientific) and SDS-PAGE was performed. Antibodies used in this study are listed as follows: Human, CREB (9197, Cell Signaling Technology, Danvers, MA), phosphorylated CREB (9198, Cell Signaling Technology), JNK (9252, Cell Signaling Technology), phosphorylated JNK (9255, Cell Signaling Technology), Caspase-12 (2202, Cell Signaling Technology), GAPDH (AP0060, Bioworld, St. Louis Park, MN), PKAcα (4782s, Cell Signaling Technology), Ki-67 (9449s, Cell Signaling Technology), Cleaved-Caspase-3 (9664s, Cell Signaling Technology), p65 (8242, Cell Signaling Technology), phosphorylated p65 (3033, Cell Signaling Technology), M1 E1 and NS3 (produced by Beijing Protein Innovation).

Harvested cells were washed twice with cold PBS and then lysed using RIPA buffer (Beyotime, Shanghai, China) containing a protease inhibitor cocktail (Selleck, Shanghai, China). The lysates were centrifuged for 15 min at 12,000×g at 4°C and the supernatant was collected. The supernatant was boiled for 5 min and then separated by 10% SDS-PAGE gels. The proteins were transferred to a PVDF membrane (Millipore, Bedford, MA, USA), blocked with 5% milk, and incubated overnight with E-cadherin (Abcam, Cambridge, MA, USA), α-SMA (Abcam), TGF-β1 (Abcam), phosphorylated ERK1/2 (Cell Signaling Technology, Boston, USA), ERK1/2 (Cell Signaling Technology), phosphorylated Smad2/3 (Cell Signaling Technology), Smad2/3 (Cell Signaling Technology) and phosphorylated JNK (Cell Signaling Technology). Membrane was then washed three times in TBST and incubated with peroxidase-labelled secondary antibodies (Boster, Inc., Wuhan, China) for 2 h at room temperature. Following three washes in TBST, bands were visualized by western electrochemiluminescence (ECL) kit (Tanon, Shainghai, China) and then exposed to X-ray film. The band densities were quantified using the AlphaEaseFC image analyzer system (Alpha Innotech, Inc., CA, USA) and the results were expressed as ratio of band density to total GAPDH.

Cells were lysed in RIPA buffer (Sigma) supplemented with 1% protease and phosphatase inhibitors (Roche) for 30 min on ice. The lysates were then centrifuged at 12,000 rpm for 30 min at 4 °C, and the proteins in the supernatant were quantified using a BCA assay kit (Sigma) according to the manufacturer’s instructions. Equal amounts of protein diluted in sodium dodecyl sulfate (SDS) loading buffer, separated by 10% SDS–polyacrylamide gel electrophoresis, and electrotransferred onto polyvinylidene fluoride membranes (EMD Millipore Billerica). The membranes were blocked with 5% skim milk in TBST at room temperature for 60 min and incubated overnight at 4 °C with primary antibodies against GAPDH, TRAP, CTSK, NFATc1, p65, phosphorylated p65, phosphorylated p38, p38, ERK-1/2, phosphorylated ERK-1/2, JNK, and phosphorylated JNK (each diluted 1:1000; Cell Signaling Technology). Afterwards, the membranes were washed three times with TBST and incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (diluted 1:3000; Santa Cruz Biotechnology) at room temperature for 60 min. The membranes were washed three times with TBST, and signals were detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore).