α tublin

Proteins from Kupffer cells and hepatocyte were homogenized with RIPA lysis buffer (Wako, Osaka, Japan) containing phosphatase inhibitor cocktail and protease inhibitor cocktail. Proteins were blotted onto PVDF membranes (Bio-Rad, Hercules, CA). The membranes were incubated with appropriate primary antibodies overnight at 4°C after blocked with skim milk for 1hr at room temperature. Afterwards, the membranes were washed three times with TBST and incubated with secondary antibodies for 1hr at room temperature. The blots were visualized by enhanced chemiluminescence. The primary antibodies were used are as follows: HO-1 (1:2000; Abcam, Cambridge, MA), Nrf-2 (1:2000; Santa Cruz Biotechnology), HIF-1α (1*2000; GeneTex), p-Akt (1:1000; Cell Signaling Technology), Akt (1:1000; Cell Signaling Technology), p-mTOR (1:1000; Cell Signaling Technology), mTOR (1:1000; Cell Signaling Technology), p-p38& p38 (1:2000; Cell Signaling Technology), p-ERK1/2& ERK1/2 (1:2000; Cell Signaling Technology), p-JNK (1:2,000; Cell Signaling Technology), JNK (1:4000; Cell Signaling Technology), α-tublin (1:4000; Santa Cruz Biotechnology) and GAPDH (1:4000; Santa Cruz Biotechnology).

For these experiments, 20, 70 - dichlorofluorescein diacetate (DCFH-DA), vitamin C, and linoleic acid (LA) were purchased from Sigma (Shanghai, China). Fetal bovine serum (FBS) and RPMI-1640 cell culture media were purchased from Thermo Fisher (Shanghai, China). The primary antibodies against Poly (ADP-ribose) polymerase (PARP), procaspase-8, cleaved caspase-3,NFκB, p65, CDK1, Cyclin D1, CDK2, IκB, p-IκB, Fas, Bcl-2, Bax, Cyto-c, and ND1 antibodies were provided by Cell Signaling Technology (Shanghai, China). α-tublin, lamin B, β-actin, GAPDH, HO-1, NQO-1, iNOS, COX-2, p53, Nrf2, keap-1 were from Santa Cruz Biotechnology (Dallas, Texas, USA). Cell cycle kit and Annexin V/PI cell apoptosis kit were from Wanleibio Company (Shenyang, Liaoning, China). All other chemicals were of analytical grade and were produced in China.

Total protein was extracted from liver tissues and analyzed with bicinchoninic acid protein concentration assay kit (Beyotime Biotech, Beijing, China). Sample protein was separated by electrophoresis in 12% SDS-PAGE with a Bio-Rad electrophoresis system (Hercules, CA, USA). The primary antibodies (rabbit anti-Smad3 antibody, UCallM biotech Co., Ltd, Wuxi, China, 1:1000 dilutions) were incubated at 4°Covernight. The corresponding horseradish-peroxidase-conjugated secondary antibodies (anti-rabbit IgG, 1:5000 dilutions) were incubated for 1 h at room temperature. The membrane containing antibody-protein complexes were visualized with an enhanced chemiluminescence detection system on radiograph film. The brands were scanned and analyzed by the software Quantity ONE (Bio-rad, Hercules, CA, USA). The expression of protein in each sample was normalized by α-Tublin(Santa Cruz Biotechnology, CA, USA).

Total protein was extracted from MIBECs and analyzed with bicinchoninic acid protein concentration assay kit (Beyotime Biotech, Beijing, China). Sample protein was separated by electrophoresis in 12 % SDS-PAGE with a Bio-Rad electrophoresis system (Hercules, CA, USA). The primary antibodies (rabbit anti-TLR4, at 1 μg/ml, NF-κB p-65 at 1 μg/ml, MyD88 at 1 μg/ml antibody, Cell Signaling biotech Co, Ltd, MA, USA, 1:1000 dilutions) were incubated at 4 °C overnight. The secondary anti-bodies (anti-rabbit IgG, 1:5000 dilutions) was incubated for 1 h at room temperature. The membrane containing antibody-protein complexes were visualized with an enhanced chemiluminescence detection system on radiograph film (Bio-rad, Hercules, CA, USA). The bands were scanned and analyzed by the software Quantity ONE (Bio-rad, Hercules, CA, USA). The expression of protein in each sample was normalized by α-Tublin (Santa Cruz Biotechnology, CA, USA).