Traf2

Liver tissues were lysed using a radioimmunoprecipitation assay buffer containing phosphatase inhibitors and phenylmethylsulfonyl fluoride. The total protein was extracted following centrifugation at 12, 500 g for 35 min, and the protein concentration was calculated using a bicinchoninic acid kit. In order to split each protein sample into 30 g for protein blot analysis, 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used. All proteins were transferred to nitrocellulose membranes to avoid non-specific staining, and for 1 h they were treated with 5% skim milk powder dissolved in 0.1% Tris-Buffed saline/Tween 20. The resultant membranes were gradually incubated with the appropriate primary antibody (TRAF2, 1:1,000, ab126758; Abcam) at 4°C for an entire night before being exposed to the secondary antibody for 2 hours at room temperature. To find the signal, a chemiluminescence imaging apparatus was employed.

The total protein were lysed in RIPA buffer and extracted. 10 % SDS polyacrylamide gel was used to separated the proteins. After blocking with 5 % fat-free milk for 1 h, the membranes were incubated with antbody of CUL1 (mouse monoclonal; Invitrogen, USA), TIMP (Rabbit monoclonal; Cell Signaling Technology, MA) or TRAF2 (Rabbit polyclonal, Abcam, USA) overnight at 4 °C. Blots were washed with PBST and incubated with the secondary antibody for 1 h. Took the photo using enhanced chemiluminescence.

Liver tissue was homogenized in RIPA lysis buffer containing protease inhibitor (Complete mini EASY pack, Roche, Basel, Swiss) to extract protein. The concentration was determined by BCA kit (CoWin Bioscience, Beijing, China) and protein was resolved with 10% polyacrylamide gel, then transferred to the PVDF membrane (Millipore, Billerica, MA, USA). After blocking, they were incubated overnight in antibody dilutions of GRP78 (1:1000) (CST, Boston, MA, USA, 3183), P-PERK (1:1000) (CST, Boston, MA, USA, 3179), P-EIF2α (1:1000) (CST, Boston, MA, USA, 3398), P-NFκB (1:500) (CST, Boston, MA, USA, 3033), P-IREα (Abcam, Cambridge, UK, ab148187), TRAF2 (1:1000) (Abcam, Cambridge, UK, ab126758) or β-actin (1:1000) (Huaan biological technology, Hangzhou, China), and secondary antibody were incubated for 2 h at room temperature. The images of blots were acquired by GBOX Chemi XT4 gel imaging system (Syngene, Cambridge, UK).

For whole cell extraction, cells and tissue samples were lysed in radioimmune precipitation assay buffer (Beyotime, Beijing, China) supplemented with protease and phosphatase inhibitor. Equal amounts of proteins were separated by 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. After being blocked with 5% nonfat dry milk, membranes were incubated overnight at 4°C with primary antibodies against VCAM-1, E-selectin, ERK, P-ERK, JNK1/2, p-JNK1/2, TNF-R1, TRAF2, NF-κB/P65, RIP, GAPDH, and beta-actin that were diluted at the indicated concentration. Then, the membranes were washed three times with TBST (10 mmol/L Tris-HCl, pH 8.0, containing 150 mmol/L NaCl and 0.1% Tween-20) and incubated with secondary antibodies (Merck Millipore, Darmstadt, Germany) for 1.5 h at room temperature. After washing for 3 × 10 min with TBST, target bands were developed using ECL (Advansta, California, USA) and exposed on a film (Kodak, NY, USA). The intensity of the protein bands was measured using Quantity One version 4.6.2 Image software (Bio-Rad, USA).
The anti-GAPDH primary antibody was purchased from Bioworld (St. Louis Park, MN, USA). The anti-p-JNK1/2, TRAF2, NF-κB/P65, and beta-actin primary antibodies were purchased from Abcam (MA, USA). All other primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Cell or liver homogenate proteins of the above groups were extracted, and the concentrations were measured with BCA Protein Assay Kit (Sangon Biotech Co., Ltd., Shanghai, China). Samples were denatured for 10 min at a temperature of 100°C. A total of 40 μg protein was loaded per lane, separated using 12% SDS-PAGE gel, and then electrotransferred onto polyvinylidene difluoride membranes. Membranes were blocked using 5% nonfat milk for 1 h and then incubated at 4°C overnight with primary antibodies against Bad (cat. no. ab32445; 1 : 1000; Abcam Inc.), Bax (cat. no. E63; 1 : 1000; Abcam Inc.), Caspase-3 (cat. no. ab2302; 1 : 1000; Abcam Inc.), Bcl2 (cat. no. ab196495; 1 :1000; Abcam Inc.), p-IRE1α (cat. no. ab48187; 1 : 1000; Abcam Inc.), IRE1α (cat. no. ab37117; 1 : 2000; Abcam Inc.), TRAF2 (cat. no. #4712; 1 : 1000; CST Inc.), p-IκBα (cat. no. #2859; 1 : 1000; CST Inc.), IκBα (cat. no. #9242; 1 : 1000; CST Inc.), p-p65 (cat. no. #3033; 1 : 1000; CST Inc.), p65 (cat. no. sc-71675; 1 : 2000; Santa Cruz Inc.), PPARγ (cat. no. ab209350; 1 : 1000; Abcam Inc.), Arg1 (cat. no. ab60176; 1 :1000; Abcam Inc.), and iNOS (cat. no. ab15323; 1 : 2000; Abcam Inc.). The membranes were blotted with species-matched secondary antibodies. Protein bands were visualized using the BioRad ChemiDoc™ XRS system (Hercules, CA). All images were analyzed using the NIH ImageJ software.

Liver tissues were fixed in 4% paraformaldehyde, dehydrated, and subjected to heat-induced antigen retrieval using citrate. Then, the sections were incubated in 1% Triton X-100 for 15 min. After eliminating endogenous peroxidase activity with 3% hydrogen peroxide for 15 min, the sections were blocked with goat serum albumin at room temperature for 1 h. Then, the sections were incubated overnight at 4°C with p-IRE1α (cat. no. ab48187; 1 : 100; Abcam Inc.), TRAF2 (cat. no. #4712; 1 : 100; CST Inc.), and p-p65 (cat. no. #3033; 1 : 100; CST Inc.). The sections were washed and incubated with species-matched secondary antibodies (1 : 200 dilution) for 1 h at room temperature. The sections were washed with PBS, treated with 3,3′-diaminobenzidine for 5 min at room temperature, stained with hematoxylin for 30 sec at room temperature, and washed with flowing water for seconds. Following dehydration, sections were sealed with neutral resin, and specific staining was visualized by light microscopy as described before [22 (link)].