Gb11142

Detection of protein expression were used according to the method described by Zhuge et al. [27 (link)]. Total protein was extracted from liver tissue and quantified by the total protein assay kit (with standard: BCA method) (A045-4-2, Nanjing Jiancheng, China). After SDS-PAGE electrophoresis and membrane transfer, the total protein samples were incubated with antibody for immune reaction. The films were then sealed with 5% skim milk (containing 0.5% TBST) on the shaker for 1 hour. Then, the primary antibodies were added and shaken slowly at 4 °C overnight. The dilution of JNK antibody (GB12001, Wuhan Servicebio Technology Co., Ltd., Wuhan, China), p65 antibody (GB11142, Wuhan Servicebio Technology Co., Ltd. Wuhan, China) and β-actin antibody (24164-1-AP, Proteintech Group, Inc, Wuhan, China) were 1:2000, 1:1000 and 1:3000, respectively. Subsequently, the membrane was washed with TBST at room temperature for 3 times (5 min each time). After adding the secondary antibody (diluted 3000 times with TBST) and mixing with the membrane, incubate at room temperature for 30 min. After incubation, the membrane was washed with TBST at room temperature for 3 times (5 min each time). Finally, chemiluminescence was carried out in the dark room. The film was scanned and analyzed by the gel image processing system to obtain the molecular weight and optical density information of the protein.

CD4⁺ T cells were lysated and proteins were extracted using a nuclear extraction reagent (Boster Biological Technology, Pleasanton, CA, USA). Proteins were quantified by the Bradford reagent (Thermo Fisher Scientific, Waltham, MA, USA), followed by 12% vertical dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were then transferred into a polyvinylidene difluoride (PVDF) membrane (Sigma-Aldrich, St. Louis, MO, USA). The PVDF membrane was blocked in 5% skim milk for 1 h at room temperature, then incubated with an antibody against P65 (GB11142, 1:1000, Wuhan Servicebio Technology Co., Ltd., Wuhan, China) or P50 (ab7971, 1:5000, Abcam, Cambridge, MA, USA) for 12-16 h at 4℃ , and followed by incubating with a mouse anti-rabbit IgG antibody (H&L) (GenScript, Piscataway, NJ, USA). Proteins were detected with an enhanced chemiluminescence (ECL) western blot detection kit (Thermo Fisher Scientific, Waltham, MA, USA). Quantification of P65 and P50 was normalized to GAPDH by densitometry.