Rabbit anti β actin

The total protein of Jianbai Xiang pig melanocytes was extracted using RIPA lysate buffer, and the protein concentration was determined using the BCA method. Subsequently, 10% SDS-PAGE gel electrophoresis was performed, and the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane obtained from Thermo Fisher Scientific, USA. The PVDF membranes were blocked with 5% nonfat dry milk in TBST (CWBIO, Beijing, China) for 3 h. Primary rabbit polyclonal antibodies, including TYR, TYRP1, and DCT (ABclonal, Wuhan, China) and rabbit anti-β-actin (ABclonal, Wuhan, China), were added at a dilution of 1:2,000 for β-actin, 1:1,500 for TYRP1, 1:1,500 for TYR, and 1:1,500 for DCT, and then incubated overnight at 4 ℃. The membranes were then washed 6 times for 5 min each, followed by the addition of goat anti-rabbit secondary antibody labeled with horseradish peroxidase (HRP) (ABclonal, Wuhan, China) at a dilution of 1:10,000, and incubated at 37℃ for 2 h. After membrane washing, the target bands were visualized using High-sig ECL Western Blotting Substrate (Tanon, Shanghai, China). The expression levels of the target proteins were analyzed by Image J software.

Primary antibodies used in this study were as follows: mouse anti-USP4 (66822; Proteintech1:1000 dilution for WB, 1:300 dilution for IHC); mouse anti-BRCA1 (sc-6954; Santa Cruz; 1:300 dilution for WB); rabbit anti-BRCA1(ab16780; Abcam;1:400 dilution for IHC); rabbit anti-BRCA1 (ab191042; Abcam;1:1000 dilution for WB); rabbit anti-BARD1 (NB100; Novus Biologicals; 1:1000 dilution for WB); mouse anti-BARD1 (sc-74559; Santa Cruz; 1:500 dilution for WB); mouse anti-Flag (F3165; Sigma; 1:5000 dilution for WB); rabbit anti-HA (3924 S; Cell Signaling Technology; 1:1000 dilution for WB); mouse anti-Myc (M4439; Sigma;1:5000 dilution for WB); mouse anti-ubiquitin (3936 S; Cell Signaling Technology; 1:1000 dilution for WB); mouse anti-α-His (2366 S; Cell Signaling Technology; 1:1000 dilution for WB); mouse anti-α-Tubulin (3873 S; Cell Signaling Technology; 1:3000 dilution for WB), rabbit anti-β-actin (AC026; ABclonal; 1:1000 dilution for WB); rabbit anti-GAPDH(5174 S; Cell Signaling Technology; 1:5000 dilution for WB). For WB, western blots were detected and analyzed using ChemiDoc Imaging system (Bio-Rad). The original WB blots were included in the supplementary information.

For western blotting, the primary antibodies used were a mouse-anti-actin antibody, mouse anti-cytokeratin 18 antibody, and mouse anti-cytokeratin 8 antibody (Abcam, Cambridge, US), rabbit anti-caspase-3, rabbit anti-ASK1, rabbit anti-Bip/GRP78, rabbit anti-XBP1, rabbit anti-eIF2α, rabbit anti-DDIT3/CHOP, rabbit anti-FADD, rabbit anti-caspase-8, rabbit anti-FasLG, rabbit anti-Bcl-2, rabbit anti-Bax, rabbit anti-Bid, rabbit anti-Mitofusin 2, rabbit anti-AIF, rabbit anti-Cystatin C, rabbit anti-TTC11/FIS1 and rabbit anti-β-actin (ABclonal, Wuhan, China). We used secondary antibodies of HRP Goat Anti-Rabbit IgG (H + L) (ABclonal, Wuhan, China), and FITC Goat Anti Mouse IgG (H + L) antibody (AS001, ABclonal, Boston, USA). The item numbers of the used primary antibodies are depicted in Table S14.

Western blot assay was performed according to our previous study [38 (link)]. Liver tissues from the four groups were homogenized in RIPA lysis buffer (phosphatase and protease inhibitor). Protein assay (BCA assay kit) was conducted, and equal amounts of protein were then separated using sodium dodecyl sulphate–polyacrylamide gels and electrophoresis (SDS–PAGE). Using transfer equipment, separated proteins were transferred into polyvinylidene difluoride (PVDF) membranes. After blocking membranes with 3% non-fat milk in 1 tris buffered saline with tween (TBST) for 1 h, primary antibodies were then incubated overnight at 4 °C. These primary antibodies were rabbit anti-interleukin-6 (IL-6, MyBiosource, San Diego, CA, USA), rabbit anti-CYP3A11 (mouse homolog of human CYP3A4) (ABclonal, Wuhan, China), and rabbit anti-β-actin (ABclonal, Wuhan, China). The PVDF membranes were incubated with the secondary antibody in 3% non-fat milk in 1 TBST for 90 min the next day. In order to identify the band signals, a chemiluminescence-detecting reagent was applied, and imaging equipment (ChemiDocTMMP-Bio-Rad, Bio-Rad, Hercules, CA, USA) was used. The quantification of protein expression was then performed using Image J program (Java 8).

Rabbit anti-α-SMA (Cat No. ab179467), Col4α1 (Cat No. ab6586), CD206 (Cat No. ab64693), CD68 (Cat No. ab125212), PPARα (Cat No. ab227074), and mouse anti-CD86 (Cat No. ab220188) antibodies were purchased from Abcam (Cambridge, MA). Rabbit anti-F4/80 (Cat No. #30325), NF-κB-p65 (Cat No. #8242), and phosphorylation of NF-κB-p65 (Cat No. #3033) antibodies were purchased from Cell Signaling Technology. Rabbit anti-β−actin (Cat No. AC028) was purchased from ABclonal (Wuhan, China). Mouse AST ELISA kit (Cat No. ab263882), ALT assay kit (Cat No. ab241035), ALP assay Kit (Cat No. ab267583), gamma-glutamyl transferase (γ-GT) assay kit (Cat No. ab241029), triglyceride assay kit (Cat No. ab65336), free fatty acid assay (Cat No. ab65341), ROS detection assay (Cat No. ab139476), caspase-3 assay kit (Cat No. ab39401), and mitochondrial complex I enzyme activity microplate assay kit (Cat No. ab109721) and mitochondrial complex III activity assay kit (Cat No. ab287844) were obtained from Abcam (Cambridge, MA). Glycyrrhetinic acid (Cat No. HY-N0375), betaine (Cat No. HY-B0710), ursolic acid (Cat No. HY-N0140), and wogonin (Cat No. HY-N0400) were purchased from MedChemExpress (Shanghai, China).