Nbp2 37412

Proteins were extracted from cells or liver tissues in the lysis buffer (Beyotime, Shanghai, China) consisting of protease inhibitor cocktail (1:100, TargetMol, MA, USA). The concentration of proteins was determined using BCA Protein Assay kit (Beyotime). The extracted proteins were separated by 10% SDS/PAGE and electro‐transferred to PVDF membranes (Millipore, Billerica, MA, USA). After blocking in 5% milk for 1 h, the membranes were probed with primary antibodies against SLC27A5 (1:1000, NBP2‐37412, Novus Biologicals, CO, USA), α‐SMA (1:1000, ER1003, Huabio, Hangzhou, China), COL1A1 (1:1000, WL0088, Wanleibio, Shenyang, China), COL3A1 (1:1000, 22734‐1‐AP, Proteintech, IL, USA), RUNX2 (1:1000, 20700‐1‐AP, Proteintech, IL, USA), EGR3 (1:500, sc‐390967, Santa Cruz Biotechnology, USA), β‐actin (1:3000, BL005B, Biosharp), or GAPDH (1:3000, AG019‐1, Beyotime, Shanghai, China) at 4 °C overnight. Membranes were then incubated with horseradish peroxidase‐conjugated secondary antibody (Abcam, Cambridge, UK). The staining was visualized using ClarityTM Western ECL Substrate (Bio‐Rad, CA, USA).

Liver specimens were fixed in 4% paraformaldehyde, embedded in paraffin and cut into 4 µm sections. Then, the specimens were deparaffinized, hydrated and stained by standard methods. To examine hepatic morphology and assess liver fibrosis, H&E and Sirius Red staining were performed. Sections were immune‐stained for SLC27A5 (1:500, NBP2‐37412, Novus Biologicals, CO, USA), α‐SMA (1:300, 19245T, CST, MA, USA), F4/80 (1:300, 70076T, CST), or RUNX2 (1:300, 20700‐1‐AP, Proteintech, IL, USA) overnight at 4 °C. Sections were then incubated with a secondary anti‐rabbit or anti‐mouse IgG (ZSGB‐BIO, Beijing, China) and stained using 3,3′‐diaminobenzidine (ZSGB‐BIO). Stained slides were scanned with a Pannoramic Scan 250 Flash and images were acquired using Pannoramic Viewer 1.15.2 (3DHistech, Budapest, Hungary). Immunostaining and Sirius Red staining were quantified by threshold analysis using the NIH ImageJ software. The immunohistochemical staining of SLC27A5 and RUNX2 was semi‐quantitatively analyzed using the immunoreactive scoring system.^[62 (link)
^]