Bs 5435r

Cellular proteins were prepared from mouse pancreas and PSCs using standard methods. Protein concentrations were measured using a BCA protein assay kit (Boster); the samples were adjusted to 4 μg/μL using 6 × loading buffer (TransGen Biotech, Beijing, China) and boiled at 95°C for 5 min. Protein extracts were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE; Bio-Rad, Hercules, CA, United States) and blotted to polyvinylidene difluoride membrane (EMD Millipore, Darmstadt, Germany). The membranes were blocked with 5% non-fat milk for 1 h, then probed with the following primary antibodies: anti-GAPDH (BM1623, Boster, 1:1,000), anti-α-SMA (bs-10196R, Bioss, 1:400), anti-NF-κB p65 (sc-8008, Santa Cruz, 1:400), anti-p-NF-κB p65 (#3033, CST, 1:1,000), anti-COL1A1 (#84366, CST, 1:1,000), TGF-βR1 (SC-402, Santa Cruz, 1:400), and p-TAK1 (bs-5435R, Bioss, 1:400), respectively, overnight at 4°C. The samples were then incubated with horseradish peroxidase conjugated second antibody (goat anti-rabbit, goat anti-mouse, or rabbit anti-goat, 1:2,000) for 1 h. A 10–250 kDa protein marker was used to determine the size of detected bands in the BeyoECL Star Chemiluminescent system (Beyotime Biotechnology, Shanghai, China).