Protein Quantification and Western Blot Analysis of Pancreas Tissues

Pancreas tissues were homogenized on ice in lysis buffer (125 mM tris-HCl [pH 6.8], 4% SDS, 4M urea) with 1× protease inhibitor cocktail (87786, Thermo Fisher Scientific), and supernatant was collected following centrifugation at 10,000g for 20 minutes at 4°C. Protein concentrations were determined using BCA Protein Assay Kit (23225, Pierce Biotechnology). Western blots were then performed as described previously (21 (link)). Densitometry analysis was performed using ImageJ software to quantify protein bands, which were then normalized against loading control GAPDH. The primary antibodies used in this study were anti-COL1A1 (NBP1-30054, Novus Biologicals), anti-fibronectin (NBP1-91258, Novus Biologicals), phospho–c-Jun (ab32385, Abcam), phospho-JNK (AF1205, Novus Biologicals), and anti-GAPDH (MA5-15738, Thermo Fisher Scientific).