Nbp1 91258

The antibodies used in this study were anti-Fibronectin (rabbit polyclonal 1:400; Novusbio NBP1-91258), and conjugated FITC-CD31 (mouse monoclonal 1:100; Biolegend c.102405). The secondary antibody was Donkey anti-Rabbit Alexa-596 (1:200). Hematoxylin and eosin stain kit (Vectors Laboratory; H-3502).

SDS-PAGE of 25 μg of protein was performed on 8% reducing gels. Proteins were transferred at 4 °C onto PVDF membranes in tris-glycine buffer containing 20% methanol. Total protein loading was measured prior to blotting using Ponceau S staining and densitometric analysis. Non-specific binding sites were blocked with 5% skim milk in tris-buffered saline supplemented with 0.1% Tween-20 (TBS-T) at room temperature. The blots were thoroughly washed in TBS-T before applying primary antibodies overnight at 4 °C with shaking. Primary antibodies were used at the following dilutions: ED-A (cellular) fibronectin (1:1000; MAB1940; MilliporeSigma, Burlington, MA; or NBP1-91258; Novus), SMemb (1:1000; ab684; Abcam), αSMA (1:5000; A2547; Sigma). Because the primary fibroblasts originated from a heterogenous population of cells, vimentin (1:2000; ab8069; Abcam) and platelet-derived growth factor receptor alpha (PDGFRα; 1:1000; ab134123; Abcam) were used as a phenotype controls on each blot. Appropriate HRP-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA) were applied at a 1:5000 dilution for 1 hour at room temperature. Protein detection was done using ECL substrate, and protein bands were visualized on blue X-ray film. Protein expression was measured by relative densitometry using Quantity One® analysis software (version 4.6.9; Bio-Rad).

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).