Protein Extraction and Western Blot Analysis of Pancreatic Tumors

Protein extraction and western blot analysis were carried out as described (Miao et al. 2017 (link)). Briefly, lysate was obtained from specimens of pancreatic tumors and peripancreatic tumor tissues. Specimens were ground using a low-speed tissue homogenizer. All operations were performed on ice. Homogenates were centrifuged (12,000 rpm, 15 min, 4 °C), and the supernatant was collected. The protein content was measured with a BCA protein assay. Subsequently, protein was denatured at 99 °C for 5 min. Equal amounts of protein (30 μg/sample) were loaded onto sodium dodecyl sulfate-polyacrylamide gels, electrophoresed, and transferred onto polyvinylidene difluoride membranes. Membranes were incubated overnight at 4 °C with mouse monoclonal anti-β-actin antibody (1:3000, Abcam, ab5694) and rabbit monoclonal anti-mast cell tryptase antibody (1:500, Abcam, ab151757), followed by anti-mouse IgG-horseradish peroxidase (HRP) (1:5000, Cell Signaling Technology, #7076) and anti-rabbit IgG-HRP (1:5000, Cell Signaling Technology, #7074) antibodies, respectively, for 2 h at room temperature. A chemiluminescence reagent kit (ECL, Bio-Rad, Hercules, CA, USA) was used to detect the immunoreactive bands. Protein bands were normalized to those of β-actin. Image-Pro Plus 6.0 software was used to quantitate the protein content.