2.11 Western blot analysis
Pancreatic cancer cells were seeded in six-well plates and incubated overnight. The cells were subsequently treated with different concentrations of TA for 24 h. The cells were harvested and lysed in the RIPA buffer (Beyotime Biotechnology, Shanghai, China) containing protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific) on ice for 20 minutes. After centrifugation for 20 min at 12 000 rpm under 4°C, the supernatants were collected. The protein concentration was then quantified by the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific Inc., San Jose, CA, USA). Equal amounts of protein (20-30 µg) were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked with Tris-buffered saline with 0.05% Tween-20 (TBST) supplemented with 5% nonfat dry milk for 1 h at room temperature. After blocking, the membranes were incubated with specific primary antibodies overnight at 4°C. After washing three times with TBST, the membranes were incubated with a IRDye 800CW secondary antibody (LI-COR Biosciences, Lincoln, NE, USA; RRID:AB_621843; 1:10 000) for 1 h at room temperature. Following washing three times with TBST, the membranes were scanned and visualized by using the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA). For the protein extraction of tissues, a small piece of tumor tissues was removed, frozen in liquid nitrogen, and homogenized in the lysis buffer described above with 5 mm stainless steel beads (QIAGEN Inc., Valencia, CA, USA) in the TissueLyser system (QIAGEN Inc.). The remaining operations were then performed as described above. Primary antibodies including p-STAT3 (Tyr705) (RRID:AB_2491009), p-STAT3 (Ser727) (RRID:AB_331589), STAT3 (RRID:AB_2629499), c-Myc (RRID:AB_1903938), Cyclin D1 (RRID:AB_2827374), Survivin (RRID:AB_2063948), Bcl2 (RRID:AB_1903909), ICAM-1 (RRID:AB_2280018), MMP-9 (RRID:AB_2798289) and GAPDH (RRID:AB_10622025) were purchased from Cell Signaling Technology (Beverly, MA, USA). The immune-related procedures used comply with the recommendations made by theBritish Journal of Pharmacology (Alexander et al., 2018).