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).