Mouse anti glyceraldehyde 3 phosphate dehydrogenase gapdh

Cell extracts were lysed in SDS buffer and a protease inhibitor cocktail (Promega). After quantifying protein concentrations using BCA reagent (Thermo Fisher Scientific), proteins were resolved by SDS-PAGE (polyacrylamide gel electrophoresis) and transferred to PVDF membrane by electroblotting. Membranes were blocked in TBS containing 5% skim milk, and probed with the following primary antibodies: mouse anti-α-SMA (1:2000, R&D Systems), rabbit anti-SM22 (1:2000), rabbit anti-type I collagen (1:2000), mouse anti-fibronectin (1:2000), mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:2000, Thermo Fisher Scientific), rabbit anti-SNAIL (1:1000, Cell signaling technology, Danvers, MA), and mouse anti-GS (1:1000, Millipore, Temecula, CA) antibodies. Horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgGs (1:4000, Jackson ImmunoResearch Laboratories, West Grove, PA) were used as secondary antibodies for chemoluminescence detection. Signals were obtained by enhanced chemoluminescence (Perkin Elmer, Waltham, MA).

Cells were lysed with 100 μL of PRO-PREP Protein Extraction Solution (iNtRON Biotechnology, Seongnam, Korea) and protein concentrations determined by Bio-Rad Protein Assay kit (Bio-Rad, Hercules, CA, USA), according to the manufacturer’s protocol. Samples and pre-stained protein markers were electrophoresed through 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, and then transferred to polyvinylidene fluoride (PVDF) membranes, using the Mini Trans-Blot Electrophoretic Transfer Cell system (Bio-Rad). The membrane was then incubated overnight at 4 °C with primary antibodies, rabbit anti-pSTAT3 (1:1000, Cell Signaling, Beverly, MA, USA), mouse anti-STAT3 (1:1000, Cell Signaling), rabbit anti-GATA6 (1:1000, Abcam, Cambridge, UK), rabbit anti-TFF1 (1:1000, Abcam), mouse anti-TFF2 (1:1000, Abcam) or mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies (1:1000, Thermo Fisher Scientific, Rockford, IL, USA), as diluted in 1× phosphate-buffered saline with Tween (PBST). The membranes were incubated at room temperature for 1 h, with secondary antibodies (Thermo Fisher, anti-mouse, 1:1000 or anti-rabbit 1:1000, diluted in 1× PBST). Proteins were detected by an enhanced chemiluminescence horseradish peroxidase (HRP) substrate detection kit (Merck Millipore) and MiniChemi^TM Chemiluminescence imaging system (Sage Creation, Beijing, China).