Bolt bis tris plus 4 12 sodium dodecyl sulfate polyacrylamide gel

Human gastric cancer cells were washed with phosphate-buffered saline (PBS). Total protein samples were then extracted, and protein concentrations were measured using the Bradford protein assay (Bio-Rad, Hercules, CA, USA). Equal quantities of total proteins were separated using Bolt Bis-Tris Plus 4–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Thermo Fisher Scientific, New York, NY, USA) and transferred onto polyvinylidene fluoride membranes. After the membranes were washed with PBS and Tween 20, they were then blocked with a blocking buffer (Bio-Rad) for 30 min at room temperature and incubated with primary antibodies GRP78 (1:1000; Cell Signaling Technology, Danvers, MA, USA), OCT4 (1:1000; Abcam, Cambridge, UK), β-actin (1:5000; Cell Signaling Technology), TGF-β1 (1:1000; Abcam), Smad2/3 (1:1000; Cell Signaling Technology), phospho-Smad2/3 (p-Smad2/3, 1:1000; Cell Signaling Technology), Arg1 (1:1000; Proteintech, Martinsried, Planegg, Germany), iNOS (1:1000; Abcam), STAT3 (1:1000; Cell Signaling Technology), and phospho-STAT3 (p-STAT3, 1:1000; Cell Signaling Technology) at 4 °C. Finally, the membranes were incubated with secondary antibodies at room temperature for 1 h and analyzed using an electrochemiluminescence detection system.

The tissues or human gastric cancer cells were washed with phosphate-buffered saline (PBS). Total protein samples were extracted, and protein concentrations were measured using the Bio-Rad Bradford Protein Assay (Bio-Rad, Hercules, CA, USA). Equal quantities of total proteins were separated through BOLT BISTRIS PLUS 4–12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Thermo Scientific, New York, NY, USA) and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with a blocking buffer (Bio-Rad, Hercules, CA, USA) for 30 min at room temperature and incubated with the primary antibodies SOX2 (1:1000; #23064; Cell Signaling, Danvers, MA, USA), GRP78 (1:1000; #3183; Cell Signaling, Danvers, MA, USA), β-actin (1:5000; #4967; Cell Signaling, Danvers, MA, USA), CREB3L1/OASIS (1:1000; ab137565, Abcam, Cambridge, UK), matrix metallopeptidase 9 (MMP-9; 1:1000; ab283575, Abcam, Cambridge, UK), and α-smooth muscle actin (1:1000; #14968; Cell Signaling, Danvers, MA, USA) at 4 °C after the membranes were washed with PBS with Tween 20. The membranes were incubated with secondary antibodies at room temperature for 1 h and then analyzed using an electrochemiluminescence detection system.