Anti phosphorylated p stat3

NC was purchased from APExBIO. Anti‐cleaved caspase‐3, anti‐ Bax, anti‐B‐cell lymphoma (Bcl)‐2, anti‐Anti‐poly(ADP‐ribose) polymerase (PARP), anti‐cleaved PARP, anti‐E‐cadherin, anti‐Snail, anti‐Twist1, anti‐Bmi1, anti‐Slug, anti‐Sox2, anti‐Oct4, anti‐vimentin, anti‐β‐catenin, anti‐N‐cadherin, anti‐JAK2,anti‐STAT3, anti‐phosphorylated (p)‐STAT3, anti‐p‐JAK2 antibodies, and anti‐β‐actin were purchased from Cell Signaling Technology. Epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and transforming growth factor (TGF)‐β1 were purchased from PeproTech. N2 (100×), B27 (50×), and GlutaMAX was purchased from Gibco.

The procedure for protein extraction was the same as in a previous study (Yi et al., 2018 (link)). Protein samples of 20–30 μg were subject to electrophoresis on SDS polyacrylamide gels. After transferring the proteins to membranes, 5% skimmed milk was used to block at room temperature for 2 h, followed by incubating at 4°C overnight with primary antibodies: anti-P2X7 (Alomone Labs, Jerusalem, Israel), anti-TACE (Novus Biologicals Co., Littleton, United States), anti-TNF-α (Boster Biological Technology, Wuhan, China), anti-NF-κB (Affinity Biosciences, Ohio, United States), anti-STAT3 (Cell Signaling Technology, Beverly, MA, United States), anti-phosphorylated (p)-STAT3 (Cell Signaling Technology, Beverly, MA, United States) or anti-β-actin (ZSGB-BIO, Beijing, China). After washing with TBST for 3 × 10 min, the membranes were incubated at room temperature for 2 h with the second antibodies: goat anti-rabbit IgG (Proteintech, Rosemont, United States), or goat anti-mouse IgG (Proteintech, Rosemont, United States). After 10 min wash with TBST thrice, the membranes were exposed and developed in a gel imaging system. Image-ProPlus 6.0 was used to analyze the results.