Anti β tubulin

RIPA buffer (Beyotime) with 1% protease inhibitor cocktail (Roche Applied Science) was used for total protein extraction. For cytoplasmic and nuclear protein fractionation, a Nuclear and Cytoplasmic Protein Extraction Kit (Thermo Scientific, Catalog Number: 78833) was utilized. Next, 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gels were used to separate proteins according to their molecular weights. Then, the proteins were transferred onto polyvinylidene fluoride (PVDF) membranes by electrophoresis. After blocking with 5% nonfat milk, the PVDF membranes were incubated with primary antibodies followed by horseradish peroxidase (HRP)-linked secondary antibodies. Enhanced chemiluminescence (ECL) reagent was used to detect the protein bands. The primary antibodies used for western blotting were as follows: anti-HOXC6 (Santa Cruz, sc-376330), anti-β-catenin (Abcam, ab32572), anti-DKK1 (Abcam, ab109416), anti-c-Jun (Cell Signaling Technology, #9165), anti-EMT antibody kit (Cell Signaling Technology, #9782), anti-RNF43 (Abcam, ab84125), anti-Axin2(CST, #5863S), anti-Histone H3 (Huabio, Hangzhou, M1306–4), and anti-β-tubulin (Huabio, Hangzhou, M1305–2). All the antibodies used in this study were detailed in Table S2.

Total proteins were extracted from tissues and cells and quantified using the bicinchoninic acid method according to the manufacturer’s instructions (Solarbio, China) and separated by electrophoresis. Extracted proteins were then resolved via 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electro-transferred onto a polyvinylidene difluoride (PVDF) membrane. The membranes were blocked with 5% skimmed milk on a shaker for 1 h. The primary antibodies used in the present study were 1:300 anti-PAQR3 (Abcam, Cambridge, United Kingdom), 1:1000 anti-NF-κB (HuaBio, China), 1:2000 anti-p53, 1:3000 anti-Bax (Proteintech, China), and 1:1000 anti-p-p53 (CST, United States). A 1:3000 anti-GAPDH (Bioss, China) and 1: 2000 anti-β-tubulin (HuaBio, China) were used as controls. Images were obtained using Image Lab^TM Software (ChemiDoc^TM XRS+, Bio-Rad Laboratories Inc., Hercules, CA, United States). These experiments were performed in triplicate.