Anti hoxc6

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.

Cells were lysed on ice using RIPA buffer (Cell Signaling Technology, USA). Protein concentration was measured by BCA kit (Beyotime, Shanghai, China). Equal amounts of total protein were resolved using 12% SDS-PAGE gel and transferred onto PVDF membranes (Millipore, Burlington, MA, USA). Membranes were incubated with anti-HOXC6 (Santa Cruz, CA, USA, dilution, 1:1,000) and anti-β-actin (Santa Cruz, CA, USA, dilution, 1:1,000) overnight at 4 °C followed by incubation with a secondary antibody. Protein bands were visualized using ECL chemiluminescence (Millipore, Billerica, MA, USA) according to the manufacturer’s recommendation.