Ab138378

The protein used for Western blotting was extracted using RIPA lysis buffer (Beyotime Biotechnology, Shanghai, P.R. China) supplemented with protease inhibitors (Roche, Guangzhou, P.R. China). The proteins were quantified using the BCA™ Protein Assay Kit (Pierce, Appleton, WI, USA). The Western blot system was established using a Bio-Rad Bis-Tris Gel System according to the manufacturer’s instructions. Primary antibodies against YEATS4 (ab205018), GAPDH (ab128915), phosphorylated β-catenin (p-β-catenin, ab138378), β-catenin (ab6302), Bcl-2 (ab32124), Bax (ab77566), c-Myc (ab152146), CDK6 (ab151247), CDK4 (ab137818), and cyclin D1 (ab137875) (all from Abcam, Cambridge, MA, USA) were prepared in 5% blocking buffer. Primary antibodies were respectively incubated with the membrane at 4°C overnight, followed by wash and incubation with secondary antibodies marked by horseradish peroxidase for 1 h at room temperature. After rinsing, the polyvinylidene difluoride (PVDF) membrane carrying blots and antibodies were transferred into the Bio-Rad ChemiDoc™ XRS System, and then 200 μl of Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA, USA) was added to cover the membrane surface.