Ab205018

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.

ChIP assay was carried out following the standard instructions (Upstate Biotechnology, Inc.). BGC-823 cells were treated with 1% formaldehyde for 10 min, cracked with SDS lysis buffer and broken by the ultrasonic wave, and then incubated with antibodies against YEATS4 (ab205018) or histone H3 acetylated at lysine 27 (H3K27Ac, ab4729) (both from Abcam). Normal human rabbit IgG was used as a negative control. After washing with high salt, low salt, and LiCl buffer, the eluent was used to harvest the chromatin fragments. Finally, de-crosslinking was performed, and the enrichment was examined using RT-PCR.