Ab213167

Cells were lysed by RIPA buffer (Beyotime Biotechnology) to isolate the proteins. Then, 30 μg were subjected to 10% sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE), electro-transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore), and blocked by 5% non-fat milk in phosphate-buffered solution (PBST) at room temperature for 1 h. Next, the membranes were incubated with the primary antibody against CBX3 (1:1,000; ab213167, Abcam, USA), B-cell lymphoma 2 (Bcl-2) (1:1,000; ab32124, Abcam), BCL2 associated X (Bax) (1:1,000; ab32503, Abcam), cleaved caspase3 (Cleaved-casp-3; 1:1,000; ab13847, Abcam), or β-actin (1:1,000; ab5694, Abcam) overnight at 4°C, and then incubated with the corresponding secondary antibody (1:2,000; ab150077, Abcam) for 1 h at 37°C. Finally, the proteins were visualized using ECL reagents (Millipore).

The protein was separated and transferred, and then incubated with 5% milk for 1 h. The protein was incubated at 4°C overnight using antibodies: PSMC4 (ab139184, Abcam), CBX3 (ab213167, Abcam), tropomodulin 3 (TMOD3, ab157215, Abcam), SUZ12 polycomb repressive complex 2 subunit (SUZ12, ab307891, Abcam), LCK proto‐oncogene (LCK, ab227975, Abcam), beta‐transducin repeat containing E3 ubiquitin protein ligase (BTRC, ab71753, Abcam), PI3 kinase p85 (PI3K, 4292S, CST, USA), phospho‐PI3K (BS‐3332R, Bioss, China), Akt (4685S, CST, USA), phospho‐Akt (4060S, CST, USA), mTOR (2983S, CST, USA), phospho‐mTOR (5536S, CST, USA), EGFR (ab52894, Abcam), Bax (ab32503, Abcam), Bcl‐2 (ab182858, Abcam), Caspase 3 (ab32351, Abcam), Cleaved‐Caspase 3 (C‐Caspase, ab32042, Abcam) and GAPDH (1:3000, AP0063, Bioworld). Then secondary antibody was performed and protein was verified using an imaging system (Tanon 5200, China).

Tissue microarrays (TMAs) were obtained from Shanghai Outdo Biotech Co., Ltd, and immunohistochemistry (IHC) was performed according a previous study.¹³ In brief, tissue samples were treated, and then incubated with PSMC4 antibody (1:100, ab196589, Abcam), CBX3 antibody (1:100, ab213167, Abcam) and Ki67 antibody (1:100, ab16667, Abcam) overnight at 4°C. Finally, the samples were stained and imaged after secondary antibodies were incubated for 1 h at 37°C. The scores for IHC were performed according to a previous study.¹³ In brief, the score of staining intensity was determined followed 0 to 3+ points. Positive percentage was recorded followed 0 to 4+ points. The scores of IHC were obtained according to the multiplied intensity and positive percentage scores. Low expression indicated a below average score, whereas high expression indicated an above average score.