Bs 3332r

qPCR and Western blot were performed as previously reported 19 (link). qPCR was carried out using the Hairpin-it^TM microRNA qPCR Quantitation Kit (GenePharma, Shanghai, China) and LightCycler 96 (Roche, Germany). U6 snRNA was used as an internal control for miR-92b, and the mRNA levels of PTEN, TNF-α and IL-6 were normalized to GAPDH. The 2^-ΔΔCt method was employed to calculate relative expression of each gene. For Western blot analysis, the protein blots were incubated with following antibodies: rabbit anti-TLR4 (1:1000, 7074, Cell Signaling Technology, USA), rabbit anti-phospho-NF-κB p65 (1:1000, 3033, Cell Signaling Technology, USA), rabbit anti-NF-κB p65 (1:1000, 4764, Cell Signaling Technology, USA), rabbit anti-β-actin (1:1000, 4970, Cell Signaling Technology, USA), rabbit anti-Bax (1:200, bs-0127R, Bioss, China), rabbit anti-Bcl-2 (1:200, bs-4563R, Bioss, China), rabbit anti-phospho-PI3K (1:200, bs-3332R, Bioss, China), rabbit anti-PI3K (1:1000, ab227204, Abcam, USA), rabbit anti-phospho-AKT (1:1000, ab8932, Abcam, USA), rabbit anti-AKT (1:500, GB111114, Servicebio, China), rabbit anti-β-catenin (1:5000, ab196204, Abcam, USA). The optical density (OD) value of each protein band was quantified using the Image-Pro Plus 6.0 (IPP 6.0) software (Media Cybernetics Inc., USA) and normalized to β-actin.

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).