Anti prb

The cells were harvested with Trypsin EDTA (Lonza) and lysed with IPH buffer containing protease and phosphatase inhibitor (Roche, Basel, Switzerland). Further, the cells were washed with 1xPBS and loaded on to 12% polyacrylamide gels (BioRad, Hercules, CA, USA) with PS11 protein ladder (GeneOn, Ludwigshafen, Germany) and blotted for 7 min with Trans-blot Turbo system (BioRad) on to 0.2 μM nitrocellulose membranes (BioRad). Membranes were blocked with 5% non-fat milk in TBS-Tween_0.05%, and visualized by anti-p53 (Sigma), anti-pRb (Cell Signaling, Danvers, MA, USA) and HRP-tagged secondary antibody (Sigma) in 1% dry milk in TBS-Tween_0.05% on Fuji-LAS 4000 (GE Healthcare Life Science, Little Chalfont, UK). As loading control, anti-Actin (Thermo Scientific, Carlsbad, CA, USA) was used as primary antibody.

Protein extraction, separation and detection were achieved as described before [8 (link)]. The following antibodies were used: anti-human E2F1 (1:500), anti-S6K1 (1:3000) and anti-Aldolase B (1:1000) from Santacruz Biotechnology (USA), anti-LC3 (1:2000) from MBL International (Germany), anti-FLAG (1:5000) from Sigma Aldrich (USA), anti-ATP6V1A (1:2000) from Thermo Scientific (USA), anti-ATP6V1C1 (1:1000) from Aviva Systems Biology (USA), anti-raptor (1:1000) from Millipore (USA), anti-pS6K1 (1:2000), anti-p4EBP1 (1:3000), anti-mTOR (1:1000), anti-pRb (1:1000) and anti-actin from Cell Signaling Technology (USA).

Protein levels of the genes were detected through western blot. Briefly, cells were lysed using RIPA buffer for 1 h on ice, the protein concentrations were determined via a BCA assay kit (Boster, Wuhan). The proteins were separated by SDS-PAGE. 50 μg of protein and 4×loading buffer were boiled for 10 min and separated by SDS-PAGE (10% separating gel and 5% stacking gel), the proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA) that were subjected to blocking by 5% skimmed milk for 1 h at room temperature, then incubated with the specific antibodies at 4 °C overnight. The goat anti-mouse and goat anti-rabbit second antibodies were got from Odyssey. The relative amount of gene product was normalized to GAPDH levels.
Proteins were detected by using anti-FAM84B (Proteintech, 18421-1-AP), anti-NPM1 (Proteintech, 60096-1-Ig), anti-CCND1 (Proteintech, 60186-1-Ig), anti-CDK4 (Proteintech, 11026-1-AP), CDK6 (Proteintech, 14052-1-AP), anti-FLAG (Cell Signaling, #14793), anti-HA (Abcam, 9110), anti-GST (Proteintech, 66001-2-Ig), anti-pRb (Cell Signaling, #8516), anti-CDKN2A (10883-1-AP), anti-E2F1 (12171-1-AP) and anti-GAPDH (Proteintech, 60004-1-Ig).

The following antibodies were used in this study: anti-Ell3 (cat no. ab67415, Abcam, Cambridge, UK), anti-β-actin (cat no. sc-47778 Santa Cruz, CA, USA), anti-p53 (cat no. 2527, Cell Signaling, MA, USA), anti-acetyl p53 (cat no. 2525, Cell Signaling, MA, USA), anti-phospho p38 (cat no. 4511, Cell Signaling), anti-p38 (cat no. 8690, Cell Signaling), anti-caspase3 (cat no. 9662, Cell Signaling), anti-BAX (cat no. 5023, Cell Signaling), anti-p21 (cat no. sc-471, Santa Cruz, CA, USA), anti-p16 (cat no. sc-468, Santa Cruz, CA, USA), anti-pRb (cat no. 2181, Cell Signaling), and anti-BCL2 (cat no. 4223, Cell Signaling). For immunoblot analysis, cells were lysed in tissue lysis buffer (cat no. 9803, Cell Signaling, Denver, USA), and total cell extracts were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and then transferred to immunoblot PVDF membranes (cat no. IPVH00010, Millipore, MA, USA). The membranes were blotted with antibodies, and immunoreactivity was detected by enhanced chemiluminescence (cat no. 34579, Thermo Fisher Scientific).

Immunoblotting was performed as described previously.^{50 (link)} Briefly, whole-cell extracts or immunopurified proteins were separated on sodium dodecyl sulfate-polyacrylamide gels (6–12%) and transferred to a HyBond ECL nitrocellulose membrane (GE Healthcare, Little Chalfont, UK). The following antibodies were used as primary antibodies: anti-ACOT7 (Abcam, Cambridge, UK), anti-pRb (Cell Signaling, Danvers, MA, USA), anti-phospho-pRb (Cell Signaling), anti-cleaved PARP (Cell Signaling), anti-p53 (Do-7; Leica, Milton Keynes, UK), anti-p21 (Santa Cruz Biotech, Dallas, TX, USA), anti-cyclin D1 (Santa Cruz Biotech), anti-CDK2 (Santa Cruz Biotech), anti-CDK4 (Santa Cruz Biotech), anti-phospho-PKCα/β II(Thr638/641) (Cell Signaling), anti-phospho-PKCδ/θ(Ser643/676) (Cell Signaling), anti-phospho-PKCζ/λ(Thr410/403) (Cell Signaling), and anti-actin (Santa Cruz Biotech).

A total of 5×10⁶ cells were washed in cold PBS prior to lyse with RIPA buffer (Pierce, Rockford, USA) supplemented with PMSF, EDTA, and protease inhibitors. Samples underwent 30 min of centrifugation at 12000×g, after which supernatants were isolated and loaded in equal protein amounts (30-50 ug) onto gels for SDS-PAGE analysis. After separation, proteins were transferred onto PVDF membranes which were then blocked with 5% skim milk in TBST for 1 h, followed by probing overnight at 4°C with anti-β-ACTIN (#3700), anti-WWTR1 (#83669), anti-PARP (#9532), anti-AKT (#4685), anti-p-AKT (#4060), anti-CDK6 (#13331), anti-Rb (#9309), anti-p-Rb (#8516), anti-E2F1 (#3742), anti-CCND1 (#2978), anti-BCL-2 (#15071), anti-p27 (#3686) (Cell Signal Technology, Beverly, USA), or anti-c-myc (Abcam, Cambridge, USA, ab32072). A peroxidase-conjugated secondary antibody was then applied to blot for 1 h, followed for four washed with TBST, after which chemi-luminescence (ECL, Pierce) was used to detect protein bands.