Anti cdk4 sc 260

Human PBMC pellets were homogenized in the presence of ice-cold lysis TNG buffer (Tris–HCl 50 mM, pH7.5, NaCl 200 mM, Tween-20 1%, NP-40 0.2%) supplemented with protease inhibitor Complete Mini cocktail, PhosSTOP Phosphatase Inhibitor Cocktail (Roche, Germany), 2 Mm PhenylMethylSulfonylFluorid (PMSF), ß-glycerol phosphate 50 mM and 200 µM Na3VO (Sigma). Protein extracts (35-50 µg) were prepared with Laemmli buffer (5 min, 95 °C) and subjected to 12% w/v polyacrylamide gel electrophoresis and western blot analysis as described [10 (link)]. The following primary and secondary antibodies were used: anti-CDK4 (sc-260, SantaCruz), anti-p21 (sc-397 SantaCruz), anti-p27 (610242, BD), anti-β-Actin (A5441, Sigma), anti-mouse IgG-HRP (sc-2005, SantaCruz) and anti-rabbit IgG-HRP (sc-2004, SantaCruz). The immunocomplexes were detected with ECL Plus (ThermoFisher).

For cytoplasmic and nuclear extracts preparation, 3T3-L1 preadipocytes at ci48 h were treated by NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific Inc., Rockford, IL, USA). The procedure was carried out according to the manufacturer's instructions. After separation, the components were prepared with 5 × SDS lysis buffer. For preparation of the whole-cell extracts, cells were rinsed with PBS and harvested with 1 × SDS lysis buffer. Protein content was quantified by a Lowry assay. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to PVDF membranes. Following transfer, the membrane was blocked in 5% BSA for 1h at room temperature and probed with antibody. The following primary antibodies were used: anti-AP2α (#3215; Cell signaling, Boston, MA, USA), anti-Dnmt3a (#2160; Cell signaling), anti-CDK4 (sc-260, Santa Cruz, CA, USA), anti-C/EBPβ (sc-150; Santa Cruz), anti-C/EBPα (sc-61; Santa Cruz), anti-PPARγ (sc-7273; Santa Cruz), anti-Lamin B (sc-6216; Santa Cruz), anti-rabbit IgG (sc-2027; Santa Cruz), anti-aP2 (AF1443; R & D Systems, Minneapolis, MN, USA), anti-Tubulin (T6199; Sigma, St. Louis, MO, USA), anti-HA (Tiangen, AB104) and anti -5-MeC (ab10805; Abcam, Cambridge, MA, USA). The Immobilon Western Chemiluminescence HRP Substrate was from Millipore Corporation (Billerica, MA, USA).

Cells were harvested and resuspended in SDS buffer (Beyotime, Shanghai, China) for preparation of total protein extracts. Briefly, total protein extract was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto an Immobilon-P transfer polyvinylidene fluoride membrane (Millipore, MA, USA). The membrane was incubated at room temperature in 5% bovine serum albumin (BSA) prepared with TBS-T buffer for 2 h. The membrane was incubated with primary antibody diluted 1∶1,000 with 1% BSA at 4°C overnight. On the following day, the membrane was incubated with secondary horseradish peroxidase (HRP)-conjugated antibody diluted 1∶5,000 with 1% BSA at 4°C for 6 h. The membrane was visualized after incubation in HRP substrate (BeyoECL plus A/B, Beyotime, Shanghai, China). The antibodies used in this study were anti-cyclin D1 (sc-8396, Santa Cruz, CA, USA), anti-CDK4 (sc-260, Santa Cruz, CA, USA), anti-β-actin (A2066, Sigma, MO, USA), HRP-conjugated anti-mouse IgG (sc-2005, Santa Cruz, CA, USA), and HRP-conjugated anti-rabbit IgG (sc-2004, Santa Cruz, CA, USA).