Anti ap2α

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).

Total protein was isolated with RIPA buffer (#P0013, Beyotime, China) and quantified with a BCA kit (#P0009, Beyotime, China). Each sample (50 μg) was separated by 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane, which was then blocked with 5% BSA and incubated with primary antibodies overnight at 4 °C. Membranes were then rinsed 3 times with PBS containing 0.1% Tween 20 (PBST) and incubated with appropriate secondary antibodies at room temperature for 1 h. Then, membranes were washed with PBST 3 times. Signals were generated with Enhanced Chemiluminescence Substrate (#NEL105001 EA, PerkinElmer) for 1 min before detection with a Bio-Rad ChemiDoc MP System (170-8280). The primary antibodies included anti-Ap-2α (#3215, Cell Signaling; the dilution ratio was 1:1000), anti-Elk-1 (#9182, Cell Signaling; the dilution ratio was 1:1000), anti-Sirpα (#13379, Cell Signaling; the dilution ratio was 1:1000) and anti-GAPDH (#5174, Cell Signaling; the dilution ratio was 1:2000) antibodies. Images were cropped for presentation.