Anti c ebpα

Proteins were isolated from ovine SVFs using a lysis buffer supplemented with protease inhibitors (Solarbio, Beijing, China), phosphatase inhibitors (Solarbio, Beijing, China), and phenylmethylsulfonyl fluoride (PMSF, Solarbio, Beijing, China). Equal amounts of protein were separated by SDS-PAGE, transferred to nitrocellulose filter membranes (Solarbio, Beijing, China), and sealed in 5% skim milk for 1 h. Membranes were then incubated with primary antibodies (anti-β-actin: Immunoway, Beijing, China; anti-MEOX2: Proteintech, Wuhan, China; anti-adiponectin: Proteintech, Wuhan, China; anti-C/EBPα: Proteintech, Wuhan, China; anti-FABP4: Proteintech, Wuhan, China; anti-PPARγ: Proteintech, Wuhan, China) and secondary antibodies (LI-COR, Lincoln, NE, USA). After washing three times, the membranes were imaged using an Odyssey Clx imaging system (LI-COR, Lincoln, NE, USA).

Western blotting experiments were performed as previously described (23 (link)). The primary antibodies used are: rabbit monoclonal or polyclonal antibodies by Cell Signaling Technology: anti-PPARγ (#2443, 1:1000), anti-Perilipin (#9349, 1:1000), anti-C/EBPα (#8178, 1:1000) and anti-ACC1 (#4190, 1:1000); rabbit monoclonal or polyclonal antibodies by Proteintech (Wuhan, China): anti-aP2 (12802-1-AP, 1:1000), anti-FASN (10624-2-AP, 1:1000), anti-SREBP1 (66875-1-Ig, 1:2000) and anti-β-actin (66009-1-Ig, 1:5000); rabbit polyclonal antibody by Abclonal (Wuhan, China): anti-TOB2 (A7223, 1:1000). Protein bands were visualized using a chemiluminescence reagent (Advansta, Menlo Park, CA) and analyzed with Image J software.

Cells or ventricular tissues were lysed using RIPA Lysis Buffer (Beyotime, # P0013B) supplemented with 1% phenylmethanesulfonylfluoride fluoride (PMSF, Beyotime, #ST506) following manufacturer’s instructions. Protein concentration was determined using Pierce BCA Protein Assay Kit (Thermo Scientific, #23225). Equal protein was loaded onto SDS-PAGE, separated and transferred to nitrocellulose membranes. Then the membranes were blocked with 5–10% skim milk in 1×TBST and incubated with primary antibodies at 4 °C overnight. After primary antibodies incubation, the membranes were incubated with secondary antibodies at room temperature for 1.5–2 h. A chemiluminescent detection system (Image Lab, Bio-Rad, US) was used to visualize and analyze the final results. Antibodies used in this study include anti-RTN3 (Abcam, #ab187764), anti-DDDDK tag (Abcam, #ab205606), anti-Myc tag (Abcam, #ab206486), anti-FABP5 (Proteintech, #12348-1-AP), anti-C/EBPα (Proteintech, #18311-1-AP), anti-CD36 (Affinity, #DF13262), anti-β-Actin (Proteintech, #66009-1-Ig), HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H + L) (Proteintech, #SA00001-2), HRP-conjugated Affinipure Goat Anti-Mouse IgG(H + L) (Proteintech, #SA00001-1), HRP-conjugated Goat Anti-Rabbit IgG HCS (Abbkine, #A25222), and HRP-conjugated Goat Anti-Mouse IgG HCS (Abbkine, #A25112).