Atp synthase subunit alpha

Antibodies list: CPT1A (A5307, ABclonal, Wuhan, China), UCP1 (A5857, ABclonal), UCP3(A16996, ABclonal), PPARα (sc-9000, Santa Cruz, Dallas, TX, USA), PPARγ (2435S, CST, Danvers, MA, USA), FAS (3189S, CST), SREBP1 (Sc13551, Santa Cruz), PGC-1α (TA319007, Origene, Rockville, MD, USA), NDUFS3 (459130, Invitrogen, Waltham, MA, USA), SDHB (459230, Invitrogen), UQCRC1 (459140, Invitrogen), COX4 (459600, Invitrogen), ATP Synthase Subunit Alpha (459240, Invitrogen), SOD2 (sc-137254, Santa Cruz), β-Actin (3700S, CST), α-Tubulin (3873S, CST).
Mice diet was provided by SLAC Laboratory Animal Co. Ltd. (Shanghai, China). Insulin was purchased from Nove Nordisk A/S. The blood glucose meter and blood glucose test strip were both purchased from ROCHE (ACCU-CHEK Active).
The Reverse Transcription System kit was purchased from Promega; SYBR green was purchased from Takara; polymerase chain reaction (PCR) primers were synthesized by Beijing Qingke biotechnology Co. Ltd. Nitrocellulose membranes used in W.B. were purchased from PerkinElmer Life Sciences. Other reagents used in this study were purchased from Sigma (St. Louis, MO, USA).

Cells were lysed and centrifuged at 13,000 g for 15 min at 4°C. The supernatants were collected, and protein concentrations were determined with the BCA Protein Assay Kit (Pierce). Equal amounts of protein samples were applied to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels, transferred to pure nitrocellulose membranes (PerkinElmer), and blocked with 5% nonfat milk for 2 hours. The membranes were then incubated with the indicated primary antibodies at 4°C overnight. Primary antibodies used in this study were β-actin from Sigma-Aldrich; Atg7, Atg5, Beclin1, and FOXO1 from Cell Signaling Technology; FOXO3 from Sigma-Aldrich; p62, Cu-ZnSOD, MnSOD, and catalase from Santa Cruz Biotechnology; Complex I Ndufs3, Complex I Ndufa9, Complex II subunit 30 kDa Ip, Complex III subunit Core 1, Complex IV Subunit I, and ATP Synthase Subunit Alpha from Invitrogen; and LC3B from Abcam. The membranes were incubated with secondary peroxidase-conjugated antibodies (Jackson ImmunoResearch) at room temperature for 1 h. Chemiluminescent detection was performed by ECL (Pierce). The results were analyzed and quantified by Quantity One Software (Bio-Rad) to obtain the optical density ratio of the target protein to β-actin. One representative result was shown from at least three independent experiments.