Cox 4

AlloP (3α-hydroxy-5α-pregnan-20-one) was purchased from Steraloids, Inc. (Newport, RI). AlloP was reconstituted in 22.5% (w/v) 2-hydroxypropyl-β-cyclodextrin, purchased from Sigma (St. Louis, MO). Soluble membrane glycoprotein (gp)130 (sgp130) was purchased from R&D Systems (#468-MG) and reconstituted in vehicle (0.1% BSA in saline) as described previously (14 (link)). For intracerebroventricular injections of IL-6 and sgp130, 1.4 μg was dissolved in 100 μl of vehicle to deliver 100 ng per day via a 14 day osmotic pump, at a pump rate of 0.25 μl per hour (Alzet, mini-osmotic pump, model 1002). BSA (0.1%) in saline was used as the vehicle control for both the sgp130 and IL-6 intracerebroventricular injection studies. IL-6 was purchased from Invitrogen and reconstituted in the vehicle (0.1% BSA in saline). Primary antibodies used for immunoblot analysis included NeuN, Calnexin (Millipore), and Cox 4 (Invitrogen), as well as p-STAT3 and STAT3 (Cell Signaling). Secondary antibodies used included goat anti-mouse IRDye 800 and IRDye 680 (LI-COR) and goat anti-rabbit IRDye 800 and IRDye 680 (LI-COR).

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).