Ab5593

Total protein from Arc and NTS were extracted by homogenization in ice-cold radioimmunoprecipitation assay buffer (phosphate-buffered saline, 1% NP40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1 mmol/l phenylmethylsulfonyl fluoride, aprotinin 5 μg/ml, leupeptin 10 μg/ml, pepstatin A 1 μg/ml and phosphatase inhibitors cocktail). Homogenates were centrifuged for 25 min at 14,000g, supernatants collected and extract normalized to total protein content. The protein concentration was measured by BCA protein assay reagent kit (Pierce, Rockford, IL, USA). Proteins were separated by 12% SDS-PAGE, transfer to nitrocellulose membrane (Bio-Rad, Hercules, CA, USA) and blots was blocked for 1 h in 5% milk. Blots were incubated with antibodies to NPY (ab91262), SCOCS3 (ab16030), POMC (ab94446), ObRb (ab5593) and pSTAT3 (ab5073) (Abcam, Cambridge, UK, 1:1000) overnight at 4C°, and then incubated with their respective secondary antibody for one hour. Expression was normalized to GAPDH (CW bio CW0266A, Beijing, China, 1:1000) and protein intensities were determined and analyzed using Odyssey® Imager (LI-COR, Lincoln, NE, USA).

Cellular proteins were extracted from ovaries and cultured granulosa cells with RIPA (Applygen Technologies Inc., Beijing, China) supplemented with 1 mM PMSF serine protease inhibitor. The protein content was measured using a BCA Protein Assay Kit according to the manufacturer’s guidelines (CWBIO, Beijing, China). Western blotting was conducted as described in a previous report [24 (link)]. A total of 20 micrograms of protein was electrophoresed on a 12% SDS-polyacrylamide gel and transferred to PVDF membranes. The levels of the Bmal1 and other proteins involved in Lepr function and E₂ synthesis were measured using specific antibodies. Antibodies against Bmal1 (1/500; sc-365645, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), Lepr (1/500; ab5593, Abcam, Inc., Cambridge, MA, USA), Fshr (1/1000; ab75200, Abcam, Inc.), Cyp19a1 (1/1000; ab124776, Abcam, Inc.), and Cyp11a1 (1/500; #14217, CST, Inc., Danvers, MA, USA) were used to detect the levels of these proteins in granulosa cells.

Protein samples from the supernatant of hepatic tissue were electrophoretically separated via SDS‒PAGE and then transferred to polyvinylidene fluoride membranes (Millipore, USA). After blocking with bovine serum albumin for 90 min at room temperature, the membranes were incubated overnight at 4 °C with the following antibodies: anti-CYP7A1 (bs-21430R, 1:1000, Bioss, Beijing, China), anti-HMGCR (ab174830, 1:5000, Abcam), anti-Leptin (bs-0409R, 1:1000, Bioss), anti-OB-Rb (ab5593, 1:2000, Abcam), anti-JAK2 (ab108596, 1:5000, Abcam), anti-phosphorylated (p)-JAK2 (ab32101, 1:5000, Abcam), anti-STAT3 (ab68153, 1:2000, Abcam), anti-p-STAT3 (ab76315, 1:1000, Abcam), and anti-GAPDH (ab181602, 1:10,000, Abcam). Subsequently, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG secondary antibody (bs-40295G-HRP, 1:10,000, Bioss) at room temperature for 1 h. An ECL kit (Solarbio, Beijing, China) was used to visualize the protein bands. The blots were imaged using an X-ray imaging system (Bio-Rad, USA). GAPDH was used as the internal reference.