Anti adipsin

Cells were lysed and tissues were homogenized by Polytron homogenizer immediately after dissection in western extraction buffer (150 mmol/L NaCl, 10% glycerol, 1% NP-40, 1 mmol/L EDTA, 20 mmol/L NaF, 30 mmol/L sodium pyrophosphate, 0.5% sodium deoxycholate, 0.05% SDS, 25 mmol/L Tris-HCl: pH 7.4) containing protease inhibitor cocktail (Roche). The lysate was then sonicated, and debris was removed by centrifugation. SDS-PAGE and western blotting were performed and detected with ECL (Thermo Scientific). Antibodies used for western blot analysis were as follows: anti-Adipsin (R&D Systems, catalog# AF5430), anti-adiponectin (Invitrogen, catalog# PA1-054), anti-C3 (Proteintech, catalog# 21337-1-AP), anti-FABP4 (Cell Signaling Technology, catalog# 2120), anti-phospho-GSK-3β (Cell Signaling Technology, catalog# 9336), anti-HSP90 (Proteintech Group, Inc, catalog# 1371-1-AP), and anti-β‐Catenin (Cell Signaling Technology, catalog# 9562).

Tissues were homogenized with a Polytron homogenizer in an IntactProein extraction buffer (GenuIn Biotech, Blacksburg, VA, USA, catalog #415). After placing on ice for 15 min, the lysates were sonicated for 10 min, and debris was removed by centrifugation at 14,000 rpm for 10 min at 4 °C. The total protein was resolved on 8% or 10% SDS-PAGE after BCA determination of the protein concentration. The Western blotting signals were detected with ECL (Thermo Fisher Scientific, Waltham, MA, USA, catalog PI32209). The following antibodies were used in this study: anti-HSP90 (Proteintech, Rosemont, IL, USA, catalog # 13171-1-AP), anti-Adipsin (R&D systems, Minneapolis, MN, USA, catalog # AF5430), anti-Adiponectin (Invitrogen, Waltham, MA, USA, catalog # PA1-054), anti-PPARγ (Cell Signaling Technology, Danvers, MA, USA, catalog # 2443), anti-SirT1, anti-UCP1 (Abcam, Cambridge, UK, ab234430), anti-PGC-1α (Abcam, Cambridge, UK, catalog # ab54481), anti-C/EBPα (Santa Cruz, CA, USA, catalog # sc-61). Western blots were quantified using densitometry via Image J.

Cells or tissues were extracted in the IntactProtein Lysis Buffer (GenuIn Biotech #415). SDS‐PAGE separation was performed using 8–10% gels. Western blotting was performed and detected with Pierce ECL Western Blotting Substrate (Thermo Scientific, catalog PI32209). Antibodies used in this study are as follows: anti‐PPARγ (Cell Signaling Technology, #2443), anti‐aP2 (Cell Signaling Technology, #2120), anti‐Perilipin (Cell Signaling Technology, #9349), anti‐ BMAL1 (Proteintech, #14268‐1‐AP, ThermoFisher Scientific, #PA1‐523), anti‐ubiquitin (Proteintech, #10201‐2‐AP), anti‐adipsin (R&D systems, #AF5430), anti‐C3 (Proteintech, #21337‐1‐AP), anti‐GAPDH (Proteintech, #HRP‐60004), anti‐HSP90 (Proteintech, #13171‐1‐AP), anti‐C/EBP𝛼 (Santa Cruz, #sc‐61), anti‐Rabbit IgG‐HRP produced in goat (Sigma, #A0545), anti‐sheep IgG (R&D, # HAF016).