Mab3608

A total of 8 μL of 1:1000 mouse serum was electrophoresed on 2% Metaphor Agarose gels (Lonza, Basel, Switzerland) for 75 minutes at 75 V. Protein was transferred onto polyvinylidene fluoride membranes (Bio-Rad, Hercules, CA) over 60 minutes at 100 V. Blocking was performed using 5% skim milk powder. A 1:10,000 dilution of primary antibody against adiponectin (MAB3608; Merck Millipore) was applied to the membranes. A 1:50,000 dilution of goat anti-mouse horseradish peroxidase antibody (Invitrogen) was applied as a secondary antibody. Visualization was performed using a Supersignal West Femto chemiluminescent kit (Thermo Fisher Scientific, Waltham, MA) on an ImageQuant LAS 500 machine (GE Healthcare Life Sciences).

4–12% Bis-Tris Bolt gels (Thermo) were loaded with 20 μg of tissue protein extract from hamster SAT or VAT, or 1 μL of hamster serum. The protein was transferred to a PVDF membrane and probed with primary antibody for adiponectin (EMD Millipore, MAB3608, 1:1000) overnight at 4 °C, followed by an incubation with horseradish peroxidase-conjugated secondary antibody. Signal detection was carried out using SuperSignal West Pico detection reagent (Thermo). Ponceau stain was used as a loading control. Band density was quantified using ImageJ.