Ab46805

Protein abundance was determined in AT samples (n = 9 per group in S vs. W, and n = 6 per group in CL vs. HS) by immune blotting method. The protein concentrations of AT were measured by Bradford assay (Bio-Rad protein quantification kit). Subsequently, under reducing conditions, SDS-PAGE was used to resolve 20 g of sample in Laemmli loading buffer, which was then transferred to nitrocellulose membrane with the following antibodies: MGLL (1:200, ab24701, Abcam, Cambridge, UK), β-actin (1:1000, ab46805, Abcam Biotech, Cambridge, UK), CB1 (1:200, ab23703, Abcam Biotech, Cambridge, UK), FAAH (1 µg/mL, ARP33121_P050, Aviva Systems Biology, San Diego, CA, USA), CB2 (ADI-905-820-100, Enzo, Farmingdale, NY, USA), PPAR-α (ab24509, Abcam Biotech, Cambridge, UK), TRPV1 (1 µg/mL, WH0007442M1, Sigma, St. Louis, MO, USA) and TNF-α (OACA04183, Aviva Systems Biology, San Diego, CA, USA). For protein detection, a 1:10,000 dilution of goat anti-rabbit HRP conjugated secondary antibody (Jackson Immunoresearch 111-035-003, West Grove, PA, USA) was used for chemiluminescence reaction. ImageJ software was used to process and analyze the data (NIH, Bethesda, MD, USA). β-actin was used to equalize the signal bands.

To validate the results of the proteomic data, protein abundance was determined in AT samples obtained at 14 d prepartum and at 4 d PP. For western blotting, the protein concentration of the sample homogenate was measured according to the Bradford assay (Bio-Rad protein quantification kit). Then, 20 μg of sample in Laemmli loading buffer was resolved by SDS-PAGE under reducing conditions, and transferred onto a nitrocellulose membrane with the following antibodies: perilipin (1:2000, #9349, Cell Signaling Technology, Danvers, MA), MGLL (1:1000, ab24701, Abcam Biotech, Cambridge, UK), and β-actin (1:1000, ab46805, Abcam Biotech). In addition, we examined the abundance of CB1 (1:200, ab23703, Abcam Biotech) and FAAH (1 μg/mL, ARP33121_P050, Aviva Systems Biology, San Diego, CA) in AT samples. Goat anti-rabbit HRP conjugated secondary antibody (Jackson Immunoresearch 111-035-003, PA, USA) at a concentration of 1:10,000 was used for an enhanced chemiluminescence reaction for protein detection. Data were processed and analyzed by densitometry using ImageJ software (NIH, Bethesda, MD). To ensure that quantitative data were obtained, chemiluminescence signals were measured after at least five consecutive exposure times to determine the linear range of the signal intensity of each antibody. Specific band signals were normalized to β-actin.