Mgat5

Total proteins were extracted from adipose tissue samples using a mixture of RIPA lysis buffer (Cell Signaling) and protease and phosphatase inhibitors (Sigma-Aldrich, Burlington, MA, USA). Proteins were quantified using BCA Protein Assays (Thermo Fisher Scientific). Total protein (10 µg) was gel electrophoresed and transferred to polyvinylidene fluoride (PVDF) membranes. PVDF membranes were then incubated overnight at 4 °C with the primary mouse monoclonal antibodies (CD147, MGAT3, MGAT4a, MGAT5, and loading control GAPDH; Abcam, Waltham, MA, USA), and then with infrared IRDye-labeled secondary antibodies (LI-COR Biosciences, Lincoln, NE, USA). Lastly, membranes were scanned with an infrared imaging system (Odyssey Clx; LI-COR Biosciences). The target protein band intensity was assessed relative to the loading control using Image Studio (LI-COR Biosciences).

Tissue and cell lysates were extracted using RIPA lysis buffer with the protease inhibitor phenylmethane sulfonyl fluoride (Merck Millipore, USA). Protein concentration was measured using the BCA protein assay kit (Beyotime Biotechnology) according to the manufacturer's instructions. Equivalent amounts of protein (30μg) were separated on 10% SDS PAGE gel and then transferred onto 0.45μm PVDF membranes (Millipore, Billeria, MA) according to the standard protocols. Membranes were blocked in 5% milk in TBST buffer for 1 hr at room temperature, followed by incubation with primary antibodies at 4°C overnight. After incubation with a secondary antibody for 1 hr at room temperature, proteins were detected using ECL regent (Millipore, Billeria, MA). Primary antibodies were as follows: Barx1 (Santa Cruz, sc-81956), β-actin (sc-47778), MGAT5 (Abcam, ab87977), and MMP9 (Cell Signaling, #13667).