Mgat3

BEAS-2B cells were lysed in RIPA lysis buffer supplied with protease and phosphatase inhibitor cocktail (Beyotime, Shanghai, China). Protein concentrations were determined using a BCA Assay kit from Beyotime. Protein specimens were electrophoresed on 10% sodium dodecyl sulfate polyacrylamide gel, and transferred onto polyvinylidene membrane. After blocking using phosphate buffered saline with tween-20 (PBST) blocking buffer (Beyotime, Shanghai, China) for 30 min, the blots were incubated with primary antibodies against E-cadherin, N-cadherin, Vimentin (Beyotime, Shanghai, China), MGAT3, and GAPDH (Abcam, Cambridge, UK) at 4 °C over night. Subsequently, the membranes underwent three consecutive washes using tris-buffered saline with tween-20 (TBST) as well as exposed to goat anti-rabbit IgG secondary antibody (Abcam, Cambridge, UK) at room temperature for 1.5 h. After washing, protein visualization and quantification were performed by enhanced chemiluminescence kit (Tanon, Shanghai, China). Protein evaluation was conducted through densitometric analysis through the ImageJ software (National Institutes of Health, Bethesda, MD, USA).

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).

Cells were lysed with RIPA lysis buffer (Beyotime, China) containing a protease inhibitor (Roche, Switzerland). Equal amounts of protein were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, USA). The membranes were blocked with 5% nonfat milk in TBS for 2 h at room temperature and then probed with antibodies against Mgat3 (Abcam, USA), GAPDH (CST, USA), and Runx2 (CST, USA). After being washed, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies. Chemiluminescent detection was performed using an ECL kit (GE Healthcare, USA). Gapdh was detected on the same membrane as a loading control. Original WB pictures were shown in Supplementary Figs 5–10.