Immunoblotting

Adipose tissue homogenates were prepared using RIPA buffer (Cell Signaling Technology) supplemented with 1 mM PMSF and phosphatase inhibitor cocktail (0.5 mM imidazole, 0.25 mM sodium fluoride, 0.3 mM sodium molybdate, 0.25 mM sodium orthovanadate, and 1.0 mM sodium tartrate). Protein concentrations were measured using a modified Lowry assay. 25–40μg protein were separated by SDS-PAGE gel (10% acrylamide) and transferred onto PVDF membrane. Membranes were blocked in 5% non-fat dry milk (w/v) for one hour then treated with primary antibody overnight at 4°C. Antibodies from Cell Signaling Technology include: eIF2α (#5324, 1:1000), phosphor-Ser51 eIF2α (#3398, 1:1000), vinculin (#4650, 1:1000), tubulin (#3873, 1:3000). Antibodies from Abcam include: OXPHOS cocktail (ab110412, 1:1000), UCP1 (ab10983, 1:2000). Antibodies from Millipore include: GAPDH (MAB374, 1:2000).