Western blotting

Cells were washed with PBS and lysed in RIPA buffer (Cell Signaling Technology) containing a protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). Cell lysates were sonicated and centrifuged at 14,000 × g for 20 min at 4 °C. Protein concentration was determined in the supernatants using the Pierce BCA kit (Thermo Fisher Scientific). Lysates were then denatured in Laemmli buffer (0.5 M Tris, 30% glycerol, 10% SDS, 0.6 M DTT, 0.012% bromophenol blue) and heated at 95 °C for 5 min. Proteins (15 to 20 μg) were loaded onto the gel and resolved by 8% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to nitrocellulose membranes (Bio-Rad) and membranes were blocked for 1 h at room temperature in Tris-buffered saline (137 mM NaCl, 20 mM Tris-HCl, pH 7.6) containing 0.1% Tween 20 (TBS-T) and 5% low-fat milk or StartingBlock™ T20 (PBS) Blocking Buffer (Thermo Scientific), followed by incubation with the primary antibody overnight at 4 °C. Primary antibodies used were produced in rabbits—HSL (Cell Signaling Technology, 4107, 1:1,000), phospho-HSL(S565) (Cell Signaling Technology, 4137, 1:1,000), ATGL (Cell Signaling Technology, 2138, 1:1,000), and α-tubulin (Cell Signaling Technology, 2144, 1:5,000)—or mice—ACE-2 (Novus Biological, NBP2-80035, 1:3,000) and Vinculin (Abcam, ab207440, 1:2,000). Protein-bound antibodies were detected with a secondary, HRP-linked anti-rabbit (Cell Signaling Technology, 7074, 1:10,000) or HRP-linked anti-mouse (Cell Signaling Technology, 7076, 1:10,000) antibody and visualized by chemiluminescence using Clarity Western ECL Substrate (Bio-Rad). To quantify band density, identically sized regions of interest were drawn on each lane and pixel density was measured using the ImageJ software. The bands of interest and their quantification are presented in the main figures, while the uncropped and unprocessed scans are included in the Source Data file.