Fig. 3. Depalmitoylation of CD36 is required for the endocytosis of FAs.

a 3T3-L1 adipocytes were pretreated and treated with oleate (100 μM) as in Fig. 1a for indicated time. Cells were harvested for Acyl-RAC assay followed by immunoblotting using indicated antibodies. b–d Control (scrambled) and APT1 knockdown 3T3-L1 adipocytes were pretreated and treated with BSA or oleate (100 μM) for 1 h. Cells were then harvested for immunostaining (b), surface biotinylation (c), and Acyl-RAC (d) assays. e–g 3T3-L1 adipocytes were pretreated and treated with ML348 (10 μM) for 1 h, followed by BSA or oleate treatment for another 1 h. Cells were then harvested for immunostaining (e), surface biotinylation (f), and Acyl-RAC (g) assays. h, i On day 0, WT and Cd36^−/− SVFs were set up at 4 × 10⁴ cells per well in a six-well plate. On day 2, cells were pretreated with serum-free medium for 4 h, and treated with ML348 (10 μM) for 1 h, followed by treatment with oleate (100 μM) and BODIPY 493/503 (0.1 μg/ml) for 4 h. h Cells were then fixed and imaged on a Zeiss LSM-780 confocal microscope. i Quantification of BODIPY fluorescent intensity was performed using ZEN 2010 software. Each value represents mean ± SEM fluorescent intensity per cell from 20 cells. The value in DMSO-treated WT cells was normalized to 1.0. Two-sided Student’s t test was performed between DMSO- and ML348-treated cells. DAPI was used to indicate the nuclei. j WT and Cd36^−/− SVFs were differentiated into adipocytes and pretreated with ML348 (10 μM) for 1 h, followed by treatment with 100 μM ³H-oleate (specific activity, 2268 dpm/nmol) for 1 h. Lipid fractions were extracted from the cells and subjected to scintillation counting. The radioactive counting was normalized to protein content. Each value represents mean ± SEM obtained from three samples. Two-sided Student’s t test was performed between DMSO- and ML348-treated cells. These experiments were repeated at least twice. Source data are provided as a Source Data file.