Fig. 5: LYN phosphorylates DHHC5 and is required for the endocytosis of FAs.

a 3T3-L1 adipocytes were pretreated with serum-free medium for 4 h and PP2 (15 μM) for 1 h, followed by treatment with BSA or oleate (100 μM) for 5 min. Cells were harvested and immunoprecipitated with anti-DHHC5 antibody to detect phosphorylation of Tyr91. b 3T3-L1 adipocytes transduced with indicated shRNA were set up and treated with oleate for 5 min. Cells were harvested to detect Tyr91 phosphorylation as in (a). c On day 0, HEK293T cells were set up at 2.5 × 10⁵ cells per 6-cm dish. On day 2, cells were transfected with 0.5 μg of WT, Y91E, or Y91F of DHHC5-Flag/pCDH-puro and/or 0.5 μg LYN/pCDNA3.3. On day 3, cells were harvested and immunoprecipitated with anti-Flag M2 beads to detect phosphorylation of DHHC5 (pY). Control and LYN knockdown adipocytes were pretreated and treated with BSA or oleate (100 μM) for 1 h, followed by immunostaining (d), surface biotinylation (e), and Acyl-RAC (f) assays. Control and CD36 knockdown 3T3-L1 adipocytes were pretreated and treated with oleate (100 μM) for indicated time (g) or 5 min (h). Cells were harvested and subjected to immunoprecipitation of LYN to detect phosphorylation of LYN (Y396). i, j WT and Cd36^−/− SVFs were set up and subjected to FA uptake as in Fig. 3h, i, except that cells were pretreated with PP2 (15 μM). Each value represents mean ± SEM obtained from 20 cells. Two-sided Student’s t test was performed between DMSO and PP2 treated cells. k WT and Cd36^−/− adipocytes were set up and subjected to ³H-oleate uptake as in Fig. 3j, except that cells were pretreated with PP2 (15 μM). Each value represents mean ± SEM obtained from three samples. Two-sided Student’s t test was performed between DMSO and PP2 treated cells. These experiments were repeated at least twice. Source data are provided as a Source Data file.