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. 2020 Sep 21;11:4765. doi: 10.1038/s41467-020-18565-8

Fig. 4. Oleate triggers phosphorylation and inactivation of DHHC5.

Fig. 4

a On day 8 of differentiation, 3T3-L1 adipocytes were pretreated with serum-free medium for 4 h, and then treated with BSA or BSA-conjugated oleate (100 μM) for 15 min. Cells were harvested and subjected to immunoprecipitation using anti-DHHC5 antibody. The bands of DHHC5 were cut out and sent for mass spectrometry analysis of the phosphorylation sites. The spectrum of pY91-containing peptide was shown. b Indication of Y91 and DHHC motif on a topology of mouse DHHC5, which was predicted at UniProt (www.uniprot.org). c Alignment of the Y91 containing regions of DHHC5 proteins from human, mouse, rat, and zebrafish. Control and CD36 knockdown 3T3-L1 adipocytes were pretreated as in (a), and treated with oleate (100 μM) for indicated time (d) or 5 min (e). Cells were harvested, immunoprecipitated with anti-DHHC5 antibody, and immunoblotted with an anti-pY antibody to detect phosphorylation of DHHC5. f On day 0, DHHC5−/− HEK293T cells were set up at 7.5 × 105 cells per 6-cm dish. On day 2, cells were transfected with 0.5 μg CD36-Flag/pCDH-puro and 0.5 μg of indicated DHHC5 WT, Y91E, or Y91F/pCDH-puro. On day 3, cells were harvested for Acyl-RAC assay and blotted with indicated antibodies. g SVFs were isolated from Rosa-CreERT2;Dhhc5f/f mice, treated with 4-OHT to induce deletion of Dhhc5, and subjected to differentiation. On day 6 of differentiation, cells were electroporated with WT, Y91E, or Y91F of DHHC5-Flag. On day 8, cells pretreated and treated with oleate as in Fig. 1c. Cells were subjected to immunofluorescence using anti-CD36 and anti-Flag antibodies. LipidTOX was used to indicate lipid droplets. Cells expressing WT, Y91E, or Y91F of DHHC5 were outlined as indicated. Scale bar, 10 μm. h DHHC5 knockdown 3T3-L1 preadipocytes were transfected with WT, Y91E, or Y91F of DHHC5-Flag. Cells were treated with oleate (100 μM) and BODIPY 493/503 (0.1 μg/ml) for 8 h, followed by immunostaining with anti-Flag M2 antibody. DAPI was used to indicate the nuclei. These experiments were repeated at least twice. Source data are provided as a Source Data file.