Figure 4. Acylation/deacylation of CD36 mediates LCFA transport.

(A) Western blotting on extracts from 4T1.2 cells nontransduced or transduced with WT CD36 or CD36 mutant lacking S-acylated cysteines. Arrow: glycosylated CD36, also expressed in 3T3-L1 adipocytes. NS, nonspecific band. (B) 4T1.2 cells transduced with WT CD36 or CD36 mutant lacking S-acylated cysteines grown in 2D were preinduced to undergo lipogenesis as in Figure 2G and then treated with BODIPY-FL-C₁₆ for 10 minutes and imaged to visualize uptake by lipid droplets (arrows). Scale bar: 50 μm. (C) Intercellular fatty acid transfer from 3T3-L1 adipocytes (not plotted) preloaded with BODIPY-FL-C₁₆ (green) to adjacent RFP⁺ 4T1 cells detected by flow cytometry with 530 nm (BODIPY) and 610 nm (RFP) lasers. Histograms at the bottom are provided to compare double-positive (BODIPY-FL-C₁₆⁺/RFP⁺) population in 4T1 cells expressing WT versus mutant CD36. (D) 3T3-L1 adipocytes treated with 300 μM palmitic acid for 0 to 120 minutes analyzed by acyl biotin exchange (ABE) assay (Supplemental Figure 4A). Extracted proteins were alkylated with hydroxylamine (HA) where indicated, de–S-acylated, biotinylated, and removed with streptavidin beads. Remaining proteins were immunoblotted with indicated antibodies. Note progressive accumulation of nonacylated glycosylated (arrow) and nonglycosylated (arrowhead) CD36 upon LCFA treatment. PHB immunoblot indicates constant PHB acylation and equal loading. Quantification is on the right. (E) bEND.3 endothelial cells treated with 300 μM palmitic acid for 0 to 120 minutes analyzed by ABE assay. Extracted proteins were alkylated (HA), de–S-acylated, biotinylated, and removed with streptavidin beads. The remaining proteins were immunoblotted with indicated antibodies. Note accumulation of nonacylated glycosylated (arrow) and nonglycosylated (arrowhead) CD36 upon LCFA treatment. ANX2 immunoblot indicates constant ANX2 acylation and equal loading. Quantification is on the right.