Fig. 3

Tat palmitoylation is performed by DHHC-20. a HEK 293 T cells were cotransfected (1/5) with the indicated myc-tagged human DHHC and Tat. Tat palmitoylation was then assessed using the acyl-RAC technique, UC unbound control, BC bound control, UH unbound hydroxylamine, BH bound hydroxylamine. Palmitoylated Tat is present in the BH fraction. Palmitoylation was calculated as BH/(BH + UH)-BC/(BC + UC), and flotillin-2 was used as a positive control. The graph shows mean ± SEM of n = 3 independent experiments, ***p < 0.001 (one-way ANOVA). b DHHC-myc overexpression level was assessed using anti-myc western blot. DHHC-5, -20, and -21 migrated at 75, 37 and 31 kDa, respectively. Transfection efficiency was 60–70%. c PC12 Cells were cotransfected with Tat-FLAG and the indicated siRNA before detecting Tat palmitoylation using acyl-biotin exchange. The efficiency of siRNA-mediated silencing is shown in Supplementary Fig. 6. d The RNA from monocyte-derived macrophages (MDMac), PC12, Jurkat or human primary CD4⁺ T-cells was extracted before quantification of DHHC-20 and GAPDH mRNAs using qRT-PCR. Results are expressed as DHHC-20/GAPDH ratio. Results for all DHHCs are shown in Supplementary Fig. 7. Mean ± SEM, n = 3 independent experiments