Journal: Nature Communications

Article Title: Cyclophilin A enables specific HIV-1 Tat palmitoylation and accumulation in uninfected cells

doi: 10.1038/s41467-018-04674-y

Figure Lengend Snippet: 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. . 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. . Mean ± SEM, n = 3 independent experiments

Article Snippet: Mouse DHHC GFP-expression vectors and human Myc-tagged DHHC plasmids were from Christophe Lamaze and Laurence Abrami, respectively.

Techniques: Control, Positive Control, Over Expression, Western Blot, Transfection, Derivative Assay, Quantitative RT-PCR

Journal: Nature Communications

Article Title: Cyclophilin A enables specific HIV-1 Tat palmitoylation and accumulation in uninfected cells

doi: 10.1038/s41467-018-04674-y

Figure Lengend Snippet: Tat interacts with CypA and DHHC-20. a HEK 293 T cells were transfected with an empty vector or Tat-FLAG (WT, 31 S, or 11Y). Cells were lysed 48 h after transfection before anti-FLAG immunoprecipitation and western blots against CypA, DHHC-5 and DHHC-20. b Cells were transfected with an empty (pCi) or Tat vector. GST or GST-CypA was added to cell extracts for GST pull-down before western blots. The graph shows the quantification of the DHHC pulled-down/input intensity ratio, setting the empty vector ratio to 100%. Representative data (mean ± SEM, n = 3 independent experiments) are shown.*** p < 0.001 (Student’s t -test between Tat and vector)

Article Snippet: Mouse DHHC GFP-expression vectors and human Myc-tagged DHHC plasmids were from Christophe Lamaze and Laurence Abrami, respectively.

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot