Fig. 4

Palmitoylation is a stable modification that enables Tat accumulation at the plasma membrane and Tat-mediated inhibition of PI(4,5)P₂-dependent membrane traffic. a PC12 cells were labeled overnight with Tat-His₆ and 17-ODYA before chasing for the indicated times, cell lysis, Tat-His₆ purification and click chemistry. Results from a representative experiment are shown. The graph (mean ± SEM of 2 independent experiments) shows Tat palmitoylation efficiency as the biotin/Tat ratio (% of the t0 value) as a function of time. The increase of labeling during the first 3 h of chase is probably due to residual intracellular 17-ODYA. b PC12 cells were labeled with Tat (WT or C31S) at 4 °C before washing, chasing for the indicated times and Tat staining by immunofluorescence with F-actin labeling using fluorescent phalloidin. Tat membrane localization was evaluated by quantifying Tat/F-actin colocalization using confocal images from 50 < n < 100 cells and Mander’s coefficient calculation. Mean ± SEM. Representative images are in Supplementary Fig. 10. c PC12 cells were transfected with human growth hormone (GH) then treated for 5 h with 100 µM 2-BP, 20 nM Tat WT or Tat-C31S as indicated. GH secretion was then triggered using 59 mM K⁺ (K59) and quantified by ELISA. Mean ± SEM (n = 3). d Macrophages (MDMs) were treated for 3 h with 100 µM 2-BP, 5 nM Tat or Tat-C31S as indicated before assaying phagocytosis of IgG-coated 3 µm latex beads. Extracellular beads were stained before cell fixation and examination using a fluorescence microscope. e CD4 + T cells were purified, stimulated then infected with a T-tropic (NL4.3) virus, bearing Tat-WT, -W11Y or -C31S as indicated, then added to Transwells into wells containing autologous MDMs. FcγR-mediated phagocytosis by MDMs was assayed after 8 days of co-culture. At this time 20–35% of T cells and 0% of macrophages were infected. Data in panels d and e are mean ± SEM of three independent experiments (counting n > 100 cells for each). The significance of differences with controls was assessed using ANOVA, one-way (d, e) or two-way (c) (***p < 0.001; **p < 0.01)