Figure 3. PI/4,5)P₂ is involved in p17 secretion.

(A) Quantification of extracellular p17 released by neomycin-treated Jurkat-Gag cells. Jurkat cells were transfected with AG49CMVGag-RTEm26CTE and cultured for 24 h in the absence or in the presence of neomycin, at the concentration of 100 μM and 500 μM. After washing, cells were added to anti-p17 mAb MBS15-coated wells for 16 h at 37 °C in the absence or in the presence of 100 μM or 500 μM of neomycin. Plate-bound p17, secreted by seeded Jurkat-Gag cells, was then quantified by ELISA. Amount of secreted p17 was calculated as mean ± SD of three independent experiments performed in triplicate. Statistical analysis was performed by one-way ANOVA, and the Bonferroni’s post-test was used to compare data (***P < 0.001). (B) Intracellular localization of p17 in Gag-expressing cells. Gag-expressing HeLa (HeLa-Gag) cells, soon after nucleofection with AG49CMVGag-RTEm26CTE, were cultured for 16 h at 37 °C in the absence (NT) or in the presence of neomycin (100 μM). Cells were then fixed and permeabilized as described in the materials and methods section. Cells were then stained with biotinylated anti-p17 mAb MBS-3 followed by Alexa488-streptavidin and 4′,6-diamidino-2-phenylindole. Analysis was performed by confocal fluorescence microscopy. Images display mAb MBS-3 signals in green and cell nuclei in blue. z-Stack sections and orthogonal z reconstitution are also shown. Scale bar, 10 μM. (C) Quantification of p17 released by Jurkat cells co-nucleofected with AG49CMVGag-RTEm26CTE and 5ptaseIV or 5ptaseIV-Δ1 mutant expression plasmids, or by Jurkat cells expressing G2A, K30T/K32T or K26T/K27T Gag mutants. Amount of secreted p17 was calculated as mean ± SD of three independent experiments performed in Triplicate. Statistical analysis was preformed by one-way ANOVA, and the Bonferroni’s post-test was used to compare data (**P < 0.01, ***P < 0.001). (D) Intracellular localization of p17 in HeLa cells co-expressing Gag and 5ptaseIV or 5ptaseIV-Δ1 and in HeLa cells expressing G2A, K30T/K32T or K26T/K27T Gag mutants. Cells were stained with biotinylated anti-p17 mAb MBS-3 followed by Alexa488-streptavidin (green) and 4′,6-diamidino-2-phenylindole (in blue). Representative median confocal sections of at least 20 cells per conditions are shown. z-Stack sections and orthogonal z reconstitution are also shown. Scale bar, 10 μM.