Fig 5.
The PI3K activity of PikA is insensitive to wortmannin. (A) The PI3K inhibitor wortmannin does not detectably inhibit the activity of PikA. The inhibitor was added to PikA-catalyzed reactions for PI3P production at the indicated concentrations. Reactions using P110α/P85α with PI or PI (4, 5)P2 were established as controls. The activity was monitored by measuring ADP using the ADP-Glo assay. Results (mean ± s.e.) shown were from three independent experiments done in triplicate. (B) Wortmannin does not impact the distribution of GFP-2xFYVEHrs induced by PikA. HeLa cells transfected to express the indicated proteins for 14 h were subjected to image acquisition and scoring after adding 1 µM Wortmannin for 2 h. Control samples received DMSO (left). Bar, 5 µm. Quantitation of cells in which the PI3P probe was localized vesicle-like structures or aggregated spots. Results (mean ± s.e.) shown were from three independent experiments done in triplicate, at least 100 cells were scored for each sample (right). ns, not significant; ***, P < 0.001.