FIGURE 2.

Apilimod inhibits Fab1 activity. A, Vacuoles were incubated with BODIPY-TMR C6-PI3P at 27°C after which reactions were quenched with acetone and lipids were extracted and resolved by TLC to detect the production of BODIPY-TMR C6-PI(3,5)P₂ by Fab1 activity. Wild type vacuoles were tested in parallel to those from fig4Δ and fab1Δ strains. Pure BODIPY-TMR C6-PI3P and C6-PI(3,5)P₂ were used as standards in lanes 1 and 2. B, Average of three experiments in panel A. C, Vacuoles were incubated with DMSO (carrier) or 500 μM Apilimod. D, Average of three experiments in panel C. E, Vacuoles were incubated under docking conditions for 20 minutes. Endogenous PI(3,5)P₂ was labeled with 2.5 μM Cy3-ML1-N and the total membranes were labeled with 1 μM MDY-64. Vacuoles were incubated with Apilimod or DMSO. Arrows point at examples of PI(3,5)P₂ puncta. Error bars are SEM (n = 3). Significant differences were in comparison wild type or DMSO at 1 minute **P < .01 (unpaired t test). Scale bar: 2 μm