Inhibition of PLD alters PIP2 distribution and results in the loss of EHD2 and caveolin1 from the plasma membrane.

<p>HeLa cells growing on coverslips were treated with DMSO or with 25 µM each of the PLD inhibitors CAY10593 and CAY10594 (CAY93/94) for 45 min. The cells were then fixed or washed 3X with DMEM and chased in complete growth medium for 2 h (CAY93/94+ washout). After fixation, the cells were stained with purified GST-PH-PLCδ1 to visualize PIP2 (A–C), or with antibodies against EHD2 (D–F) or caveolin1 (G–I), or with phalloidin-488 to visualize actin (J–L). After incubation with the appropriate primary and secondary antibodies, the cells were analyzed by confocal microscopy. The images showing PIP2 staining (A–C) are z-series stacks, while cells stained for EHD2, caveolin1 or actin (D–L) were imaged as single optical sections. The images shown in D–F and J–L are the same set of cells that were co-stained for EHD2 and actin. Bar, 10 µm. The confocal micrographs are representative of three independent experiments.