Acute PLD inhibition reduces EHD2 protein levels, but does not dissociate EHD2 from membranes.

<p>(A) HeLa cells were treated with DMSO (control), or 15 µM each CAY10593 and CAY10594 for 4 h, or 25 µM each CAY10593 and CAY10594 for 45 min. The cells were then lysed, and equal amounts of proteins were separated by SDS-PAGE and subjected to immunoblotting with antibodies against EHD2, caveolin1 or actin. The immunoblots are representative of seven independent experiments. (B) HeLa cells were pre-treated with 100 µM leupeptin or with 10 µM lactacystin for 3 h. The cells were then treated with fresh 100 µM leupeptin or 10 µM lactacystin along with either DMSO, 15 µM each CAY10593 and CAY10594 (for 4 h), or 25 µM each CAY10593 and CAY10594 (for 45 min). Following treatment, the cells were lysed, and equal amounts of proteins were separated by SDS-PAGE and subjected to immunoblotting with antibodies against EHD2, caveolin1 or actin. The immunoblots are representative of three experiments conducted with leupeptin, and two experiments done with lactacystin. (C) HeLa cells were treated as in (A). The cells were then homogenized and post-nuclear supernatants were separated into cytosol and membrane fractions by ultracentrifugation. The samples were resolved by SDS-PAGE and immunoblotted with antibodies against EHD2, caveolin1 or cytochrome C (cytosolic fraction control). The immunoblots are representative of two independent experiments.