PLD1 and EGFR Endocytosis Figure 2. PLD1 associates with the m2 subunit of AP2 in a PA- dependent manner. (A) HeLa cells were treated with EGF (20 nM) and cell extracts were immunoprecipitated with anti-PLD antibody and then immunoblotted with the indicated antibodies. (B) The kinetic of EGF-induced PLD1 activation in HeLa cells. (C) HeLa cells were treated with EGF (20 nM) for 1 min in the absence or presence of either 1- butanol (0.4%) or t-butanol (0.4%) as shown and then the cell lysates were immunoprecipitated as in (A). Alcohols were added 5 min prior to EGF treatment. doi:10.1371/journal.pone.0007090.g002 Figure 3. Association with PA enhances the direct interaction between PLD1 and m2. (A) Purified PLD1 immobilized with anti-PLD antibody (PLD) or with pre-immune serum (CTL) was incubated with purified GST alone or GST-m2. (B) HeLa cells expressing vector, wild type, R304A, or R304K PLD1 were grown in DMEM containing 10% fetal bovine serum. Cells were lysed and immunoprecipitated with anti-PLD antibody. (C) Purified PLD1 immobilized with anti-PLD antibody was incubated with purified GST alone or GST-m2 in the absence or presence of C6-PA at the indicated concentrations. (D) Vector, wild type, K898R, or R158Q PLD1 transiently expressed in HeLa cells was immunoprecip- itated and incubated with purified GST-m2 in the absence or presence of 20 nM of C6-PA. doi:10.1371/journal.pone.0007090.g003 PLoS ONE | www.plosone.org 3 September 2009 | Volume 4 | Issue 9 | e7090