PKD-1 mediates LPA-induced down-regulation of CD36. (A) HMVECs were exposed to LPA (5μM) or vehicle control for 30 minutes, and then cell lysates were prepared and analyzed by Western blot using antibodies specific for PKD-1 phosphorylated (P-PKD-1) at Ser916 or Ser738/742. Blots were stripped and reprobed with an antibody to total PKD-1 as a loading control. The bar graph shows relative phosphorylation levels (mean ± SEM) from 3 independent experiments. The blot is a representative image of a time course of Ser916 phosphorylation. Relative band densities are shown below the blot. (B) MVECs were treated with the LPA₃ antagonist DGPP (2-20μM) or the LPA_1,3 antagonist Ki14625 (1-2μM) for 30 minutes followed by LPA (5μM) for 30 minutes. Western blots for phospho–PKD-1 (Ser738/742) were performed as in panel A. Relative band densities are shown above the blot. (C) MVECs were pretreated with a selective PKD inhibitor, CID 755673 (CID; 25μM), or C3 transferase (C3*; 5 μg/mL) for 30 minutes followed by LPA (5μM) for 24 hours. Quantitative real-time PCR for CD36 mRNA was then performed as in Figure 1, and relative CD36 expression was normalized with PPIA. (D) Lysates from MVECs transfected with pmaxGFP (GFP), scrambled RNAi plasmids (Scramble), or pSUPER PKD-1 RNAi were analyzed by Western blot using an antibody to PKD-1. Blots were stripped and reprobed with anti–β-actin as the loading control. (E) MVECs transfected with RNAi plasmids as in panel D were treated with LPA (5μM) for 24 hours and analyzed by Western blot for CD36 expression. Blots were stripped and reprobed with anti–β-actin as the loading control (CTL).