Fig. 1

Activation of zebrafish Pld1 in HEK293 cells. Recombinant human PLD1 (hPLD1) or zebrafish Pld1 (zPld1), was transiently expressed in HEK293 cells, and activity was measured, in vivo (A) and in vitro (B). Cells were transfected with an empty pEGFP-C1 vector, or a vector encoding wild-type hPLD1 (hPLD1 wt), or zPld1 wt. In addition, catalytically inactive mutants of hPLD1 K989R and zPld1 K846R were transfected. (A) In vivo activity was measured as the transphosphatidylation of radiolabeled endogenous substrate to 1-butanol, in response to 100 nM PMA compared to basal. (B) In vitro PLD activity was measured using 20 mg total cell lysates for either basal or with the following recombinant protein additions: Arf1 (275 nM), PKCα (1 μM), or Arf1 (275 nM) + PKCα (1 μM) for each transfection condition. Data are expressed as mean ± SEM (*, p < 0.03 when compared to the basal activity of each transfection) of triplicate samples from three independent experiments. (C) Representative Western blot analysis of the transiently expressed N′-GFP tagged PLD1 proteins (GFP-PLD) or GFP vector control (GFP) used in the PLD activity assay. Equal loading of the protein was standardized by GAPDH.