Figure 6

Effect of catalytically active and inactive PKCζ mutants on PLD activity. A, Representative Western blot showing the transfection of pCMV-FLAG-PKCζ constructs using an anti-FLAG antibody. Samples were immunoprecipitated with anti-FLAG antibody and probed with the same antibody; 1: untransfected, 2: pCMV-FLAG wt PKCζ, 3: pCMV-FLAG T410A PKCζ, 4 and 5: pCMV-FLAG T410E PKCζ. B, Cells were transiently transfected with wt PKCζ, kinase-deficient T410A PKCζ and constitutively active T410E PKCζ for 48 h and PLD activity was determined as described in Methods. Data are expressed as the change in PLD activity as a fraction of basal activity (non transfected cells). Values are the mean ± S.E. of three independent experiments performed in duplicate on different batches of cells. * Value significantly different from basal p < .01. C, PHE-induced increase in PLD activity was adjusted for basal variations in the absence of PHE (shown in panel B) for each treatment (vehicle, wt, T410A, T410E). PLD activity was calculated as [PKCζ construct + PHE / [PKCζ construct alone]. This data presentation allows the elimination of non specific variations of basal PLD activity due to the overexpression of the PKCζ constructs. Values are the mean ± S.E. of three independent experiments performed in duplicate on different batches of cells. * Value significantly different from vehicle, p < .01.