Figure 3

Time series experiments. (A) HEK293T cells were transfected with a plasmid encoding GFP-tagged PI4KIII β and cultured for 24 h. Cells were stimulated with PDBu for the indicated time points, lysed, and phosphorylation and expression of PI4KIII β were analyzed by Western blot analysis using a pS294- and a GFP-specific antibody, respectively. Autophosphorylation of endogenous PKD was detected using the pS910-specific antibody. Detection of tubulin served as a loading control. Shown is a representative experiment, n = 3. (B) HEK293T cells were transfected with a control vector or a plasmid encoding Flag-tagged CERT and cultured for 24 h. Control cells were left untreated, whereas Flag-CERT transfected cells were treated with the PKD selective inhibitor kbNB142-70 for the indicated time points. Afterwards cells were lysed, and phosphorylation and expression of CERT were analyzed by Western blot analysis using a pS132- and a Flag-specific antibody, respectively. Autophosphorylation of endogenous PKD was detected using the pS910-specific antibody. Detection of tubulin served as a loading control. Shown is a representative experiment, n = 3.