Figure 2. Agonist-specific activation patterns for PKD1, PKD2 and PKD3 in cardiac fibroblasts.

Cardiac fibroblasts were challenged with S1P (5 μM), Thr (1 U/ml), or PDGF (50 ng/ml) for 5 min or 200 nM PMA for 20 min (Panel A). Treatment with these agonists as well as ET-1 (100 nM), or H₂O₂ (at the indicated concentrations) was for the indicated intervals following a pretreatment for 45 min with vehicle or 10 μM GF109203X (GFX) in Panels B and F. Lysates were subjected to Laemmli SDS-PAGE followed by immunoblot analysis for PKD isoform expression and phosphorylation in Panel A. Proteins were separated according to the Mn²⁺-Phos-tag SDS-PAGE method for the PKD1, PKD2, and PKD3 blots in Panels B and D, for the PKD1 blot in Panel E, and for all blots except PKD1 in Panel F. Labels in shaded boxes in this Figure (as well as in Figs 3–5) denote immunoblots performed following protein separation by Mn²⁺- Phos-tag SDS-PAGE. In each panel, the results are from a single gel (or a set of gels run in parallel, in Panel B) exposed for a uniform duration. The results are representative of data obtained in separate experiments on three separate culture preparations. Panel C: Lysates from cardiomyocytes infected with adenoviral vectors that drive expression of PKD1, PKD2, or PKD3 were probed with PKD isoform-specific antibodies. Panel F: PKD1 dephosphorylation was accomplished by incubating lysates from PMA-treated cardiac fibroblasts (0.5 mg) in a reaction mixture containing 20 mM Tris HCl, 1 mM MgCl₂ and bovine intestinal mucosa alkaline phosphatase (0.5 μg/ml) for 12 hr at 37°C. Lysates were then subjected to immunoblot analysis for PKD1 protein and Ser⁷⁴⁴/Ser⁷⁴⁸ phosphorylation.