Fig. 5.

Mutation of DAG-binding sites blocks tyrosine phosphorylation of PKD1. (A) Cells were transfected with vector control, wild-type PKD1 or PKD1.P157G.P281G, and were stimulated with H₂O₂ (10 mM, 10 minutes) where indicated. PKD1 was immunoprecipitated (α-HA) and resolved by SDS-PAGE. Western blots were performed against PKD1 phosphorylated at Y95 (α-pY95). Total PKD1 was detected by stripping and re-probing for PKD1. (B) Cells were co-transfected with vector control, PKD1 or PKD1.P157G.P281G and either empty vector or constitutively active Src (Src.Y527F). PKD1 was immunoprecipitated (α-HA) and resolved by SDS-PAGE. Western blots were performed with α-pY95, re-probed with α-pS738/S742 and α-PKD1. Immunoblotting was performed against Src (α-Src) to control overexpression. All experiments were performed three times and similar results were obtained.