Rho activation is not involved in thrombin-induced permeability changes mediated by PKCζ (A) Blocking PKCζ activation does not block thrombin-induced Rho activation. ECs were infected with pAdEasy-1 constructs encoding dominant-negative PKCζ or constitutively active PKCζ. DN indicates cells overexpressing dominant-negative PKCζ with no treatment; DN + T, cells overexpressing dominant-negative PKCζ and treated with thrombin; CA, cells overexpressing constitutively active PKCζ with no treatment; CA + T, cells overexpressing constitutively active PKCζ treated with thrombin. One milligram of each total cell lysate was analyzed for active Rho (top panel). Total Rho is shown in the bottom panel. (B) Inhibition of Rho does not inhibit thrombin-induced PKCζ phosphorylation. ECs were infected with a pAdEasy-1 construct encoding wild-type PKC-ζ, or pretreated with the Rho inhibitor Clostridium botulinum C3 transferase followed by thrombin. Nil indicates untreated; T, thrombin alone; C3, C3 transferase alone; C3 + T, pretreated with C3, then treated with thrombin. Proteins were separated by SDS-PAGE before transfer to PVDF membrane, and probing with an antibody directed against phosphorylated Thr410 of the activation loop of PKCζ (top panel). Membranes were stripped and reprobed with an anti-PKCζ antibody (bottom panel).