Figure 4.
Figure 4. Angiopoietin-1 inhibits thrombin-induced PKCζ activation. (A) Ang-1 inhibits thrombin-stimulated EC permeability increases. Cells were untreated (Nil), treated with Ang-1 (Ang-1; 0.1 μg/mL) for 30 minutes, treated with thrombin (T; 0.2 U/mL) for 15 minutes, or pretreated with Ang-1 (0.1 μg/mL) for 30 minutes followed by thrombin (Ang-1 + T). FITC-dextran passage (measured in μg/mL) during 30 minutes is shown. Data shown are means ± SEM of a pool of 3 experiments where each group from each experiment was performed in triplicate. *P < .02 compared with Nil; †P < .0001 compared with thrombin alone. (B) EC monolayers were stained to demonstrate changes in PKCζ localization following: (i) no treatment (Nil); treatment with thrombin (0.2 U/mL) either alone (ii; Thrombin), after pretreatment with Ang-1 (iii; 0.1 μg/mL; Ang-1+ T), or after pretreatment with PKC inhibitor bisindolylmaleimide I (iv; 6 μM; BIS + T). Counterstaining for VE-cadherin is shown in panels v-viii. (C) Dominant-negative PKCζ inhibits thrombin-stimulated PKC phosphorylation. ECs were infected with pAdEasy-1 constructs encoding dominant-negative PKCζ (DN) or constitutively active PKCζ (CA). 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. 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). (D) Ang-1 blocks thrombin-stimulated PKCζ phosphorylation. ECs were infected with pAdEasy-1 constructs encoding wild-type PKCζ. Cells were lysed after no treatment (Nil), treatment with thrombin (0.2 U/mL) either alone (T) or after pretreatment with Ang-1 (Ang-1 + T; 0.1 μg/mL) or bisindolylmaleimide I (Bis + T; 6 μM). 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).

Angiopoietin-1 inhibits thrombin-induced PKCζ activation. (A) Ang-1 inhibits thrombin-stimulated EC permeability increases. Cells were untreated (Nil), treated with Ang-1 (Ang-1; 0.1 μg/mL) for 30 minutes, treated with thrombin (T; 0.2 U/mL) for 15 minutes, or pretreated with Ang-1 (0.1 μg/mL) for 30 minutes followed by thrombin (Ang-1 + T). FITC-dextran passage (measured in μg/mL) during 30 minutes is shown. Data shown are means ± SEM of a pool of 3 experiments where each group from each experiment was performed in triplicate. *P < .02 compared with Nil; †P < .0001 compared with thrombin alone. (B) EC monolayers were stained to demonstrate changes in PKCζ localization following: (i) no treatment (Nil); treatment with thrombin (0.2 U/mL) either alone (ii; Thrombin), after pretreatment with Ang-1 (iii; 0.1 μg/mL; Ang-1+ T), or after pretreatment with PKC inhibitor bisindolylmaleimide I (iv; 6 μM; BIS + T). Counterstaining for VE-cadherin is shown in panels v-viii. (C) Dominant-negative PKCζ inhibits thrombin-stimulated PKC phosphorylation. ECs were infected with pAdEasy-1 constructs encoding dominant-negative PKCζ (DN) or constitutively active PKCζ (CA). 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. 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). (D) Ang-1 blocks thrombin-stimulated PKCζ phosphorylation. ECs were infected with pAdEasy-1 constructs encoding wild-type PKCζ. Cells were lysed after no treatment (Nil), treatment with thrombin (0.2 U/mL) either alone (T) or after pretreatment with Ang-1 (Ang-1 + T; 0.1 μg/mL) or bisindolylmaleimide I (Bis + T; 6 μM). 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).

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