Effect of kinase-defective mutant of Syk on thrombin-induced NF-κB activity. HUVEC were transfected with NF-κBLUC construct in combination with pcDNA3-Syk-KD using DEAE-dextran method as described under “Experimental Procedures.” After 12 h, cells were challenged with thrombin for 10 h. Cell extracts were prepared and assayed for Firefly and Renilla luciferase activities. Data are means ± S.E. (n = 6 for each condition). ^**, p < 0.01 compared with un treated control;##,p<0.01 compared with thrombin-stimulated control.

Effect of kinase-defective mutant of Syk on thrombin-induced transactivation potential of RelA/p65. HUVEC were co-transfected with Gal4-LUC and Gal4-p65 constructs in combination with pcDNA3-Syk-KD using DEAE-dextran method as described under “Experimental Procedures.” After 12 h, cells were challenged with thrombin for 10 h. Cell extracts were prepared and assayed for firefly and Renilla luciferase activities. Data are means ± S.E. (n = 4-5 for each condition). ^**, p < 0.01 compared with untreated control; ##, p < 0.01 compared with thrombin-stimulated control (p < 0.01).

A and B, inhibition of PKC-δ prevents thrombin-induced phosphorylation of Syk. Confluent HUVEC monolayers were pretreated with chelerythrine (2 μm) (A), rottlerin (10 μm) (B), or transfected with the construct pcDNA3-PKCδ-KD encoding kinase-defective mutant of PKC-δ (PKCδ-KD) (C) and then challenged with thrombin for 1 h. Total lysates were separated by SDS-PAGE and immunoblotted with an anti-phospho-Syk (Tyr^525/526) antibody. The blots were subsequently stripped and reprobed with an anti-Syk antibody to monitor loading. Results in A are representative of two independent experiments. The bar graph in B represents the effect of rottlerin on thrombin-induced Syk phosphorylation. The phosphorylated Syk level normalized to the total Syk level is expressed relative to the untreated control set at 1. Data are mean ± S.E. (n = 3-4 for each condition). ^*, p < 0.05 compared with untreated control; #, p < 0.05 compared with thrombin-stimulated control. C, effect of kinase-defective mutant of PKC-δ on thrombin-induced phosphorylation of Syk. HUVEC were transfected with the construct pcDNA3-PKCδ-KD encoding kinase-deficient mutant of PKC-δ (PKC-δ-KD) or vector alone by electroporation as described under “Experimental Procedures.” After 24 h, cells were challenged with thrombin (5 units/ml) for 1 h. Total lysates were separated by SDS-PAGE and immunoblotted with an anti-phospho-Syk (Tyr^525/526) or anti-PKCδ antibody. The blots were subsequently stripped and reprobed with an anti-Syk antibody to monitor loading. The bar graph represents the effect of PKC-δ-KD on thrombin-induced Syk phosphorylation. The phosphorylated Syk level normalized to the total Syk level is expressed relative to the untreated control set at 1. Data are mean ± S.E. (n = 3-4 for each condition). ^*, p < 0.05 compared with untreated control; #, p < 0.05 compared with thrombin-stimulated control.

A, inhibition of PKC-δ prevents thrombin-induced phosphorylation of c-Src. HUVEC were transfected with the construct pcDNA3-PKCδ-KD encoding kinase-deficient mutant of PKC-δ (PKC-δ-KD) or vector alone by electroporation as described under “Experimental Procedures.” After 24 h, cells were challenged with thrombin (5 units/ml) for 1 h. Total lysates were separated by SDS-PAGE and immunoblotted with an anti-phospho-c-Src (Tyr⁴¹⁶) antibody or anti-PKCδ antibody. The blots were subsequently stripped and reprobed with an anti-c-Src antibody to monitor loading. The bar graph represents the effect of PKC-δ-KD on thrombin-induced c-Src phosphorylation. The phosphorylated c-Src level normalized to the total c-Src level is expressed relative to the untreated control set at 1. Data are mean ± S.E. (n = 4 for each condition). ^**, p < 0.01 compared with untreated control; ##, p < 0.01 compared with thrombin-stimulated control. B, inhibition of c-Src prevents thrombin-induced phosphorylation of Syk. Confluent HUVEC monolayers were pretreated with Src family kinase inhibitor PP2 (10 μm) and then challenged with thrombin for 1 h. Total lysates were separated by SDS-PAGE and immunoblotted with an anti-phospho-Syk (Tyr^525/526) antibody. The blots were subsequently stripped and reprobed with an anti-Syk antibody to monitor loading. The bar graph represents the effect of PP2 on thrombin-induced Syk phosphorylation. The phosphorylated Syk level normalized to the total Syk level is expressed relative to the untreated control set at 1. Data are mean ± S.E. (n = 3 for each condition). ^**, p < 0.01 compared with untreated control; ##, p < 0.01 compared with thrombin-stimulated control.

Effect of kinase-defective mutant of Syk on thrombin-induced IκBα degradation, RelA/p65 nuclear translocation, and DNA binding. HUVEC were transfected with the construct pcDNA3-Syk-KD encoding kinase-deficient mutant of Syk (Syk-KD) or vector alone by electroporation as described under “Experimental Procedures.” After 24 h, cells were challenged with thrombin (5 units/ml) for 1 h. A, cytoplasmic extracts were separated by SDS-PAGE and immunoblotted with anti-IκBα and anti-Syk antibodies. Low exposures of the Syk blots were taken to allow the visualization of overexpressed Syk-KD but not the endogenous Syk protein. Actin levels were used to monitor loading. The bar graphs represent the effect of Syk-KD on thrombin-induced IκBα degradation. IκBα level normalized to actin level is expressed relative to the untreated control set at 1. Data are mean ± S.E. (n = 3 for each condition). ^***, p < 0.001 compared with untreated control. B, nuclear extracts were separated by SDS-PAGE and immunoblotted with an anti-RelA/p65 antibody. Actin levels were used to monitor loading. Results are representative of two independent experiments. C, nuclear extracts were assayed for RelA/p65 DNA binding activity by EMSA as described under “Experimental Procedures.” Results are representative of two independent experiments.

A, effect of kinase-defective mutant of Syk on thrombin-induced tyrosine phosphorylation of RelA/p65. Total cell lysates from the experiment in A were immunoprecipitated (IP) with an antibody to RelA/p65 or IgG as described under “Experimental Procedures.” The immunoprecipitates were then immunoblotted with an anti-phosphotyrosine antibody or an anti-RelA/p65 antibody. The bar graph represents the effect of Syk-KD on thrombin-induced tyrosine phosphorylation of RelA/p65. The phosphorylated RelA/p65 level normalized to the total RelA/p65 level is expressed relative to the untreated control set at 1. Data are mean ± S.E. (n = 3 for each condition). ^***, p < 0.001 compared with untreated control; ##, p < 0.01 compared with thrombin-stimulated control. B and C, thrombin induces association of Syk with RelA/p65. HUVEC were transfected with the construct pApuro-Syk-WT encoding wild type Syk (Syk-WT) or vector alone by electroporation as described under “Experimental Procedures.” After 24 h, cells were challenged with thrombin (5 units/ml) for 1 h. B, total cell lysates were immunoblotted with an antibody to Syk. Low exposure was used to allow the visualization of overexpressed Syk but not the endogenous Syk protein. C, total cell lysates were subjected to immunoprecipitation with an antibody to RelA/p65 or IgG. The immunoprecipitates were then immunoblotted with an antibody to Syk or RelA/p65. Results are representative of two independent experiments.

Effect of kinase-defective mutant of Syk on PKC-δ-induced NF-κB activity and ICAM-1 expression. A, HUVEC were co-transfected with the constructs NF-κBLUC and pcDNA3-PKCδ-CAT encoding constitutively active PKC-δ (PKCδ-CAT) in combination with pcDNA3-Syk-KD using DEAE-dextran method as described under “Experimental Procedures.” After 12 h, cells were challenged with thrombin for 10 h. Cell extracts were prepared and assayed for Firefly and Renilla luciferase activities. Data are mean ± S.E. (n = 6 to 12 for each condition). ^**, p < 0.01 compared with vector (pcDNA3)-transfected control; ##, p < 0.01 compared with PKCδ-CAT-transfected control. B, HUVEC were co-transfected with the constructs pcDNA3-PKCδ-CAT and pcDNA3-Syk-KD using Lipofectamine 2000 as described under “Experimental Procedures.” After 24-36 h, total lysates were immunoblotted with an antibody to ICAM-1, Syk, or PKC-δ. Low exposure of the Syk blot was taken to allow the visualization of overexpressed Syk-KD but not the endogenous Syk protein. PKCδ-CAT corresponds to ∼47 kDa as it contains only the catalytic domain and therefore can be distinguished from the endogenous PKC-δ (76 kDa). Actin levels were used to monitor loading. The bar graph represents the effect of Syk-KD on PKC-δ-CAT-induced ICAM-1 protein expression. ICAM-1 protein expression normalized to actin level is expressed relative to vector (pcDNA3) alone control set at 1. Data are mean ± S.E. (n = 3 for each condition). ^***, p < 0.001 compared with vector (pcDNA3)-transfected control; #, p < 0.05 compared with PKCδ-CAT-transfected control.

A, thrombin induces phosphorylation of Syk in endothelial cells. Confluent HUVEC monolayers were challenged with thrombin (5 units/ml) for the indicated time periods. Total cell lysates were separated by SDS-PAGE and immunoblotted with an antibody to the phosphorylated (Tyr^525/526) form of Syk. The blots were subsequently stripped and reprobed with an antibody to Syk. The bar graph represents the level of Syk phosphorylation induced by thrombin at different time points. Syk phosphorylation normalized to total Syk is expressed relative to the untreated control set at 1. Data are mean ± S.E. (n = 3 for each condition). ^*, p < 0.05; ^**, p < 0.01; and ^**, p < 0.001 compared with untreated control. B and C, effect of piceatannol on thrombin-induced ICAM-1 mRNA and protein expression. B, confluent HUVEC monolayers were pretreated with indicated concentrations of piceatannol for 1 h prior to stimulation with thrombin for 3 h. Total RNA was isolated and analyzed by RT-PCR for ICAM-1 mRNA expression. GAPDH mRNA expression was used as an internal control. Results are representative of two independent experiments. C, confluent HUVEC monolayers were pretreated with piceatannol (10 μm) for 1 h prior to stimulation with thrombin for 6 h. Total cell lysates were separated by SDS-PAGE and immunoblotted with an antibody to ICAM-1. Actin levels were used to monitor loading. The bar graph represents the effect of piceatannol on thrombin-induced ICAM-1 protein expression. ICAM-1 protein expression normalized to actin level is expressed relative to the untreated control set at 1. Data are means ± S.E. (n = 3-4 for each condition). ^***, p < 0.001 compared with untreated control; #, p < 0.05 compared with thrombin-stimulated control. D, effect of kinase-defective mutant of Syk on thrombin-induced ICAM-1 protein expression. HUVEC were transfected with the construct pcDNA3-Syk-KD encoding kinase-deficient mutant of Syk (Syk-KD) or vector alone by electroporation as described under “Experimental Procedures.” After 24-36 h, cells were challenged with thrombin (5 units/ml) for 6 h. Total cell lysates were immunoblotted with anti-ICAM-1 and anti-Syk antibodies. Low exposures of the Syk blots were taken to allow the visualization of overexpressed Syk-KD but not the endogenous Syk protein. Actin levels were used to monitor the loading. Results are representative of two independent experiments.