PMC

PLCϵ is localized to the Golgi. KO astrocytes were infected with 150 moi of PLCϵ-mCherry adenovirus. The nucleus was stained with DAPI, and the Golgi was stained with GM-130.

PKD is activated at the Golgi in a PLCϵ-dependent manner. Following transfection of Golgi-DKAR (1.5 μg) in WT astrocytes (A) or in KO astrocytes (B), the FRET response (CFP/FRET) was measured over time after the addition of thrombin (5 nm). Data quantitated as the mean ± S.E. (n = 8) of four independent experiments. C, plasma membrane-DKAR (1.5 μg) was transfected into WT astrocytes. The FRET response (CFP/FRET) was measured over time after the addition of thrombin (5 nm). Data are quantitated as the mean ± S.E. (n = 8) of four independent experiments.

The CDC25 and RA2 domains are required for IL-6 mRNA expression. A, IL-6 mRNA levels in primary WT and PLCϵ KO astrocytes treated with thrombin (5 nm) or vehicle (control) for 1 h were assessed by q-PCR. Fold increase is expressed relative to the WT or KO averaged controls. Data shown are the mean ± S.E. of values (n = 6) from three independent experiments. B, IL-6 mRNA levels were measured by q-PCR in PLCϵ KO astrocytes infected with WT PLCϵ, CDC25Δ, or RA2 mutant (K2150E) adenovirus followed by thrombin (5 nm) treatment for 1 h. Fold increase is expressed relative to its own control. Data shown are the mean ± S.E. of values (n = 6) from three independent experiments. *, p < .05 and **, p < .01 between control and thrombin treatment; #, p < .05 between thrombin treatments, one-way ANOVA.

PKD activation and COX-2 expression require the RA2 domain of PLCϵ. A, PKD phosphorylation (p-PKDS916) was measured in PLCϵ KO astrocytes infected with WT PLCϵ or RA2 mutant (K2150E) adenovirus followed by thrombin (5 nm) treatment for 1 h. The p-PKDS916 protein levels were normalized to total PKD and expressed relative to their own averaged control. Representative Western blots and data quantitated as the mean ± S.E. (n = 6) of three independent experiments. B, COX-2 protein levels were measured in PLCϵ KO astrocytes infected with WT PLCϵ or RA2 mutant adenovirus followed by treatment with thrombin (5 nm) for 1 h and 6 h. COX-2 protein levels were normalized to GAPDH and expressed relative to its own control. Representative Western blots and data quantitated at the 6 h time point as the mean ± S.E. (n = 6) of three independent experiments. *, p < .05 and **, p < .01 between control and thrombin treatment; #, p < .05 and ##, p < .01 between thrombin treatments, one-way ANOVA.

Rap1 activation is sustained and requires the CDC25 domain of PLCϵ. A, Rap1 activation was assessed by measuring levels of Rap1-GTP after Rap1 pull-down and Western blot in WT and PLCϵ KO astrocytes treated with vehicle or thrombin (5 nm) for 15 min, 1 h, and 6 h. Rap1-GTP was normalized to total Rap1 and data normalized to its own control. Representative Western blots are shown and data quantitated as the mean ± S.E. (n = 6) of three independent experiments. B, Rap1 activation was assessed in KO astrocytes that were infected with either WT PLCϵ adenovirus or mutant PLCϵ adenovirus that lacks the CDC25 domain (CDC25Δ) followed by vehicle or thrombin (5 nm) treatment for 1 h and 6 h. Rap1-GTP was normalized to total Rap1 and data normalized to each of its own control. Representative Western blots are shown and data quantitated as the mean ± S.E. (n = 9) of three independent experiments. *, p < .05 and **, p < .01 between control and thrombin treatment; #, p < .05 and ##, p < .01 between thrombin treatments, one-way ANOVA.

PKD activation and COX-2 expression require the CDC25 domain of PLCϵ. A, PKD phosphorylation (p-PKDS916) was measured in PLCϵ KO astrocytes that were infected with WT PLCϵ or CDC25Δ adenovirus followed by vehicle or thrombin (5 nm) treatment for 1 h. The p-PKDS916 protein levels were normalized to total PKD and expressed relative to its own averaged control. Representative Western blots are shown and data quantitated as the mean ± S.E. (n = 9) of four independent experiments (control error bars are small but present). B, COX-2 protein levels were measured in PLCϵ KO astrocytes infected with WT PLCϵ or CDC25Δ adenovirus followed by thrombin (5 nm) treatment for 1 h and 6 h. COX-2 protein levels were normalized to GAPDH and expressed relative to its own control. Representative Western blots are shown and data quantitated at the 6 h time point as the mean ± S.E. (n = 6) of three independent experiments. *, p < .01 between control and thrombin treatment; #, p < .01 between thrombin treatments, one-way ANOVA.

Intact Golgi is necessary for Rap1 activation, PKD activation, and COX-2 expression. A, BFA (5.0 μg/ml) was used to disrupt the Golgi. B, following pretreatment with BFA (5.0 μg/ml), WT astrocytes were treated with vehicle and thrombin (5 nm) for 1 h and 6 h. Rap1 activation was assessed by measuring Rap1-GTP after Rap1 pull-down and Western blot. Rap1-GTP was normalized to total Rap1 and expressed relative to its own averaged control. Representative Western blots are shown and data quantitated as mean ± S.E. (n = 8) of four independent experiments. C, following pretreatment with BFA (5.0 μg/ml), WT astrocytes were treated with thrombin (5 nm) for 1 h, and PKD phosphorylation (p-PKDS916) was assessed via Western blot. The p-PKDS916 protein levels were normalized to total PKD and expressed relative to its own averaged control. Representative Western blots are shown and data quantitated as mean ± S.E. (n = 8) of four independent experiments. D, COX-2 mRNA levels were measured after pretreatment with BFA (5.0 μg/ml) and subsequent treatment with thrombin (5 nm) for 1 h. Fold increase is expressed relative to the averaged ± inhibitor controls. *, p < .05 and **, p < .01 between control and thrombin treatment; #, p < .05 and ##, p < .01 between thrombin treatments, one-way ANOVA.