Phosphorylation at Ser 727 Increases STAT3 Interaction with PKCε Regulating Neuron–Glia Crosstalk via IL-6-Mediated Hyperalgesia In Vivo and In Vitro

Figure 7

Ser727 of STAT3 increased its interaction with PKCε. (a) Endogenous PKCε was immunoprecipitated from cell lysates, and immune complexes and total cell lysates were analyzed by Western blot analysis with PKCε and STAT3 antibodies. Endogenous immune PKCε/STAT3 complexes were detected in the rat spinal cord tissues. (b) IL-6 promoter-firefly luciferase reporter plasmid (0.5 μg), PKCε (1 μg), and STAT3 (1 μg) were cotransfected overnight into HEK293 cells. The transfected cells were incubated with 1 μg/mL lipopolysaccharide (LPS) for 12 h. Interleukin-6 promoter activity increased by STAT3 was further enhanced by PKCε and STAT3, indicating that PKCε improved the ability of STAT3 to bind to IL-6 promoter. Data are shown as (). , , ; one-way ANOVA followed by Bonferroni tests. (c, d) HKE293 cells were transfected with GFP, GFP-PKCε, Flag, Flag-STAT3, and phosphomimetic and dephosphomimetic mutants of STAT3, and then, immunoprecipitants were assayed. Protein complexes were detected using an anti-GFP antibody (c), and then, relative PKCε binding to STAT3 was quantified (d). Data are presented as (). , , ; one-way ANOVA followed by Bonferroni tests.

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