FIGURE 4.

Protein kinase C and PDLIM2 phosphorylation. a, pharmacological inhibitors of PKC, p38, PKA, PI3K, and MEK1/2 were incubated with LPS-stimulated RAW264.7 cells. IP was performed for PDLIM2 and then blotted with anti-phosphoserine Ab to determine serine phosphorylated PDLIM2. Immunoblot for PDLIM2 was performed as a control. Blot is representative of three experiments. b, PKC pharmacological inhibitors, G06850 (general PKC inhibitor), G06976 (PKCα and PKCβ inhibitor), rottlerin (PKCδ and PKCθ inhibitor), and pseudosubstrate peptide (PKCζ inhibitor), were incubated with LPS-stimulated RAW264.7 cells. IP was performed for PDLIM2 and then blotted with anti-phosphoserine Ab to determine serine-phosphorylated PDLIM2. Immunoblot for PDLIM2 was performed as a control. Blot is representative of three experiments. c, in vivo ubiquitination assay. RAW264.7 cells were cotransfected with WT-PDLIM2, HA-Ub, and FLAG-STAT1 vectors. The PKC pharmacological inhibitors, G06850 (general PKC inhibitor), G06976 (PKCα and PKCβ inhibitor), rottlerin (PKCδ and PKCθ inhibitor), and pseudosubstrate peptide (PKCζ inhibitor), were utilized. After 24 h, cells were treated with MG132 (10 μm) and stimulated with LPS (50 ng/ml) for 6 h. The Ub-STAT1 complex was immunoprecipitated and subjected to Western blot analysis using anti-HA Ab. The blot is representative of three experiments. d, in vitro phosphorylation assay. PDLIM2, S137A, and S137D were immunoprecipitated from RAW cells and incubated in the presence and absence of classical PKCα and [γ-³²P]ATP. S137A and S137D were used as controls. Blot is representative of two experiments.