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. Author manuscript; available in PMC: 2011 Apr 1.
Published in final edited form as: J Immunol. 2010 Mar 1;184(7):3866–3877. doi: 10.4049/jimmunol.0903417

FIGURE 5.

FIGURE 5

Regulation of transcription and phosphorylation of C/EBPα by triptolide. A, MPM were incubated for 15 h with (dotted line) or without (solid line) 10 ng/ml triptolide and subsequently cultured for different times in the presence of 1 μg/ml LPS or absence (gray line and gray dotted line). B, A mouse C/EBPα promoter-luciferase construct, which spans 809-bp upstream and 187-bp downstream of the transcription initiation site, was transfected transiently into RAW264.7 cells. Cells were incubated for 15 h with (filled bars) or without 10 ng/ml triptolide (open bars), then stimulated with or without IFN-γ (15 h), followed by LPS (7 h). Cell lysates were assayed for luciferase activity. Data are summaries of four separate experiments. C, Western blotting was performed to detect Ser21- and Thr222/226-phosphorylation of C/EBPα in MPM after incubation with or without triptolide, and stimulation with LPS (1 h). D, Stably expressed siRNA against C/EBPα efficiently inhibited expression of the endogenous C/EBPα at both mRNA (left) and protein (right) levels in RAW264.7 cells. E, Mouse C/EBPα promoter-luciferase construct was cotransfected with WT or various phosphorylation mutants of C/EBPα expression vector into RAW264.7 cells depleted of endogenous C/EBPα by stable siRNA expression. Cell lysates were assayed for luciferase activity 36 h after transfection. F, IL-12p40 luciferase reporter was cotransfected with WT or various phosphorylation mutants of C/EBPα expression vector into RAW264.7 cells depleted of endogenous C/EBPα. Luciferase activity was measured from cell lysates after stimulation of RAW264.7 cells with IFN-γ and LPS in the presence or absence of triptolide (15 h). G, Western blot analysis of recombinant C/EBPα and phosphorylation mutants expressed in transfected RAW264.7 cells that had been depleted of the endogenous C/EBPα via siRNA, then stimulated with IFN-γ/LPS in the presence of triptolide. Expression of GAPDH was analyzed as a loading control. All transfection results represent three independent experiments showing mean ± SE. *p < 0.05.