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. 2006 Feb;26(4):1223–1234. doi: 10.1128/MCB.26.4.1223-1234.2006

FIG. 3.

FIG. 3.

CREB transcriptional activity is required for cAMP-mediated inhibition of p38 activation. (A) Rat-1 cells were treated with or without emetine or actinomycin D (ActD) (1 μg/ml each, 30 min) prior to FSK treatment (20 μM, 30 min), followed by stimulation with TNF-α (5 ng/ml, 15 min), or left untreated. Phosphorylation and expression of p38 were determined. (B) Rat-1 cells were cotransfected with Gal4-LUC (0.2 μg) and Gal4-CREB (0.05 μg). After 30 h, cells were treated with the transcription inhibitor KG501 (10 μM, 30 min) prior to stimulation with or without FSK (20 μM, 10 h). LUC activity was determined as described previously (35). (C) Rat-1 cells were treated with or without KG501 (10 μM, 30 min) prior to FSK treatment (20 μM, 30 min), followed by stimulation with or without TNF-α (5 ng/ml, 15 min). Phosphorylation and expression of p38 were determined. (D) Rat-1 cells were cotransfected with CREB siRNA or the control scramble siRNA (200 nM each) and mammalian expression vector encoding the CREB-LUC reporter gene (EVX-LUC) (0.4 μg). After 36 h, cells were treated with or without FSK (20 μM, 10 h). LUC activity was determined as described previously (35). (E) Rat-1 cells were transfected with CREB siRNA or the control scramble siRNA (200 nM each). After 48 h, cells were pretreated with FSK (20 μM, 30 min), followed by stimulation with TNF-α (5 ng/ml, 15 min), or left untreated. Phosphorylation of p38 and expression of CREB, β-actin, and p38 were determined. (F) Rat-1 cells were transfected with the CREB-LUC reporter gene (EVX-LUC, 0.4 μg), followed by infection with adenovirus ACREB (Ad/ACREB) or the control adenovirus green fluorescent protein (Ad/GFP) (multiplicity of infection = 300 each). Cells were treated with or without FSK (20 μM, 10 h). LUC activity was determined as described previously (35). (G) Rat-1 cells were infected with Ad/GFP or Ad/ACREB as described for panel F. After 48 h, cells were treated with FSK (20 μM, 30 min) prior to stimulation with TNF-α (5 ng/ml, 15 min) or left untreated. The activity and phosphorylation of p38 and expression of M2-ACREB were analyzed by immune complex kinase assays and immunoblotting, respectively. Ctrl, control.