FIG. 4.
Upregulation of dynein light chain by cAMP via CREB is essential for cAMP-mediated inhibition of p38 activation. (A) Rat-1 cells were treated with or without FSK (20 μM, 30 min) prior to stimulation with TNF-α (5 ng/ml, 15 min) or left untreated. Expression of p150Glued, p50 dynamitin, DHC, dynein intermediate chain (DIC), or DLC was analyzed by immunoblotting. (B and C) Rat-1 cells were treated with or without FSK (20 μM) (B) or CTX (100 ng/ml) (C) for various times as indicated. Phosphorylation of CREB and expression of DLC, CREB, and β-actin were analyzed by immunoblotting. (D) Rat-1 cells were infected with Ad/GFP or Ad/ACREB as for Fig. 3F. 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. Phosphorylation of p38 and expression of p38, M2ACREB, and DLC were determined. (E) Rat-1 cells were transfected with CREB siRNA or the control (Crtl) scramble siRNA (200 nM each). After 48 h, cells were pretreated with or without FSK (20 μM, 30 min), followed by stimulation with or without TNF-α (5 ng/ml, 15 min). Phosphorylation of p38 and expression of p38, CREB, β-actin, and DLC were determined. (F) Rat-1 cells were transfected with DLC siRNA or the control (Crtl) scramble siRNA (100 nM each). After 48 h, cells were treated with or without FSK (20 μM, 30 min) prior to stimulation with TNF-α (5 ng/ml, 15 min) or left untreated. Phosphorylation of p38 and expression of DLC, β-actin, and p38 were determined. (G) Rat-1 cells were transfected with a mammalian expression vector encoding Xpress-DLC (4 μg). After 24 h, cells were stimulated with or without TNF-α (5 ng/ml, 15 min). The activity and phosphorylation of p38 and expression of Xpress-DLC were analyzed by immune complex kinase assays and immunoblotting, respectively.