Fig. 8. Characterization of MSK1^–/–/MSK2^–/– MEFs. MSK1^–/–/MSK2^–/– and wt cells were treated with 2000 IU/ml TNF for the indicated times. Nuclear and total cell extracts were prepared to visualize nuclear import of p65 and IκBα degradation, respectively (A) and NF-κB DNA-binding capacity (B). (C) MSK1/MSK2 double knockout and wt MEFs were transiently transfected with Gal4-p65 expression vector together with p(gal4)₂hu.IL6-luc+. At 48 h post-transfection, cells were left untreated or were treated with 2000 IU/ml TNF for 6 h. Corresponding cell lysates were analyzed for luciferase activity and normalized for transfection efficiency by quantifying β-galactosidase levels expressed upon co-transfection of pPGKβgeobpA. (D) Confocal microscope images of TNF-induced wt or MSK1^–/–/MSK2^–/– MEFs showing Ser276-phosphorylated p65 (green) and PI-stained (red) nuclei. (E) L929sA, wt and MSK1^–/–/MSK2^–/– MEFs were left untreated or were treated for 4 h with 2000 IU/ml TNF, either pre-treated or not with 10 µM SB203580 and 10 µM U0126 for 2 h. Total RNA was isolated and analyzed using GEArray technology according to the manufacturer’s instructions. Specific mRNA expression was normalized for loading differences.