Journal: The EMBO Journal
Article Title: Glucocorticoids inhibit MAP kinase via increased expression and decreased degradation of MKP-1
Figure Lengend Snippet: Fig. 3. Glucocorticoids induce MKP-1 gene expression. RBL-2H3 mast cells ( A ), mouse bone marrow-derived mast cells ( B ) and NIH-3T3 fibroblasts ( C ) were treated for the indicated time with dexamethasone (Dex, 0.1 µM) or solvent alone. Total RNA was extracted and subjected to northern blotting using cDNA probes for MKP-1 and GAPDH. ( D ) COS-7 cells were transiently co-transfected with an MKP-1 promoter firefly luciferase construct (-1716MKP-1luc) and a Renilla luciferase construct as an internal control, together with either wild-type glucocorticoid receptor (GRwt) or two different dimerization/transactivation-deficient GR mutant [A458T or hGR(D4X)] expression vectors. Treatment with dexamethasone (Dex, 0.1 µM) or solvent alone was performed 4 h after the transfection and the cells were harvested 48 h later for luciferase activity measurements. The results are expressed as the level of MKP-1 promoter-driven firefly luciferase expression after correcting for the transfection efficiency by Renilla luciferase measurements (relative luciferase activity), and are presented as the mean ± SD of three independent experiments.
Article Snippet: A 3 µg aliquot of poly(A)+ RNA was used for first and second strand cDNA synthesis using an oligo(dT) primer harboring a T7 polymerase promoter sequence (Superscript Choice System; Life Technologies, Karlsruhe, Germany).
Techniques: Expressing, Derivative Assay, Northern Blot, Transfection, Luciferase, Construct, Mutagenesis, Activity Assay