Mutation of Thr75 within the p38 MAP kinase site of C/EBPϵ affects C/EBP-ϵ–specific transactivation of a myeloid-specific promoter in the presence of activated NFkappaB. (A) The C/EBP-ϵ expression constructs for the wild-type (T75) or mutant (A75 and D75) proteins were transfected into Jurkat cells along with Mim promoter in a luciferase reporter vector. Half of the transfected cells were activated by addition of TPA and calcium ionophore. Cells were harvested 48 hours after transfection and luciferase (LUC) activity was analyzed using dual luciferase assay kit (Promega). Cotransfected renilla-luciferase expression plasmid was used as internal control for transfection efficiency. Results are presented in relative luciferase units (RLUs) as a mean of 3 independent experiments (▪), adjusted for transfection efficiency, with standard deviation (brackets). (B) Western-blot analysis of nuclear accumulation of p65RelA prior to (lane1) and after Jurkat cell activation with TPA and calcium ionophore (lane 2). (C) Level of C/EBP-ϵ transcriptional activation is dependent on the presence of p65RelA. Western-blot analysis of nuclear expression of p65RelA in parental MiaPaca2 cells (lane1) and after lentiviral vector–driven siRNA knock down of p65RelA expression in 3 different clones. Clones with maximal suppression of RelA expression, analyzed in lanes 3 and 4, were used for reporter assay experiments. (D) The C/EBP-ϵ expression constructs for either the wild-type (T75) or mutant (A75 and D75) proteins were transfected into MiaPaca cells that express high levels of nuclear RelA (Rel+) or the MiaPaca cells in which RelA expression was suppressed by specific siRNA (Rel-). C/EBP-dependent myeloid Mim luciferase reporter was cotransfected with either expression plasmids or control vector (pcDNA3.1). Each experimental point was done in triplicate and error bars represent standard deviation (SD).