CM stimulates the transcriptional potential of C/EBPβ. (A) Effect of CM or cytokines on C/EBPβ transcriptional activity in ANA-1 macrophages. Cells were transiently transfected with (DEI)4-alb-LUC and pMEX-C/EBPβ (upper panel) or pGL2-promoter (lower panel), a control promoter-luciferase construct. The cells were treated with the indicated agents 16 hours before harvesting. UCM and CM were preincubated for 1 hour at room temperature with 10 μg/mL -IL-6 or 10 μg/mL TNF- neutralizing antibodies. Recombinant human IL-6, murine TNF-, and LPS were used at concentrations of 10 ng/mL, 20 ng/mL, and 10 μg/mL, respectively. Luciferase activities were measured and normalized to protein concentration in each lysate. The data represent the average of at least three independent experiments. (B) C/EBPβ expression in transfected ANA-1 cells. The cells were transfected with pMEX-C/EBPβ-F, a derivative of the pMEX-C/EBPβ vector in which the C/EBPβ leucine zipper was replaced by the FLAG epitope.48Whole cell extracts were prepared and analyzed by Western blotting using the anti-FLAG M2 monoclonal antibody. CRM, cross-reacting material.
