TNF- in CM induces C/EBPβ expression. Kinetics of C/EBPβ protein and mRNA expression were assessed in control and CM-treated IC-21 macrophages. Cells were washed with PBS and given fresh medium 2 hours before the start of the experiment. At time 0, the cells were washed again and given fresh medium containing either concentrated P388D1(IL1) CM or concentrated unconditioned medium (control). In lanes 10 through 13, CM was preincubated with anti–TNF- neutralizing antibody for 1 hour before addition to the cells. Cells were harvested at the indicated times and divided into two aliquots for protein and RNA analysis. Nuclear extracts were prepared by detergent cell lysis and C/EBPβ protein was analyzed by immunoblotting using an anti-C/EBPβ antibody. C/EBPβ mRNA was analyzed by Northern blotting and levels were quantitated and normalized to actin. The fold increase in C/EBPβ mRNA was determined by comparison to the 0 hour time point.