Altered C/EBPβ isoforms in macrophage extracts. (A) EMSA of C/EBPβ binding activities in P388D1(IL1) macrophage nuclear extracts. Cells were cultured for 3 days without medium change (0 hour), washed with PBS, and fed with fresh medium. Nuclear extracts were prepared at the indicated times (1 to 24 hours) and analyzed by EMSA using a consensus C/EBP binding site probe. (B) Western blot analysis of truncated C/EBPβ polypeptides. Analysis of C/EBPβ proteins in P388D1(IL1) nuclear extracts. A total of 15 μg of each nuclear extract from the time course shown in (A) were analyzed by Western blotting using an antiserum specific for the basic region (panCRP28). Bacterially expressed forms of C/EBPβ (full-length [p34], LIP [p20], and the DNA-binding domain [DBD]; lanes 8 through 10) were included as standards. Asterisks indicate weak bands that correspond to p34-C/EBPβ and p20-C/EBPβ.