(A) Antibody supershift analysis of the fast and slow-migrating C/EBPβ complexes. P388D1(IL1) nuclear extracts from cells grown without medium change (Extract #1) or from freshly fed cells (Extract #2) were incubated with normal rabbit serum or peptide antibodies directed against the C/EBPβ N terminus (C/EBPβ-N) or C terminus (C/EBPβ-C) before adding the binding site probe. Purified recombinant C/EBPβ was included as a control. The two slow-migrating complexes in extract #1 (p34-C/EBPβ) that are supershifted by the N-terminal antibody represent C/EBPβ homodimers (faint upper band) and a heterodimeric complex formed with an unidentified partner (lower band). (B) Truncated C/EBPβ species are dependent on the cell lysis procedure. Two hours before harvesting, P388D1(IL1) cells were washed with PBS and fed with fresh medium. The harvested cells were divided into two pools and nuclear extracts were prepared by either the hypotonic lysis or detergent lysis procedures (see Materials and Methods) and C/EBPβ species analyzed by EMSA.