FIG. 4.

Physical association between C/EBPβ and MH2 or p65. (A) Gel electrophoretic mobility shift assay (EMSA) was performed using 10 μg of nuclear extracts from U-87MG cells transfected with C/EBPβ (lanes 1–5) alone, with C/EBPβ + MH2 (lanes 2, 4, and 5), or with C/EBPβ + p65 (lane 5). For supershift assays, antibodies directed against C/EBPβ (lanes 3–5) were mixed with nuclear proteins for 1 h at 4°C prior to the addition of the probe. The positions of the C/EBPβ-DNA complexes are indicated with brackets. (B) GST pull-down. Whole cell extract from cells transfected with 2.5 μg CMV-C/EBPβ was incubated with either glutathione-S-transferase (GST) bound to glutathione beads (lanes 2 and 5) or full length GST-C/EBPβ fusion protein bound to glutathione beads (lanes 3 and 6). Lanes 1 and 4 represent direct analysis of the input protein extract by Western blot, used as a positive control for migration of C/EBPβ on the gel.