C/EBPα blocks the coactivation of PU.1 by c-Jun.

(A) Transient transfection in F9 cells with reporter constructs of p(PU.1)4TK, p(mutated PU.1)4TK, and expression plasmids for PU.1, C/EBPα, and c-Jun. p(C/EBP)2TK was used as a positive control reporter for C/EBPα. Gal4-VP16 and a pGal4-luc reporter construct were used as a negative control. Luciferase activities were measured 24 hours after transfection. (B) C/EBPα displaces c-Jun from binding to PU.1. (³⁵S) Met-labeled in vitro–translated C/EBPα (lane 1) and c-Jun were incubated with GST-PU.1 (lanes 2, 3) or with GST protein (lanes 4, 6, 8), respectively. Both proteins were incubated with GST-PU.1 (lanes 5, 7). In vitro–translated C/EBPα was run alone in lane 1. (C) C/EBPα binds to the β3-β4 region of the PU.1 DNA-binding domain. (³⁵S) Met-labeled in vitro–translated C/EBPα and c-Jun were incubated with GST- β3-β4 PU.1 (lanes 2, 4) or with GST alone (lanes 1, 3), respectively. (D) The DNA-binding domain of C/EBPα interacts with PU.1. (³⁵S) Met-labeled in vitro–translated PU.1 (lane 1) was incubated with the GST–DNA-binding domain of C/EBPα (lane 2) or GST alone (lane 3).