FIG. 5.
Differential binding of C/EBPα mutants to DNA. (A) In vitro-translated FLAG-C/EBPα WT, BRM2 (B2), or BRM5 (B5) were incubated with radiolabeled probes containing a palindromic C/EBP binding site of the cMGF promoter (51). Competition assays were performed with increasing amounts of cold C/EBP probe (triangle: competitor). Binding to radiolabeled probes was examined by gel shift assay and detected by autoradiography. Bar graphs represent the signal quantification of the autoradiogram. The expression of C/EBPα mutants was controlled with anti-C/EBPα serum (inset). The lanes were run on the same gel but were noncontiguous. IB, immunoblot. (B) Reduced DNA binding affinity of BRM2. ITC data for C/EBP DNA site binding to the bZIP-domain of C/EBPα are presented. The panels show the sequential heat pulses and integrated heat data, fitted to a two-site binding model, for the WT protein (left) and for the BRM2 variant (right), respectively. Dissociation constant (Kd) values derived from these measurements (lower panels) correspond to 1/K (K, association constant).