Figure 7
The bHLH transcription factor NeuroD co-immunoprecipitates and physically interacts with C/EBPα and β. (a) Co-immunoprecipitation (IP) assay and western blot analysis. Q2bn cells were transfected as indicated, and lysates were subjected to a Co-IP assay with an anti-Flag antibody and were immunoblotted (WB) with anti-Myc and anti-Flag antibodies. Direct WB analysis was used to monitor protein expression levels. (b) WB, using an anti-NeuroD antibody, of TEV-released IgG pull down of 5 mg nuclear extracts from E16.5 forebrain of either CebpaTAP/+(T/+, lane 1) or +/+ (lane 2) mice; 100 μg of input lysate (+/+) was loaded in lane 3. Note the specific presence of NeuroD (50 kDa) in the T/+ pull down. The blot was reprobed with an anti-C/EBPα antibody showing the specific pull down of C/EBPα (42 kDa and 30 kDa isoforms) in the T/+ sample (lane 1). C/EBPα in 100 μg of nuclear extract was below detectable levels by WB (lane 3). (c) NeuroD does not associate with CEBPα upon mixing lysates from myc-NeuroD transfected cells with lysates from flag-C/EBPα transfected cells.(d) Direct interaction of NeuroD to C/EBPα and β. In vitro translated NeuroD was subjected to a pull down assay using the indicated GST fusion proteins: GST-alone, GST-C/EBPβ-bZIP. GST-C/EBPα-bZIP., GST-C/EBPα-TAD. Autoradiograph revealed that NeuroD preferentially bound the bZIP domain of C/EBPα or β. *Radiolabeled.