Figure 4.

The affinity of Id1b for Hes1 is more potent than that of Id1a. (A) The Id1 coding sequence consists of two exons: the 5′ exon of 426 bp, including the HLH region, and the 3′ exon of 42 bp. These exons are separated by an intron of 239 bp. From the sequence of the introns, Id1b is generated by skipping the 5′ splice donor signal following the first exon. This skipping adds 24 bp to the translatable sequence. (B) Mammalian two-hybrid analysis of the ability of Id1a or Id1b to dimerize with bHLH transcription factors. Cells were transfected with the indicated plasmids and assayed for luciferase production at 48 h post-transfection. The value is normalized to Renilla luciferase activity. All error bars are mean ± S.D., n = 3. *P < 0.05, compared with GAL4-Id1a. (C) Electrophoretic mobility shift assays to identify differential Id1a and Id1b protein interaction. The assays were performed with increasing amounts (20, 30, 40, 50, 60 μg) of cell extracts from Id1a- or Id1b-transfected cells, then mixed with a constant amount cell extracts from Hes1-transfected cells. The same amount of cell extracts from untransfected cells (Ctr) was used as a negative control.