The helix-loop-helix domain of ID2 is the protein-binding site necessary for inhibition of CLOCK-BMAL1 transactivation. The effect of co-transfection of CLOCK and BMAL1 with ID2 expression plasmids on transactivation of the mPer1 promoter. NIH3T3 cells were co-transfected with mPer1:luciferase promoter reporter, β-gal, and the indicated expression plasmids. A, amino acid maps of full-length and truncated ID2 proteins encoded by DNA plasmids, with deleted residues noted in parentheses. B, mean ± S.E. luciferase reporter measurements normalized to β-gal, with five samples per treatment group. All groups, except pcDNA3.1 empty vector, were transfected with equal amount of CLOCK and BMAL1 expression vector (125 ng each). Values obtained for cells transfected with empty vector were set to 1.0. Differences determined by one-factor ANOVA with Bonferroni post hoc t tests. ***, p < 0.001 versus CLOCK-BMAL1-pcDNA3 group. Data are representative of four independent experiments. Results for inhibition of transativation by full-length ID2 is consistent with previous results (27). C, Western blot analysis of transfected cell lysates revealing protein bands for full-length ID2, ID2-ΔHLH, and ID2-ΔN truncated plasmids. Plasmid combinations for each lane are as described above in B.