[A] Schematic representation of the E-boxes (consensus: CANNTG) located within genomic region 5′ to Exon 1 (ExI) of the Cx40 gene. Amplicons of the distal (Cx40 [−1144 – −814]) and proximal (Cx40 [−655 – −283]) primer sets (arrows) used for ChIP assays are denoted. [B] ChIP using a Hand2-specific antibody co-immunoprecipitates DNA fragments overlapping the proximal (Cx40 [−655 – −283]), but not distal (Cx40 [−1144 – −814]), primer sets, indicating that Hand2 binds to the Cx40 promoter within this region. [C] Luciferase-reporter transactivation assays in HEK 293 cells show that Hand2 and E12 cooperatively up-regulate expression of a Cx40-luciferase reporter (Cx40-pX2P) ~2.2 fold over baseline (n = 3, p < 0.001). Individually, Hand2 and E12 do not significantly influence Cx40-pX2P expression.