Fig. 6. Gata4 regulates Erbb3 expression. (A) qRT-PCR of control and mutant AVC samples. Gene expression was normalized to Gapdh. ^*P<0.05, n=3 per group. (B) In situ hybridization demonstrating downregulation of Thbs1, Plxnc1 and Erbb3 in Gata4^T2del EC. Arrowheads indicate AV cushions. (C) Downregulation of Erbb3 protein in AVC whole cell lysates by western blotting. Each sample contained AVCs pooled from 10 hearts. Expression was quantitated by densitometry and normalized to Gapdh. (D) Adenoviral expression of Gata4 upregulated Erbb3 in HUVEC cells, compared with GFP expression from a control virus. Erbb3 expression was measured by qRT-PCR and normalized to Gapdh. The graph shows the average of three independent experiments. (E) BT20 cells were co-transfected with an EGFP expression vector and either Gata4DBD-engrailed or an empty expression vector. After sorting for transfected (GFP-expressing) cells, Erbb3 expression was measured by qRT-PCR and normalized to Gapdh. Results are representative of two independent experiments. (F) Constructs containing Erbb3 promoter (-1356 to +214) and intron 1 enhancer (+104 to +3076) sequences driving luciferase were cotransfected with Gata4 or Gata4^ex2 expression constructs into BT20 cells. Luciferase activity was normalized to pRL-null. Results are the average of five (enhancer) or eight (promoter) independent experiments (^*P<0.05).