Fig. 7. Molecular analysis of embryonic Carp-Lbh transgenic hearts. (A-H) In situ hybridization showing downregulation of Anf mRNA (white arrow) in Carp-Lbh transgenic hearts. Serial sections of E9.5 wild-type (wt; A,C,E,G) and transgenic (Tg; B,D,F,H) hearts were hybridized with radioactive probes for Nkx2.5 (A,B), Tbx5 (C,D), Gata4 (E,F) and Anf (G,H). (I,J) Lbh inhibits activation of Anf-human growth hormone (Anf-hGH) reporter by cardiac transcription factors in NIH 3T3 cells. (I) Dose-dependent suppression of Anf activation by Nkx2.5, Tbx5 and Gata4 with increasing amounts of Lbh expression plasmid. Values are expressed as percentage of total Anf-hGH reporter transactivation by Nkx2.5, Tbx5 or Gata4 respectively. *P<0.001 (n=3) versus Nkx2.5; **P<0.001 (n=3) versus Tbx5; ***P<0.05 (n=3) versus Gata4. (J) Inhibitory effect of Lbh on synergistic transactivation of Anf by Nkx2.5 and Tbx5. *P=0.008 (n=4) versus Nkx2.5 + Tbx5.