Fig. 3.
Canonical Hh signaling in cardiomyocytes requires Gi protein activation. A: expression of the hemagglutinin (HA)-tagged GiCT peptide in NRVMs transduced with AdV-GiCT or control AdV-LacZ (multiplicity of infection: 100). Representative Western blots of HA and β-actin (loading control) are shown. B: NRVMs were cultured in the presence of the indicated adenoviral particles at a multiplicity of infection of 100 for 24 h in complete medium and then switched to low serum medium supplemented with 2.5 mg/l Shh or vehicle. After 24 h, total RNA was isolated and processed for quantitative RT-PCR of Gli1 and the housekeeping gene Pol2. Results are means ± SE of the normalized ratio of Gli1 to S15 from 3 independent experiments; n = 3. *P < 0.05. C: quantitation of intracellular cAMP levels in NRVMs transduced with control AdV-LacZ or AdV-GiCT stimulated for 5 min with vehicle or 10 μmol/L isoproterenol (Iso) in combination with 2.5 mg/l Shh or 5 μmol/l PUR with or without the smoothened inhibitor KAAD-CP (0.5 μmol/l). Results are means ± SE of 3 independent experiments calculated against a standard curve; n = 3. *Vehicle vs. Iso, P < 0.001; *Iso vs. Iso + Shh or Iso + PUR, P < 0.05. NS, not significant.