Attenuation of Gli1 Expression in Primary Tumorspheres by PACAP was Associated with PKA Activation and was Reversed by the PKA Inhibitor H89. (A) Western blot analysis of PKA activity in tumorsphere cultures treated with vehicle, 10 nM PACAP, 5 μM forskolin, 30 μM H89, 10 nM PACAP plus 30 μM H89 or 5 μM forskolin plus 30 μM H89. Blot was incubated with a rabbit phospho-PKA-substrate antibody (top) or a mouse β-actin antibody (bottom). The blot shown is representative of 3 experiments. (B) Gli1 gene expression in primary tumorspheres treated with either vehicle, 10 nM PACAP, 30 μM H89, or the combination of PACAP and H89. Experiments were performed at least 3 times and with at least n = 3 for each treatment (***P < 0.001).