Figure 7

PKA inhibitors H-89 and Rp-cAMPS block phosphorylation of CREB. After a 30 min pre-incubation with serum-free DMEM alone or with 100 nM of H-89 or 10 μM of Rp-cAMPS, UROtsa cells were stimulated with 100 nM isoproterenol in serum-free DMEM for the indicated times and immunoblotted with an antibody specific to CREB phosphorylated at S129 and S133 (pCREB). Without inhibitor pretreatment, isoproterenol induced CREB phosphorylation within 5 min. Significant phosphorylation was still observed 15 min. after drug addition with banding intensity patterns similar to basal after 60 min. Preincubation with H-89 or Rp-cAMPS significantly decreased the phosphorylation of CREB at the 5 and 15 min time points in UROtsa cells, demonstrating the effectiveness of PKA inhibition for these compounds. As reported by the antibody suppliers, pCREB is identified as a band running at 43 kD. The second band seen on the autoradiographs represents a previously-reported alternative splice variant of CREB. Autoradiographs are representative immunoblots of n = 3 independent cell treatments.