Figure 5

cAMP and its analogues modulate mRNA and protein expression. HUVEC were stimulated with cAMP or its derivates for up to 72 h and changes in transcript amount and protein expression were determined by real-time qPCR and western blotting. (A) real-time qPCR revealed a strong induction of CLDN5 and OCLN mRNA synthesis after stimulation with pCPT-cAMP and 8-Br-cAMP/Na over the whole time period whereas cAMP and 8-Br-cAMP induced only a temporal increase in CLDN5 transcript amount and obviously had no effect on OCLN mRNA expression. Untreated cells showed no differences in CLDN5 expression but a significant increase in OCLN transcript amount after 48 h. (n = 3). (B) Shown are western blots of HUVEC treated for 24 to 72 h with different cAMP derivates. Protein lysates of HUVEC were separated by electrophoresis, blotted and OCLN and CLDN5 were detected with specific antibodies. Protein amounts of tubulin served as loading controls. (n = 5); (*p < 0.01 compared to untreated controls).