Figure 11

Impact of Epac silencing on Rap1 activation and bradykinin-induced IL-8 release. hTERT-airway smooth muscle cells were transfected for 72 hrs with control siRNA, Epac1 or Epac2 specific siRNAs (each 200 pmol). Thereafter, expression of membrane-associated Epac1 or cytosolic Epac2 was evaluated and normalized to the content of the cell fraction-specific marker proteins caveolin-1 and β-actin, respectively. Representative immunoblots are shown on the left with the respective densitometric quantifications on the right. Results are expressed as mean ± SEM of separate experiments (n = 5-7). Transfected cells were treated with 100 μM 8-pCPT-2'-O-Me-cAMP (8-pCPT) or 500 μM 6-Bnz-cAMP for 5 min and the amount of GTP-Rap1 was determined as described in Material and Methods. Shown is a representative immunoblot. In C, transfected cells were incubated with 10 μM bradykinin alone or in combination with 100 μM 8-pCPT-2'-O-Me-cAMP (8-pCPT) or 500 μM 6-Bnz-cAMP for 18 hrs. IL-8 release was then assessed by ELISA. Results are expressed as mean ± SEM of separate experiments (n = 3-7). **P < 0.01, ***P < 0.001 compared to unstimulated control; ^§ P < 0.05, ^§§ P < 0.01 compared to control siRNA.