Simultaneous knockdown of AKAP12 and AKAP220 mRNA impaired cAMP-mediated Rac1 activation.

<p>(A) Subconfluent monolayers of MyEnd cells were transfected either with single AKAP siRNA or with a pool of AKAP12 and AKAP220 specific siRNAs. Basal Rac1 activity was tested 48 hours after initial siRNA depletion. For evaluation of cAMP-mediated Rac1 activation, transfected cells were subjected to F/R for 1 hour. Non-target siRNA (n.t. siRNA) alone or in combination with F/R (1 hour) was used as a respective control. Bars represent mean values ± SEM. Basal activity of Rac1 was unaffected after single or combined silencing of AKAPs. In contrast, cAMP-mediated Rac1 activation was significantly impaired following simultaneous AKAP12 and AKAP220 mRNA knockdown. (B) Successful knockdown was verified by Western blot 48 hours after initial siRNA transfection; ß-actin was used as an internal gel-loading control. ** indicates p≤0.01 versus cells transfected with n.t. siRNA treated for an hour with F/R.