Gene silencing was performed in PNTs and H727 cells using species-specific human FLNA pre-designed siRNA (#4392420, Invitrogen) and Rap1 siRNA (#sc-36384 Santa Cruz), using Lipofectamine 2000 transfection reagent (#11668-019, Invitrogen) according to the manufacturer's instruction, for 72h and 48h of incubation, respectively.
In order to obtain the best efficiency of FLNA silencing three different human FLNA silencer select pre-designed siRNAs were tested.
Preliminary experiments to determine the optimal concentration of siRNAs and the kinetics of silencing of FLNA and Rap1 were performed. A negative control siRNA (C- siRNA) (AM4611, Invitrogen), a non-targeting sequence without significant homology to the sequence of human, mouse or rat transcripts, was used in each experiment. Western blotting was performed in each experiment to control the expression level of FLNA and Rap1 in silenced cells.
In order to obtain the best efficiency of FLNA silencing three different human FLNA silencer select pre-designed siRNAs were tested.
Preliminary experiments to determine the optimal concentration of siRNAs and the kinetics of silencing of FLNA and Rap1 were performed. A negative control siRNA (C- siRNA) (AM4611, Invitrogen), a non-targeting sequence without significant homology to the sequence of human, mouse or rat transcripts, was used in each experiment. Western blotting was performed in each experiment to control the expression level of FLNA and Rap1 in silenced cells.