Exportin-5 knockdown leads to reduced silencing efficiency

Copyright information:Taken from " fluorescence analysis demonstrates active siRNA exclusion from the nucleus by Exportin 5"Nucleic Acids Research 2006;34(5):1369-1380.Published online 6 Mar 2006PMCID:PMC1390680.© The Author 2006. Published by Oxford University Press. All rights reserved () An outline of the experiment is illustrated. First the cells were transfected with different amounts of siExp5-1 and NegsiRNA and were incubated for 48 h. Then the cells were transfected with the dual luciferase reporter plasmids pGL2-Control (Firefly luciferase) and pRL-TK (Renilla luciferase) together with 1 nM siGL2 or NegsiRNA as a control for 24 h. () Dual luciferase results were obtained 24 h after transfection of the pGL2-Control (Firefly luciferase) and pRL-TK (Renilla luciferase) reporter plasmids together either with siGL2 or the NegsiRNA. Results were grouped together for each siRNA concentration used for 48 h. The ratios of target to control luciferase were normalized to the NegsiRNA control transfected for 48 h and for the luciferase assay indicated in black. The transfection of first the NegsiRNA for 48 h followed by siGL2 is shown in light grey, whereas the siExp5-1 transfection for 48 h followed by NegsiRNA is shown in dark grey and the siExp5-1 followed by siGL2 in white. () After 48 h total protein extracts from siExp5-1 transfected cells were immunoblotted with antibodies against Exp5 and GAPDH-HRP was used as a loading control. The plotted data were averaged from four independent experiments ±SD.