TbAGO1 is required for RNAi in transient transfection assays. A. Wild-type trypanosomes were first electroporated with dsRNA corresponding to the indicated genes, returned to culture for 10 h and then transfected with PFRA dsRNA. Samples were fixed 15 h later and cells exhibiting PFRA silencing were identified by immunofluorescence using an anti-PFRA specific monoclonal antibody. The experiment was repeated six times, yielding similar results. B. FACS analysis of trypanosomes transformed with plasmid pGFPTbAGO1430 grown in the absence (non-induced; thin green line) or in the presence of tetracycline (induced; thick lines). Induced cells were not transfected (thick green line) or transfected with TbAGO1 dsRNA (thick red line) or with TbGRP1 dsRNA as negative control (thick blue line). GFP::TbAGO1 protein levels were monitored by FACS analysis. Only TbAGO1 dsRNA was able to reduce GFP::TbAGO1 fluorescent protein levels (notice shift of thick red line compared to blue or green thick lines). Cells were transfected with the PFRA dsRNA 10 h later and PFRA silencing was monitored as above (inset). This experiment was carried out twice and yielded similar results.