Figure 11. Cap-donor dose-response for in vitro transcription of recombinant RNPs.

Cultures of HEK293T cells were transfected with SFPQ/PSF-specific (S1) or control siRNAs (siCTRL) as described under Materials and Methods. When the expression of SFPQ/PSF was known to be down-regulated, the cultures were further transfected with plasmids expressing the polymerase subunits and the NP, as well as with a genomic plasmid expressing a deleted NS RNA segment (clone 23) [2] in negative polarity. Extracts of these cultures were used for in vitro transcription using increasing amounts of ß-globin mRNA as a cap donor and the transcripts were analysed by denaturing polyacrylamide gel electrophoresis. (A) Denaturing polyacrylamide gel electrophoresis of in vitro transcripts. M denotes a mock-reconstitution of recombinant RNPs in which pcDNA3 plasmid was transfected. The concentrations (µg/ml) of ß-globin mRNA used as cap-donor are indicated to the top. The mobility of a genome-size marker of 248 nt is indicated to the right. (B) The quantisation of the results presented in A is presented as percent of maximal value in each series. Blue line: Control-silenced extract. Red line: SFPQ/PSF-silenced extract. (C) The down-regulation of SFPQ/PSF by silencing was verified by Western-blot with specific antibodies. The level of expression of PA and NP was ascertained by Western-blot.