Expression of NS1 protein rescues gene expression defect observed with mutant del20NLS-NP at protein level. 293 T cells were transfected with plasmid mixes to express recombinant vRNPs containing influenza RdRP with either WT-NP, del20NLS-NP, or no NP, and FLAG-M vRNA as template in the presence or absence of NS1 and NS2 or NS1 only as indicated. A. Total protein was separated by SDS-PAGE and analyzed by Western blotting with anti-FLAG antibody to visualize FLAG-M products. Anti-tubulin detects tubulin and serves as a loading control. B. Total RNA was isolated, DNase treated, and 1 μg subject to reverse transcription with oligo dT and PCR for 23 cycles with either M or NS gene specific primers. Asterisk represents aberrant product observed with FLAG-M vRNA templates. Shown are representative experiments, repeated in triplicate.