Mutant del20NLS-NP is severely defective for gene expression from M vRNA but less so from NS vRNA. 293 T cells were transfected with plasmid mixes to express reconstituted vRNPs containing influenza RdRP with either WT-NP, del20NLS-NP, or no NP, and either FLAG-NS vRNA or FLAG-M vRNA template, as indicated. A. Total protein was separated by SDS-PAGE and analyzed by Western blot. Reconstituted vRNP with WT-NP but no PB1 protein serves as an additional negative control. Anti-FLAG antibody detects NP-FLAG and FLAG-NS1, FLAG- NS2, FLAG-M1, and FLAG-M2 proteins. Tubulin serves as loading control. Shown is a representative experiment, repeated more than three times. B. Total RNA was isolated, DNase treated, and 1 μg subject to reverse transcription with oligo dT and PCR with gene specific primers for M and NS as indicated. Asterisks represents aberrant product observed with FLAG sequence containing templates. Shown is a representative experiment, repeated in triplicate.