Mutant del20NLS-NP defect is not specific to the M segment. 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 either HA vRNA or PB1-GluGlu vRNA template, as indicated. A. Total protein was separated by SDS-PAGE and analyzed by Western blot. Anti-FLAG antibody detects NP FLAG, anti-HA detects HA, anti-GluGlu detects PB1-GluGlu, and anti-tubulin detects tubulin and serves as loading control. B. Total RNA was isolated, DNase treated, and 1 μg subject to reverse transcription with oligo dT and PCR with gene specific primers for HA and PB1-GluGlu; pHH21-HA and pHH21 PB1-GluGlu DNA plasmids serve as positive controls while pcDNA PB1 untagged DNA plasmid serves as negative control, in addition to no RT. Shown is a representative experiment, repeated in triplicate.