FIG 3.

The half-life of PA-X varies among influenza A virus strains. Protein and RNA samples were collected from HEK293T cells expressing catalytically inactive (D108A mutant) C-terminal myc-tagged PA-X from the indicated strains at the indicated time points after treatment with 200 μg/ml of cycloheximide (CHX) to inhibit translation. (−) denotes vehicle-treated cells collected at 120 minutes (A to F) or 6 hours (G to L). (A, D, G, J) Representative Western blots using anti-myc antibodies to detect myc-tagged PA-X and tubulin as a loading control. (B, E, H, K) Plots of the relative levels of PA-X after cycloheximide treatment. For each replicate, PA-X protein levels were normalized to tubulin protein levels and reported as the log₂-transformed ratio from time zero. The line represents the linear regression, and the shaded region its 95% confidence interval. n ≥ 3 for each variant. The half-life (t_1/2) is calculated as the negative inverse of the slope and reported in minutes in the figure and/or in Table 1. For (K), the P value is calculated relative to PR8 PA-X (PR8 PA-X: slope = −0.64, confidence interval = −0.75 to −0.53; pH1N1 PA-X: slope = −0.29, confidence interval = −0.36 to −0.22). (C, F, I, L) Levels of PA-X mRNA were measured by RT-qPCR. After normalization to 18S rRNA, levels were plotted as amounts relative to time zero. n ≥ 3.