FIG 5.

PA-X aa 220 plays a role in protein turnover. (A) Alignment of the X-ORF of PA-X from PR8, Udorn, HK06, Perth, and pH1N1. The zoomed in region shows aa 17 to 41 of the X-ORF (PA-X aa 208 to 232). Residue 220 is boxed. “*,” same residue; “:,” strong similarity; “.,” weak similarity. (B to G) Protein and RNA samples were collected from HEK293T cells expressing the indicated catalytically inactive C-terminal myc-tagged PA-X variant 2 hours after treatment with 200 μg/ml of cycloheximide (CHX) to inhibit translation (+) or vehicle control (−). (B, E) Representative Western blot using anti-myc antibodies to detect myc-tagged PA-X with tubulin as a loading control. (C, F) For each replicate, PA-X protein levels were normalized to tubulin protein levels and reported as the amount normalized to vehicle control for each variant. ***, P < 0.001; ns, P > 0.05; 2-way ANOVA followed by Sidak’s multiple-comparison test. n ≥ 3. (D, G) Levels of PA-X mRNA were measured by RT-qPCR. After normalization to 18S rRNA, levels were plotted as amounts normalized to vehicle control-treated cells expressing the wt variant. n ≥ 3.