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. 2017 Apr 21;8:692. doi: 10.3389/fmicb.2017.00692

FIGURE 2.

FIGURE 2

Mapping motif affecting PB1-F2 half-life. (A) Individual expression plasmid for FLAG-tagged PB1-F2 of HK156 and PR8, as well as for chimeric PB1-F2 (HP1), PB1-F2 (HP2) and PB1-F2 (HP3) was transfected into HEK293T cells for 16 h before cycloheximide (50 μg/ml) was added. Cells were harvested at 0, 1, 2, 4, and 8 h post-cycloheximide treatment. Western analysis was carried out using anti-FLAG antibody. β-actin served as a loading control. (B) Plasmids expressing FLAG-HK156-ILVF and FLAG-PR8-TQDS were transfected into HEK293T cells. Cycloheximide chasing assay and Western analysis were performed as in (A). The shorter and longer exposures were both shown. V, vector control. (C) An amino acid sequence alignment between the last third of PB1-F2 (HK156) and PB1-F2 (PR8) was shown in left panel. Regions of PB1-F2 (PR8) and PB1-F2 (HK156) contained in chimeric PB1-F2, as well as sequence of amino acid 68–71 presenting in wild-type and mutant PB1-F2 were shown in right panel.