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

FIGURE 3.

FIGURE 3

Sensitivity to MG132 of PB1-F2 variants. (A) Individual expression plasmid for FLAG-tagged PB1-F2 of HK156 and PR8, as well as for chimeric PB1-F2 (HP3) and PB1-F2 (HP1), was transfected into HEK293T cells for 6 h. MG132 (5 μM) was added for 16 h before cells were harvested for Western analysis using anti-FLAG antibody. (B) Individual expression plasmid for EGFP-tagged PB1-F2 of HK156 and PR8, as well as for mutants HK156-ILVF and PB1-F2-TQDS was transfected into HEK293T cells for 8 h. Cells with or without MG132 (5 μM) treatment were harvested 16 h later. Western analysis was carried out using anti-EGFP antibody. GAPDH served as loading controls. Chemiluminescence intensity for bands specific to EGFP-PB1-F2 and GAPDH were captured and measured. Signals with (+) or without MG132 (–) treatment were compared after normalized with GAPDH. Intensity of EGFP-PB1-F2 (MG132 (–)) after normalized with GAPDH was set as 1×. Constructs of chimeric PB1-F2 and sequence swapping mutants used in the experiments were shown.