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. 2012 Nov 2;7(11):e43596. doi: 10.1371/journal.pone.0043596

Figure 1. The efficiency of H5pp production does not correlate with protein expression level in producer cells.

Figure 1

HEK293T cells were transfected with a lentiviral vector containing gag/pol/luciferase reporter gene, and a plasmid coding for H5-HA from different clades: A/Indonesia/5/2005 (Ind); A/Bar-headed goose/Qinghai/60/2005 (Qin); A/Anhui/1/2005/01 (Anh); A/Cambodia/408008/2005 (Cam). Bacterial neuraminidase from Vibrio cholerae (NAvb) was added 16 hr post transfection where indicated. Supernatant containing H5pp was harvested at 48 hr and used to transduce MDCK target cells. (A) HA protein expression in cell lysate was analysed using anti-FLAG antibody (upper panel). Cyclophilin B antibody was used as the loading control. Luciferase activity in target MDCK cells was measured 72 hr post transduction (lower panel). Results are shown as means ± SD (n = 4 independent experiments); *p<0.01 compared with H5Cam by the unpaired Student's t-test. (B) H5Anh-pp and H5Cam-pp were concentrated by ultracentrifugation. Incorporation of HA into H5pp was determined by western blotting of H5pp pellets using anti-FLAG antibody, as described under Materials and Methods (upper panel). Luciferase activity in MDCK cells was measured 72 hr post transduction (lower panel) and results are shown as means ± SD (n = 4 independent experiments); *p<0.01 by the unpaired Student's t-test.