Quantifying Virus Infectivity and Protein Expression

The culture supernatant harvested at 48 hours post-transfection was centrifuged to remove cells and produce virus suspensions. The infectivity of virus suspensions was measured by TZM-bl assay as previously described25 (link). Briefly, 100 μl of the virus solution was inoculated into TZM-bl cells in 96-well plate (Nunc), and the β-galactosidase activity was measured by using the Galacto-Star mammalian reporter gene assay system (Applied Biosystems) and a 2030 ARVO X multilabel counter instrument (PerkinElmer) according to the manufacturers’ procedure. Western blotting was performed as previously described25 (link) by using the following antibodies: anti-p24 polyclonal antibody (ViroStat), anti-KGC antibody (clone 21B10; Medical and Biological Laboratories, Inc.), anti-HA antibody (3F10; Roche), and anti-alpha-Tubulin (TUBA) antibody (DM1A; Sigma).

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Takeuchi J.S., Ren F., Yoshikawa R., Yamada E., Nakano Y., Kobayashi T., Matsuda K., Izumi T., Misawa N., Shintaku Y., Wetzel K.S., Collman R.G., Tanaka H., Hirsch V.M., Koyanagi Y, & Sato K. (2015). Coevolutionary dynamics between tribe Cercopithecini tetherins and their lentiviruses. Scientific Reports, 5, 16021.

Publication 2015

96-well plate Galacto-Star mammalian reporter gene assay system 2030 ARVO X multilabel counter instrument anti-HA antibody (3F10; anti-alpha-Tubulin (TUBA) antibody (DM1A;

Alpha tubulin Anti antibody Antibodies Assay Biological Cells Clone Mammalian Reporter gene Transfection Virus Virus infectivity β galactosidase

Corresponding Organization :

Other organizations : Kyoto University, Tokyo Medical and Dental University, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Tokushima University, Japan Wildlife Research Center, University of Pennsylvania, Japan Science and Technology Agency

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Variable analysis

independent variables

Time point of virus suspension harvest (48 hours post-transfection)

dependent variables

Infectivity of virus suspensions measured by TZM-bl assay
Protein expression levels measured by Western blotting

control variables

Cell line used (TZM-bl cells)
Assay methods (Galacto-Star mammalian reporter gene assay system, 2030 ARVO X multilabel counter instrument)
Antibodies used for Western blotting (anti-p24, anti-KGC, anti-HA, anti-alpha-Tubulin)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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