FIG 2.

Confirmation of the NS1-GAPDH interaction by direct binding ELISA and a pulldown assay. (A) Microtiter plates were coated with purified GAPDH (10 μg/ml), and increasing amounts of recombinant DENV2 NS1 were added. The bound NS1 was detected using an anti-NS1 polyclonal antibody. BSA was used as a negative control. The error bars indicate the standard deviations from three independent experiments, and the asterisks indicate significant differences from the control using two-way ANOVA and the Bonferroni posttest. ***, P < 0.001. (B) For the pulldown assay, the rNS1 and GAPDH proteins at a concentration of 2.5 μM each were incubated overnight at 4°C with anti-NS1 polyclonal antibody covalently coupled to an amino-linked agarose resin. Elution was carried out with the elution buffer provided in the co-IP kit (Pierce). The control reaction was carried out following the same protocol described above, except that only GAPDH was incubated with the resin. The same procedure was performed using control IgG coupled to an amino-linked agarose resin. (C) As described in the legend to panel B, except that the GAPDH protein was replaced by the HPX protein, which does not interact with NS1. (D) Pulldown assay using extracts of mock- and DENV2-infected BHK-21 cells. IN, input; FT, flowthrough; E1 and E2, 1st and 2nd elution fractions, respectively; C+, positive control (rNS1 and GAPDH). The results presented here are representative of those from three independent experiments.