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. 2014 Dec 3;4:7242. doi: 10.1038/srep07242

Figure 4. CH25H interacts with HCV NS5A.

Figure 4

A: NS5A interacts with CH25H. Expression vectors for Flag-tagged NS5A and HA-tagged CH25H were transfected into HEK293T cells. After 36 h, the cells were harvested and lysed, and then the whole cell extracts were subjected to Co-IP assay using an anti-Flag M2-agarose. B: Colocalization of CH25H with NS5A. HEK293T cells were co-transfected with Flag-tagged NS5A and HA-tagged CH25H. After 36 h, the cells were fixed and stained with anti-Flag or anti-HA antibody, followed by TRITC- or FITC-conjugated anti-mouse or anti-rabbit secondary antibody, then analyzed by confocal microscopy and the Pearson's Coefficient is 0.86473. C: CH25H and CH25H-M interact with domain I of NS5A. Expression plasmids for HA-tagged CH25H or CH25H-M were co-transfected with Flag-tagged NS5A truncation mutant constructs. Co-IP assays were performed using cell lysates from cells expressing the indicated constructs. D: Both of TM and FA domains of CH25H interact with NS5A. Expression plasmids for HA-tagged CH25H TM or FA domain were co-transfected with Flag-tagged NS5A. Co-IP assays were done as in C. All results were confirmed by three independent experiments. All the gels were run under the same experimental conditions as detailed in the Methods section, and full-length blots were cropped for final display. Full-length blots are included in the supplementary information.