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. 2015 Jan 8;11(1):e1004573. doi: 10.1371/journal.ppat.1004573

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© 2015 Poenisch et al

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A) Immunoprecipitation of HA-HNRNPK. Huh7.5 cells stably expressing unaltered, HA-tagged HNRNPK, or HA-tagged mutants ΔKH1, ΔKH2 or ΔKI were electroporated with genomic Jc1 (HCV) RNA. Genomic Dengue Virus (DENV) RNA was used as a control. Cells were harvested 48 h post electroporation, lysates were used for HA-specific immunoprecipitation and captured complexes were analyzed by Western blot. Input was analyzed with a HNRNPK and a HA-specific antibody (upper and middle panel, respectively); GAPDH served as loading control. Immunocomplexes (corresponding to 10-fold the amount of the input) were analyzed with the HA-specific antibody (lower panel). Note that the ΔKH1 mutant is not recognized by the HNRNPK-specific antibody, but reacted with the HA-specific antibody. (B) Quantitative analysis of co-precipitated HCV and DENV RNA. Co-precipitated viral RNA was determined by RT-qPCR and normalized to input RNA. Fold enrichment of captured viral RNA was calculated by comparing the HA-tagged to the untagged HNRNPK-fraction (set to 1; dotted horizontal line). Bars represent the mean ±SD of at least three independent experiments.