Fig 3. Nuclear localization is required for NS5-PAF1C interaction.
(A) NLS mutants, including single mutant NS5central (REE396-398AAA) and NS5C-term (RR890-891AA) and the double mutant NS52xNLS (REE396-398AAA and RR890-891AA). (B) Subcellular localization of 2xStrep II tagged NS5s (yellow) and colocalization with PAF1 (cyan) was determined by immunostaining and confocal microscopy. Nuclei were stained with Hoechst (magenta). Scale bar represents 10 μm. (C) The protein interaction between NS52xNLS and PAF1 was tested biochemically. Plasmids encoding NS5s and GFP were transfected and affinity purified in HEK293T cells via a 2xStrep II tag. Immunoblot analysis of lysate and purified (Strep-AP) fractions was performed against PAF1, Strep and GAPDH (loading/negative control). Abbreviations: Strep-affinity purified (Strep-AP).