FIGURE 2.
NS5B/CINP interaction. 293T cells were grown in 6-cm dishes and transfected with the pCMV/myc-CINP or pcDNA3.1/myc-His-3×FLAG-NS5B (genotype 1b) plasmids. Forty-eight hours later, the cells were harvested as described under “Experimental Procedures,” and the supernatant was divided into two equal parts and incubated with GST or GST-NS5BΔ21 beads (A) or GST or GST-CINP beads (B) for 2 h. Protein-bound glutathione-Sepharose 4B beads were washed and boiled for 5 min, and CINP and NS5B, respectively, were detected by Western blot using an anti-myc antibody (A) or an anti-FLAG antibody (B). C, Coomassie-stained GST and GST-fused proteins are shown. D, equal amounts of pCMV/myc-CINP and pcDNA3.1/myc-His-3×FLAG-NS5B plasmids were co-transfected into 293T cells grown in 10-cm dishes. Forty-eight hours post-transfection, protein complexes were isolated by anti-FLAG immunopurification, and co-precipitating proteins were detected by Western blot with an anti-myc antibody. E, cell lysates were immunoprecipitated by an anti-myc antibody, and the co-precipitated protein was identified with an anti-FLAG antibody. The same experiment was performed in Huh7 cells, and the results are shown in F. G, pCMV/myc-CINP was co-transfected with GFP- or GFP-NS5B-expressing plasmids into 293T cells. Forty-eight hours post-transfection, cells were fixed for immunofluorescence analysis. The green represents empty GFP vector or NS5B, and CINP was detected using an anti-myc primary antibody and goat anti-mouse Cy3 secondary antibody (red). Representative cells from the same field for each experimental group are shown. H is the quantification of G. A minimum of 200 GFP-positive cells per sample were counted, and the percentage of cells with cytoplasmic CINP in GFP-positive cells was calculated. Results represent the means of data from three independent experiments. Error bars indicate the S.D. IP, immunoprecipitation; IB, immunoblot.