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. 2011 May 31;286(30):26603–26615. doi: 10.1074/jbc.M111.225672

FIGURE 4.

FIGURE 4.

Analysis of NS5B/CINP binding regions. A, schematic diagram of CINP predicted by SMART software. The green bar represents the coiled coil region (amino acids 72–108), and gray bars represent unknown regions. B, 293T cells were grown in 10-cm dishes and transfected with FLAG-NS5B-expressing plasmids. The GST pulldown assay was carried out using purified beads that contained GST, GST-CINP full length, GST-CINP (aa 1–107), and GST-CINP (aa 108–212). Precipitated NS5B was detected by Western blot using an anti-FLAG antibody. C, Coomassie-stained GST and GST-fused proteins are shown. D, deletion mutants of myc-tagged NS5B were transiently transfected into 293T cells and detected by Western blot (left panel). Proteins pulled down by GST-CINP were examined by Western blot with an anti-myc antibody (right panel). E, after induction by isopropyl β-d-thiogalactopyranoside, pET28bHCR6NS5BΔ21 and its deletion mutants (Δ(84–95), Δ(455–464), and Δ(84–95)&(455–464)) were examined by Western blot with anti-His antibody. F, approximately 0.2 μg of His-NS5BΔ21 and mutants were mixed with purified GST- or GST-CINP-containing beads. After washing with PBS with Tween 20, each bound protein was fractionated by SDS-10% PAGE and subjected to Western blot analysis with anti-His monoclonal antibody. IB, immunoblot.