Fig 4.

Nature of p14. (A) Effect of 8 M urea on p14. Huh-7 cells, transfected with either a mock plasmid or a plasmid encoding NS4A, or Huh-9-13 replicon cells were treated with ACH-806 (2 μM) overnight. Transfected Huh-7 cells (top panel) were lysed with sample buffer or sample buffer plus 8 M urea. For replicon cells (bottom panel), RCs were isolated and solubilized with sample buffer or sample buffer plus 8 M urea. All samples were subjected to Western blot analyses with anti-NS4A antibody. Molecular masses are indicated on the right in kilodaltons. (B) Dimeric form of NS4A. Two plasmids, one encoding NS4A and the other encoding NS4A-HA, were transfected alone or together in 1:1 ratio to Huh-7 cells. After overnight treatment with ACH-806 (2 μM), cells were lysed, and lysates were subjected to immunoprecipitation with anti-NS4A antibody, followed by a Western blot analysis with anti-NS4A antibody. The diagram above the Western blot illustrates the three expected dimer species if NS4A and NS4A-HA can form dimers. NS4A, open box; NS4A-HA, open box with a shaded box at its C terminus representing the HA tag; species a, NS4A homo-dimer; species b, NS4A and NS4A-HA heterodimer; species c, NS4A-HA homodimer. The asterisk on the Western blot indicates species b in the cotransfected cells. Molecular masses are indicated on the right in kilodaltons.