FIG. 6.

Immunodetection of NS proteins. (A) Detection by Western blot of purified MBP-NS-3 fusion protein (lane 2) or NS-3 protein alone after digestion with factor Xa (lane 3) by using a 1/250 dilution of the anti-NS-3 polyclonal antiserum prepared against a 17-mer synthetic oligopeptide as described in Materials and Methods. Lane 1, MBP control protein. (B) Dilutions of MBP-NS-3 fusion protein used to evaluate the strength of the antiserum. (C) Immunodetection of NS-3 in pBRJ-transfected Ld 652 cells. At various times (in hours) posttransfection, cells were labeled for 3 h with [³⁵S]methionine-[³⁵S]cysteine, and then NS-3 was immunoprecipitated by using 50 μl of anti-NS-3 antiserum and protein A, as described in Materials and Methods. The relative amounts of NS-3 were estimated by using the UTHSCSA ImageTool program. (D and E) Comparative analysis by Western blot of NS-1 and NS-2 expression in mock-transfected (lanes 1), pBRJ-transfected (lanes 2), and pJNS3ΔNsiI-transfected Ld 652 cells (lane 3). Three days posttransfection, cells were harvested, pelleted, and treated for analysis by SDS-PAGE (12% gel). Proteins were transferred onto nitrocellulose membranes, and NS-1 and NS-2 were revealed by using specific anti-NS-1 (D) and anti-NS-2 (E) antibodies.