Fig. 5

NS38 associates with and degrades N and P proteins. A NS38 localizes in the cytoplasm. EPC cells were plated onto coverslips in 6-well plates and transfected with indicated plasmids (1 μg each). After 24 h, the cells were fixed and subjected for confocal microscopy analysis. Scale bar, 5 μm. B, C NS38 associates with N and P proteins. EPC cells seeded in 10-cm² dishes overnight were transfected with the indicated plasmids (5 μg each). After 24 h, cell lysates were immunoprecipitated (IP) with anti-Myc/Flag affinity gels. Then, the immunoprecipitates and cell lysates were analyzed by immunoblotting (IB) with indicated antibodies (Abs). D–F NS38 degrades N and P proteins and barely affects G protein expression. EPC cells were plated onto coverslips in 6-well plates and transfected with indicated plasmids (1 μg each). After 24 h, the cells were fixed and subjected for confocal microscopy analysis. Green or red signals represent overexpressed N, P, and G proteins (original magnification 10×; non-immersion objective). Scale bar, 300 μm. The fluorescence intensity (arbitrary unit, a.u.) was recorded by the LAS X software, and the data were expressed as mean ± SD, n = 5. Statistical analysis was performed by the Student's t-test. Asterisks indicate significant differences from control (^∗P < 0.05). IB with indicated Abs was used to detect the expression level of N/P/G proteins. G–I NS38 degrades N and P proteins on a dose-dependent manner. EPC cells were seeded in 6-well plates and transfected with 1 μg N-Flag or P-Flag and 1 μg NS38-Myc (G) or 0.5, 1.0, or 1.5 μg NS38-Myc (H, I). At 24 h post-transfection, the cells were harvested for IB with the indicated Abs.