Figure 1

Expression profile of RGNNV B1 protein in cytoplasm and identification of nuclear translocation in RGNNV infection in GF-1 cells. (a) Western blotting analysis of B1 protein expression in RGNNV-infected GF-1 cells at different time points. Lanes 1–3 show B1 protein expression at 0 h, 24 h and 48 hpi in RGNNV-infected GF-1 cells. Actin was used as an internal control. (b) Immunostaining with B1 antibody and DAPI to determine B1 protein localization in GF-1 cells infected with RGNNV (MOI = 1) at 24 h and 48 hpi. Panels a-d and m (enlarged image) show the normal control; panels e-h and n (enlarged image) show RGNNV-infected GF-1 cells at 24 hpi; panels i-l and o (enlarged image) show RGNNV-infected GF-1 cells at 48 h. Fixed cells were stained with anti-RGNNV protein B1 polyclonal antibody (1:500) and FITC-labeled anti-rabbit secondary antibody. The staining was concentrated in the cytoplasm and only minor staining was observed in the nucleus (panels g, h and enlarged image [n]) at 24 hr; expression was then mainly localized to the nucleus at the 48 hr incubation (panels k, l and enlarged image [o]; indicated by arrows). Samples were stained with DAPI. Panel b (normal control); panel f (RGNNV-infected cells at 24 h); panel j (RGNNV-infected cells at 48 h). Merged images are shown in panel d (normal control); panel h (RGNNV-infected cells at 24 h); and panel l (RGNNV-infected cells at 48 h). Scale bar = 10 μm. (c) Quantitative analysis of B1 protein localizations in cytoplasm or targeting into the nucleus by immunofluorescence staining with anti-B1 polyclonal antibody in RGNNV-infected GF-1 cells. The localizations of cytoplasm and nucleus targeting cells were determined in each sample by counting 200 cells. Each result is expressed the mean of 3 independent experiments ± SEM. The data were analyzed using either the paired or unpaired Student’s t-test, as appropriate. A value of p < 0.05 was considered a statistically significant difference between group mean values.