FIG. 1.

Generation and testing of viral protein and NF45-specific antisera. (A) To test the specificity of generated viral protein, DF-1 cells were either infected with the IBDV strain D78 (2) or left uninfected (1). The obtained cellular lysates were subjected to Western blot analysis. The membranes were incubated with either already-established rabbit sera (anti-VP1 R [3] anti-IBDV R [30]) or the newly established chicken anti-VP1 serum (anti-VP1 ch) and a rabbit anti-VP3 serum (anti-VP3 R). The locations of the IBDV proteins (VP) are indicated by arrows. (B) For monitoring the purification of ckNF45-His, samples were taken at different stages (L, lysate; S, supernatant after centrifugation; E, eluate) and subjected to SDS-PAGE. The gel was stained with Imperial protein stain. The eluate was monitored by using an HRP-conjugated anti-His monoclonal antibody and the rabbit ckNF45-His serum. The reactivity of the rabbit anti-ckNF45His serum was tested by Western blotting of cellular lysates from different species, namely, chicken (DF-1 and CEC), quail (QM-7), dog (MDCK), monkey (Vero), human (293T), and hamster (BHK 21). The molecular masses (in kDa) of protein markers (M) are indicated on the left. (C) BHK21 cells were transfected with a mammalian vector expressing ckNF45-FLAG (see Materials and Methods). Fixed cells were incubated with rabbit anti-ckNF45-His antiserum (NF45) and a mouse anti-FLAG MAb (FLAG) followed by incubation with appropriate conjugates (goat anti-mouse-Cy3 or goat anti-rabbit-FITC). The cellular nuclei were visualized by PI staining. An overlay of the confocal pictures is shown (merge).