Duck enteritis virus UL21 is a late gene encoding a protein that interacts with pUL16

Fig. 1. Expression, identification, and purification of recombinant UL21. A: Expression analysis of the recombinant protein. B: Immunoreactivity analysis of the recombinant protein by WB. C: Purification analysis of the recombinant protein. D: DEV UL21 was recognized by a purified polyclonal antibody

Fig. 2. Genotype analysis of UL21 in DEV-infected cells. A: Transcriptional analysis of the DEV UL21 gene. The DEV UL21 gene expression data are presented as fold changes. The transcript levels of the DEV UL21 gene were normalized to those of a reference gene (β-actin). B: Comparison of transcriptional patterns between UL21 and the control genes UL54, UL13 and US2. C: The genotype of UL21 was authenticated with antiviral drug inhibition experiments. GCV represents DEV-infected cells treated with ganciclovir, and CHX represents DEV-infected cells treated with cycloheximide. (−) represents non-infected negative control cells, and (+) represents the positive control infected cells. The full image is shown in Additional file 1: Figure S1. D: DEV pUL21 expression. Proteins isolated from mock- or DEV-infected cells at different times were subjected to WB with anti-UL21 and anti-β-actin antibodies. E: A greyscale level analysis of UL21 protein expression at each time point compared with the level of β-actin protein expression

Fig. 3. Localization of DEV UL21 in infected and transfected DEFs. A: DEV-infected cells on coverslips were fixed. The samples were incubated successively with rabbit anti-UL21 IgG and goat anti-rabbit IgG conjugated with Alexa Fluor 594. We captured the images by fluorescence microscopy using a 40× objective. B: the DEFs were transfected with pUL21 to observe localization. The samples were incubated successively with rabbit anti-UL21 IgG and goat anti-rabbit IgG conjugated with Alexa Fluor 488. The images were captured under fluorescence microscopy using a 40× objective

Fig. 4. UL21 structural protein verification. Virions purified from DEF cells were separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against the UL21 protein and β-actin. Total mock-infected or infected cell lysates were also included as antibody controls

Fig. 5. Colocalization of pUL16 and pUL21. DEV-infected cells on coverslips were fixed at 60 hpi. Colocalization of pUL16 (green) and pUL21 (red) in DEFs was assessed. The cell nuclei were stained with DAPI, and the images were captured under fluorescence microscopy using a 20× objective

Fig. 6. Colocalization of pUL16 and pUL21 in HEK 293 T cells. A: Localization of pUL21 (red) and pUL16 (green) alone with TGN46 (green) and GRP78 BiP (red) as cell markers in HEK 293 T cells. Images were captured under fluorescence microscopy using a 40× objective. B: Colocalization of pUL16 and pUL21 in HEK 293 T cells with pCAGGS-UL21-Flag (red) and pCMV-Myc-UL16 (green). Images were captured under fluorescence microscopy using a 40× objective

Fig. 7. Interaction between pUL16 and pUL21. A: DEFs were infected with DEV. The anti-UL16 and anti-UL21 polyclonal antibodies were used for IP, and the anti-UL21 and anti-UL16 polyclonal antibodies were used for WB. B: HEK 293 T cells were transfected with UL21 and UL16 expression plasmids. Myc and Flag monoclonal antibodies were used for IP, and Flag and Myc polyclonal antibodies for WB