Article Title: The alphaherpesvirus conserved pUS10 is important for natural infection and its expression is regulated by the conserved Herpesviridae protein kinase (CHPK)
Figure Lengend Snippet: Replication of rMDVs in tissue culture cells and expression of pCHPK and pUS10. (A) Plaque areas were measured for rMDVs reconstituted from each parental virus (vCHPKwt, vCHPKmut, vΔCHPK) and HA-tagged US10 virus (vCHPKwt/10HA, vCHPKmut/10HA, vΔCHPK/10HA). There were no significant differences between all groups using One-way ANOVA ( P = 0.71, n = 300) and Levene’s Test ( P = 0.84, n = 300). (B) Representative plaques induced by each virus stained with anti-MDV, -Flag, and -HA antibodies. Included in each image is Hoechst 33342 staining of nuclei for reference. (C) Confocal microscopy of vCHPKwt/10HA-, vCHPKmut/10HA-, or vΔCHPK/10HA-infected CECS. Cells were stained for Flag (CHPK) and HA (US10). (D) Immunoblotting of total protein collected from mock-infected CECs or CECs infected with vCHPKwt/10HA, vCHPKmut/10HA, or vΔCHPK/10HA. To examine CHPK and US10 expression, proteins were electrophoresed under non-reducing conditions and blotted with anti-Flag (CHPK) and -HA (US10) antibodies. Protein was also run under reducing conditions (*) and blotted with anti-β-actin and -MDV pp38 as protein loading and infection level controls, respectively. (E) Confocal images of CECs infected with vCHPKwt/10HA were used to quantify the colocalization of pCHPK and pUS10. A representative image of a plaque stained with anti-Flag (pCHPKwt) and -HA (pUS10) and merged. Hoechst was used to identify cell nuclei. A single z-section with linear ROIs labeled and the profile plot for ROI-1, and its Pearson Correlation Coefficient R is shown. The average Pearson Correlation Coefficient R between pCHPKwt and pUS10 was 0.8859 ± 0.0638. (F) Uninfected CECs (Mock) or CECs infected with vCHPKwt/10HA were used for protein extraction. Total cell lysate was immunoblotted for pCHPKwt (Flag), pUS10 (HA), VP5 (non-specific control), and β-actin (protein loading control). Total lysate was immunoprecipitated with anti-Flag Fab or anti-HA combined with protein A/G-beads, electrophoresed on an SDS-PAGE gel, and then immunoblotted using anti-Flag, -HA, or VP5 antibodies.
Article Snippet: Anti-HA treated samples were then incubated with a pre-washed 50 μl slurry of protein A/G agarose beads (MilliporeSigma) for 2 h at 4°C.
Techniques: Expressing, Staining, Confocal Microscopy, Infection, Labeling, Protein Extraction, Immunoprecipitation, SDS Page