A poxvirus pseudokinase represses viral DNA replication via a pathway antagonized by its paralog kinase
Fig 7
B12 nuclear localization is distinct from chromatin bound proteins.
(A) CV1 cells were transfected with no mRNA, HA-GFP mRNA, or HA-B12 (GenScript) mRNA. Cells were either fixed then permeabilized to detect HA-tagged proteins or (B) prepermeabilized, fixed and then permeabilized again for detection of HA-tagged proteins remaining in cells following washes to remove unbound protein. The DAPI nuclear stain (blue) and αHA antibody (green) for detection of HA-GFP and HA-B12 were used. The scale bars in 7A and 7B represent 200μm. (C) Subcellular fractionation of CV1 control or HA-B12 (GeneArt) stably expressing cells was completed to separate cells into cytoplasmic extract (Cyto.), membrane extract (Memb.), soluble nuclear extract (Nuc.), chromatin-bound extract (Chrom.), and cytoskeleton extract (Cytoskel.). Lamin A/C, GAPDH and BAF protein detection were used as fractionation controls and HA was used to detect HA-tagged B12 protein.