Rta relocalizes from the nucleoplasm to the cytoskeleton during replication of an LF2-positive EBV genome. (A) B95-8 cells that stably express Zta-HT and GFP or Flag-LF2 were induced for replication by treatment with 4HT for 24 or 72 h and fractionated into cytoplasm, membrane, nucleoplasm, chromatin-bound, and cytoskeleton fractions. Equal relative amounts of the nucleoplasm (N), chromatin-bound (Ch), and cytoskeleton (Cs) fractions were analyzed by Western blotting for Rta and LF2. Fraction purity was assessed by detection of nucleoplasmic and chromatin-associated BRG1 protein and the nuclear matrix/cytoskeleton component lamin B. (B) LF2 Western blot results with anti-LF2 polyclonal rabbit sera. B95-8 or P3HR1 cells that stably express Zta-HT (P3 or B95) were treated with 4HT for 72 h and lysed in SDS loading buffer. Detection of lamin B served as a loading control. (C) P3 and B95 cells were treated with 4HT for 24, 48, and 72 h and fractionated as described for panel A. Equal relative amounts of the N, Ch, and Cs fractions were analyzed by Western blotting for the indicated EBV proteins. Fraction purity was assessed by detection of the subcellular markers BRG1 and lamin B.