FIG 10.

LANA isoforms migrating with high and low relative molecular masses are isolated from the cytosolic fraction of KSHV-infected Vero cells treated with BacK50. Vero cells undergoing a primary KSHV infection (24 hpi) (A to C) and puromycin-selected AGS.219 cells undergoing a long-term latent infection (D) were superinfected with BacK50, as described in Fig. 7. The ProteoExtract fractionation system was used to purify subcellular fractions from the cytosol (lanes I), membrane/organelles (lanes II), nucleus (lanes III), and cytoskeleton (lanes IV) of the infected cells. These fractions were screened by Western blotting for LANA isoforms (A and D) and nuclear lamin β1 (C), as described in Materials and Methods. Dots indicate multiple LANA isoforms ranging from 118- to 230-kDa relative molecular masses in the cytosol fraction I of the induced Vero cells (A) and in the nuclear and cytoskeleton fractions III and IV of the latent AGS.219 cells (D). An asterisk indicates nuclear lamin β1 isoforms in the nuclear and cytoskeleton fractions III and IV of the induced Vero cells (C). (B) Proteins in the cytosol fraction I of the infected Vero cells were further purified on a LANA affinity column for mass spectrometry analysis and screened by Western blotting for LANA isoforms. M, molecular mass standards.