Fig. 6. LANA associates with Sel10. (A and B) Immunoprecipitation analysis with mouse anti-Myc antibody or rabbit anti-GFP antibody showed that LANA-myc was directly immunoprecipitated with Sel10-GFP in 293T (A) and DG75 (B) cells. Fifteen million of each cell type were cotransfected with 10 mg of Sel10-GFP and 10 mg of LANA-myc expression vectors or transfected with these vectors 24 h after transfection; cell lysates were used for IP analysis. IN, 5% total cell lysate input; PC, preclear; IP, immunoprecipitate. (C) Endogenously LANA interacts with Sel10 in KSHV-infected pleural effusion lymphoma cells, BCBL1, and BC3. Immunoprecipitation analysis with polyclonal rabbit Sel10 antibody showed that LANA was directly immunoprecipitated with LANA in BCBL1 and BC3 cells. For this experiment, 30 million cells were harvested for preparation of cell lysates, which were use for IP analysis. KSHV-negative BJAB cells were used as a control.

Fig. 7. Carboxyl-terminal LANA associated with Sel10 in vivo. Fifteen million 293T cells were cotransfected with 10 mg of Sel10-GFP and 10 mg of LANA-myc expression vectors or transfected with these vectors 24 h after transfection; cell lysates were used for IP analysis. IN, 5% total cell lysate input; PC, preclear; IP, immunoprecipitate.

Fig. 8. Full-length LANA competes with ICN for Sel10 binding. LANA and ICN were in vitro-transcribed and translated. The ³⁵S-labeled products were incubated with GST-Sel10. A constant amount of ICN and increasing amounts of LANA were used. Pulldown products were electrophoresed on 10% SDS/PAGE gels, dried, and exposed to PhosphorImager plates. Input controls of 10% for LANA and for ICN were run as well. The quantification of each band is shown at the bottom of each binding.

Fig. 9. CFSE staining showing the mitosis of cells. (A Upper) JSC1 cells were transfected with LANA siRNA or Luciferase siRNA expression vectors and selected for stable clones. These cells along with untransfected JSC1 cells were stained with CFSE and incubated. Five days after transfection, cells were harvested, fixed, and subjected to FACS analysis. (B Upper) JSC1, LANA siRNA-transfected JSC1, and LANA siRNA/Sel10 dominant-negative cotransfected JSC1 cells were compared for mitosis as described above. (A and B Lower) An example showing the equal labeling of CFSE on different groups of cells. When CFSE staining experiments were performed, the same method was used to label each cell, and an aliquot of each labeled cell was routinely collected and checked at the beginning of the study.