Fig 5. The NNLS domain of LANA interacts with Bub1 and rescues the Bub1 Kinase activity in the presence of LANA.

A, B, HEK293 were transfected with indicated plasmids. 48 hours later, cells were collected and lysed. 2ug of Myc antibody was used for IP and western blot was done with indicated antibodies. C, A reverse CO-IP was performed between Bub1, EQ, pRich, and NNLS domain. GFP antibody was used to do IP and western blot was done with indicated antibodies. D, E, HEK293 were transfected with indicated plasmids. 48 hours later, cells were collected and lysed. Bub1 antibody was used to do IP and western blot was done with indicated antibodies. F, HEK293 cells were transfected with increasing amounts of NNLS plasmids. 48 hours later, cells were collected and lysed. Bub1 antibody was used to do IP and western blot was done with indicated antibodies. G, HEK293 were transfected with indicated plasmids. 48 hours later, cells were collected and lysed. Bub1 antibody was used to do IP and western blot was done with indicated antibodies. H, I, HEK293 cells were transfected with indicated plasmids. 48 hours post-transfection, cells were harvested and immunoprecipitated with Bub1 antibodies. The immune complexes were then incubated with purified GST-tagged H2A or Cdc20 in kinase reaction buffer at 30°C for 30 min. the autoradiograph result showed that NNLS can rescue LANA-mediated inhibition of the Bub1 kinase activity (compare lanes 3 and 4, left and right panel respectively). J, HEK293 cells were transfected with indicated plasmids. Then western blot was used to validate the in vitro results. K, HEK293 cells were transfected with indicated plasmids. 48 hours post transfection, cells were collected and lysed for western blot.