Tax shows different ubiquitin modifications and stabilities in the cytoplasm and in the nucleus. (A) 293 cells transiently transfected with Tax were CHX chased at the indicated time (in hours), followed by cytoplasmic and nuclear extractions. For the 4-hour time point, the cells were also chased in the presence of 10 μM MG132 (lanes 4 and 8). The expression levels of Tax, lamin B, and Hsp90 in these 2 fractions were detected by IB. The Tax levels were also quantitated by densitometry. (B) C8166 and MT-4 were used for Tax stability assays as described in panel A. (C) C8166, MT-4, and SLB cells were left untreated or treated with MG132 for 3 hours, followed by cytoplasmic and nuclear extractions. The ubiquitinated Tax proteins were then analyzed by IP using Tax antibody and IB using ubiquitin antibody. The levels of Tax in the extractions were analyzed by direct IB. (D) 293 cells transfected with Tax plus the indicated HA-tagged ubiquitin mutants were left untreated or treated with MG132 for 3 hours, followed by the ubiquitination assays as described in panel C.