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. 2004 Dec 27;102(1):63–68. doi: 10.1073/pnas.0406424101

Fig. 3.

Fig. 3.

Tax causes premature activation of APCCdc20. (A) Increased APC activity in HTLV-I-transformed cells. Jurkat and HTLV-I-transformed (MT4, C8166, C91PL) T cells were incubated in complete RPMI medium 1640 containing 10 μM MG132 for 24 h. Cell lysates were prepared, immunoprecipitated with a cyclin B1 antibody, and immunoblotted with ubiquitin (Upper) and Cdk1 (Lower) antibody, respectively. (B) Tax activates APC in 293T cells. 293T cells transfected with a plasmid encoding HA-tagged human ubiquitin together with a tax-expression plasmid, CMV-Tax (Left) or an empty vector plasmid (Right) were arrested at the G1/S border by a single thymidine treatment, released in complete DMEM containing 10 μM MG132 for 5 h, immunoprecipitated with a cyclin B1 antibody (supporting information), and immunoblotted to detect polyubiquitination with the HA antibody (Upper). The same samples were also immunoblotted for Cdk1 as a control (Lower). (C) Tax activates APC during S phase. HA-tagged ubiquitin was transfected into HeLa cells by using the FuGENE 6 transfection reagent. The transfected cells were then synchronized with a double-thymidine treatment. At the start of the second thymidine treatment, cells were also infected (at an MOI of 5) with Ad-Tax (Ad-Tax “+” lanes) or Ad-tTA (a control adenoviral vector that contains the tetracyclin transactivator gene, Ad-Tax “-” lanes). The cells were then released from the cell cycle arrest (for 0, 4, 5, 6, and 8 h, from left to right) in complete DMEM containing 10 μM MG132. Immunoprecipitation and immunoblots were carried out as in B. (D) APC activities of Ad-Tax- and Ad-tTA-infected HeLa cells assayed in vitro. HeLa cells were arrested at the G1/S border, infected with either Ad-Tax (denoted as Tax) or Ad-tTA (denoted as tTA) at a MOI of 5, and released from the arrest for 4 (left two lanes) and 5 (right two lanes) h as described (12). The APC was then purified from cell lysates and assayed as in Materials and Methods. Polyubiquitinated c-Myc-tagged-cyclin B1 in each reaction was resolved by SDS/PAGE and detected by anti-c-Myc immunoblot.