Figure 6

S. cerevisiae -viable tax mutants are attenuated in APC/C activation. (A) KY630 cells carrying a gene encoding CLB2-3XHA integrated at the CLB2 locus were transformed with either Gal10-wild-type tax or Gal10-mutant tax. KY630/Gal10-tax cells were cultured in SC medium containing 2% raffinose at 30°C overnight for 12 h, then diluted to 0.3 A₆₀₀, grown to mid-log phase, and induced for tax expression by the addition of 2% galactose. Cell lysates were prepared after 120 minutes. Immunoblots were carried out with anti-HA and 4C5 monoclonal antibodies. (B) 293T cells were transfected with a plasmid encoding HA-tagged human ubiquitin together with an expression plasmid for wild-type tax or mutant tax. The cells were arrested at the G₁/S border by a single thymidine treatment, released into complete DMEM containing 10 μM MG132 for 5 h, immunoprecipitated with cyclin B1 antibody and immunoblotted with an HA antibody to detect polyubiquitinated cyclin B1.