Fig. 5. Inhibition of Clr6 HDAC stimulates the formation of the 20S APC/C complex. (A) Effects of TSA treatment on cellular levels of APC/C subunit proteins. Wild type, cut4-533 and cut9-665 were cultured at 26°C and TSA (12.5 µg/ml) was added into the cultures. Cells were harvested at the indicated time points and the whole cell extracts were examined by imunoblotting to determine the concentrations of APC/C subunit proteins. -tubulin was detected by TAT1 antibody as loading control. (B) Wild type and cut4-533 mutants were cultured at 26°C and cells were harvested after 12 hour incubation in the absence or presence of 12.5 µg/ml TSA. The cell extracts were fractionated by 15-40% sucrose gradient centrifugation and APC/C subunits Cut4, Cut9 and Nuc2 were detected by immunoblotting. The fractions corresponding to 4.5S and 16.5-19S were determined using bovine serum alubmin and thyroglobulin A, respectively. (C, left panel) Sucrose gradient centrifugation was performed as in B using the cell extracts of wild-type, cut4-533 and cut4-533clr6-1 cells that were cultured at 26°C or 36°C for 4 hours. A condensin subunit Cnd2 was shown as a marker indicating the fraction corresponding to 13S. (C, right panel) The aliquots of the whole cell extracts of each strain were blotted. (D) Wild-type strains were cultured at 26°C and TSA (12.5 µg/ml) was added. Cells were harvested after 12 hour incubation at 26°C. The cell extract was fractionated and analysed as in B. (E) Wild type (no tag) and apc2HA strain were cultured at 26°C in the absence or presence of TSA (12.5 µg/ml) for 12 hours. Apc2HA protein was immunoprecipitated with anti-HA antibody. Cut4, Cut9 and Nuc2 co-immunoprecipitated with Apc2HA were detected by their specific antibodies.