(A) RPN8 and RPN11 under control of the tetO2 were silenced by addition of 20 μg/ml tetracycline to growth media. At indicated time points, WCE was resolved by 4% non-denaturing (native)-PAGE (top) and 12% SDS/PAGE (bottom). Proteasome activity was traced by in-gel peptidase activity. Effect of tetracycline gene repression on cellular levels of proteasome lid subunits was monitored by immunoblotting specific antibodies as indicated. Majority of proteasomes in untreated cells migrated as doubly and singly capped 26S proteasomes (RP2CP, RP1CP respectively) and free 20S CP. After 6-h-treatment, faster migration species becomes apparent (top panel), concomitant with ablation of the target gene product in WCE (Rpn8 or Rpn11 accordingly; bottom panels). Composition of this new species was confirmed as base-CP lacking all lid subunits (Table 3). (B) Rpn12 ejected from tetO2RPN8 and tetO2RPN11 proteasomes. Eight hours after tetracycline treatment, WCE was resolved by native-PAGE and immunoblotted for presence of Rpn12 in proteolytically-active species. (C) Six hours following tetracycline treatment, heterogeneous proteasome species were resolved by fractionating native WCE (as in panel A) through a 10%–40% glycerol gradient. Each fraction was assayed for proteasome subunits (top panels) or proteolytic activity (bottom).