Kinetics of p27 degradation in MCL.

Purified recombinant p27 was incubated for the indicated intervals with the extracts from three representative MCL cases (a, high p27 expression; b, low expression of p27 by the tumor cells, but the lymphoid tissue sample contained a relative large number of p27+ reactive T cells; c, low expression of p27 by the tumor cells, and the lymphoid tissue sample contained a small number of p27+ reactive T-cells) (A). To demonstrate that p27 degradation is mediated by proteasome the MCL lysates were incubated with and without hemin (B). p27 was polyubiquitinated before proteosome degradation (C). Purified recombinant p27 was incubated for 1 hour with the cell extract (HeLa) in the presence of HA-ubiquitin and subsequently was immunoprecipated with an anti-HA or anti-ubiquitin mAbs. After transfer, polyubiquitinated p27 forms were identified using anti-p27 mAb.