Figure 3.

Induction of p21 and p27 by proteasome inhibitors. MDA-MB-157 tumor cells were treated with (A) 40 μM of either lovastatin mixture (open and closed rings), lovastatin closed β-lactone ring (pro-drug), or pravastatin for 36 hr, (C) 10 μM LLnL for 0–36 hr, or (D) indicated concentrations of lactacystin for 24 hr and subjected (50 μg/lane) to Western blot analysis with the indicated antibodies. Brackets in A indicate the high molecular weight laddering of p21 and p27, diagnostic for poly-ubiquitination. (B) For turnover studies, cells were treated with 40 μM pro-drug (or no drug) for 36 hr at which point the cells were incubated for 4 hr with [³⁵S]methionine and [³⁵S]cysteine (pulse) and subsequently incubated in the presence of an excess of nonradioactive methionine and cysteine for the additional times indicated (chase). p21 and p27 were precipitated from nondenatured protein extracts with polyclonal antibodies (p27-C19 and p21-C19-G, Santa Cruz Biotechnology), separated by SDS/PAGE, and detected by autofluorography.