Fig. 3.

Radicicol treatment does not act at preproteasomal or proteasomal steps. (A) PIK23 cells were treated with 10 μM radicicol or DMSO for 7 h. Cells were metabolically labeled with ³⁵S for 1 h and chased for time points as indicated. Cells were lysed for immunoprecipitation with anti-Akt (Left) or anti-GFP (Right) antibody. Immunoprecipitated material was resolved by SDS/PAGE and autoradiographed. The band intensity was measured and plotted taking the initial time point (0 h) as 100%. (B) 4T1 cells were treated with the indicated doses of lactacystin, radicicol, or DMSO for 12 h and lysed, resolved by SDS/PAGE, and immunoblotted for ubiquitin. (C) 4T1-proSensor-76B, a clone stably expressing the pZsProSensor-1, was treated with the indicated doses of lactacystin or radicicol for 12 h and analyzed for expression of GFP by FACS. Error bars in C represent standard deviation of a single experiment performed in triplicate. Results are representative of three independent experiments.