FIG. 4.

HSP27 association with the 26S proteasome is required for HSP27-induced proteasome activity. (A) Immunoprecipitation (IP) performed with control- and HSP27-transfected U937 cells and an anti-HSP27 or an anti-HSP60 antibody was followed by immunodetection of HSP27, HSP70, and the PA700 subunit of the 26S proteasome. ch., chain. (B) Immunodetection of PA700 after immunoprecipitation of HSP27 in control (Co) and heat-shocked (HS; 1 h at 42°C and then 6 h at 37°C) U937 cells. (C) HSP27 was incubated in vitro with the purified 26S proteasome before immunoprecipitation of HSP27 or HSP60 and immunodetection of the indicated proteins. (D) Immunodetection of PA700 after HSP27 immunoprecipitation of REG cells transfected with an empty vector (Control) or a vector containing either wild-type hsp27 or the indicated hsp27 deletion mutations. As a control, cell lysates were immunoprecipitated with nonrelevant immunoglobulin G1. (E) The REG cells shown in panel D were treated with etoposide (VP16, 100 μM, 24 h) before measurement of the ability of cell lysates to cleave the substrate Suc-LLVY-AMC as described in the legend to Fig. 1 (fold induction is relative to that in untreated cells). Results are the means and standard deviations for three independent experiments.