Figure 2.
UBLCP1 impairs RP–CP association. (a) UBLCP1 knockdown enhances the 26S proteasome integrity. Proteasome integrity analysis was performed by GST-UBLhRad23B pull-down methods in HEK293T stable cell lines stably expressing shUBLCP1 and shControl. (b) UBLCP1, but not DDAA, impairs the 26S proteasome integrity. Proteasome integrity analysis was performed by GST-UBLhRad23B pull-down methods in HEK293T stable cell lines following western blotting with indicated proteasome subunit antibodies. (c) GST-UBLCP1 prevents RP from activating CP. Free RP was purified from HEK293T cells stably expressing HTBH-tagged Rpn11. Free CP was purified from HEK293T cells transfected with FLAG-tagged β4. RP was pretreated with GST-UBLCP1 and GST-DDAA, respectively. The proteasome was reconstituted by incubating CP with RPs. Reconstituted proteasome was incubated with Suc-LLVY-AMC substrate. AMC release was further detected at 460 nm after 380 nm excitation using a fluorescence plate reader. The values were normalized with the amount of CP. All data are the means (±s.e.) of three independent experiments performed in triplicate; **p < 0.01, paired Student's t-test. (d) GST-UBLCP1 does not affect the 26S proteasome activity. The 26S proteasome holoenzyme was purified from HEK293T cells stably expressing HTBH-tagged Rpn11, and then incubated with purified GST-UBLCP/DDAA in vitro. Then proteasome activity assay was performed as for (c).