Fig. 8.

NEDDylation compromises substrate ubiquitination and targeting to the proteasome. a U2OS cells were first transfected with control or NEDD8 siRNA and 24 h later with His₆-ubiquitin and HA-RPL7 plasmids as indicated. Twenty-four hours later cells were heat-shocked for 45 min and extracts were used for Ni-NTA pull-downs. Western blotting on purified His₆-ubiquitin conjugates and total cell extracts was performed for the indicated proteins. b Similar experiment as in (a) but cells were cotransfected with the HUWE1 siRNA as indicated. c HEK293 cells stably expressing RPN11-HTBH were transfected with control or NEDD8 siRNA and 24 h later with RPL7 expressing construct. Twenty-four hours later cells were heat-shocked for 45 min and proteasomes were purified with streptavidin beads. Total cell extracts or biotin pull-downs were blotted against the indicated proteins. d Similar experiment as in (c) with the exception that Flag-NUB1 or pcDNA3 (5 μg) constructs were transfected instead