Fig. 4.

UBA1-dependent NEDDylation of RPL7 upon proteotoxic stress. a HCT116 cells stably expressing His₆-NEDD8 were heat-shocked or treated with MG132 (25 µM, 2 h) and then subjected to a subcellular fractionation. Pellet fractions were solubilized and then NEDDylated substrates were isolated by Ni-NTA pull-down as described in Methods. The isolated NEDDylated substrates were blotted against the indicated proteins. b Experiment was performed as above but cells were pretreated with MLN4924 (500 nM 15 h) and MLN7243 (500 nM, 3 h) before MG132 treatment. Ni-NTA pull-down was performed from total cell extracts and western blot analysis as indicated