Fig. 3: RecQL4 regulates AURKB stability.

a Enhanced proteasome-dependent degradation of AURKB post RecQL4 knockdown. RecQL4 was first silenced by adenovirus-mediated RecQL4 shRNA, and then treated with MG-132 for 1, 3, and 6 h. Cell lysates were harvested and levels of AURKB and RecQL4 were determined by Western blotting. b Decreased ubiquitination level of AURKB in the presence of RecQL4. RecQL4 was first silenced in U2OS cells and then transfected with His-Ub and Flag-AURKB currently with or without Flag-RecQL4 reconstitution. Cells transfected with Flag-AURKB and Flag-RecQL4 without His-Ub were used as control. AURKB ubiquitination level was determined in His pull-down complex by Western blotting. c RecQL4 helicase domain-mutation affects AURKB ubiquitination. Native RecQL4 expression was first silenced by adenoviral RecQL4 shRNA and then reconstituted by His-Ub and Flag-AURKB with either WT RecQL4 or its mutants (Q253H/K503M). AURKB ubiquitination level was examined in His pull-down by Western blotting. d RecQL4 enhances AURKB protein level. MGC-803 cells expressing a low level of native RecQL4 were transfected with control vector and Flag-tagged wild-type (WT) or its mutants (Q253H/K508M) expressing vectors. AURKB protein level was determined by Western blotting