Absolute quantification analysis of ubiquitin chains conjugated to GP8 using an in vitro ubiquitination reaction.

To examine whether ARIH1-UBE2L3 is involved in the atypical ubiquitination of Nrf1 by SCFFBS2, we compared the ubiquitin linkage types between three reactions using GP8: E1/UBE2D1/SCFFBS2, the complete reaction including E1/UBE2D1/SCFFBS2/Nedd8 sys/ARIH1/UBE2L3, and the complete reaction in the absence of UBE2D1.