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. 2015 Oct 5;6(33):34342–34357. doi: 10.18632/oncotarget.5326

Figure 8. Zn-RING-Zn domain undergoes N-terminal ubiquitination.

Figure 8

A. In vitro ubiquitination was performed using E1, E2 (UbcH5b), Zn-RING-Zn domain of LNX2, Ub-KO ubiquitin (all Lys are mutated to Arg) and in the presence (+) or absence (−) of ATP. The samples were then resolved by 12.5% SDS-PAGE, followed by gel staining using coomassie brilliant blue B. In vitro ubiquitination was performed using E1, E2 (UbcH5b), ATP, 6xHistidine tagged Zn-RING-Zn domain of LNX2 and Ub-KO ubiquitin. After overnight cleavage with precission enzyme, the mixture was incubated with Ni-NTA beads followed by washing for three times. The Flow thru, washes and beads were separated by 12.5% SDS-PAGE (Supplementary Figure S8B) and subjected to western blotting. Shown is the immunoblot using anti-Ubiquitin (top) and anti-His (bottom) antibody. FT- Flow thru.