Figure 3. In vitro ubiquitination assay.

Recombinant 6× His-PUB20 protein was expressed using E. coli and incubated at 28°C for 1 h in the presence or absence of E1 (UBA1) and E2 (UbcH5b). The samples were separated by 7.5% SDS-PAGE and subjected to immunoblot analysis by anti-Ub antibody (top) and HisProbe (bottom). The numbers in the right indicate molecular sizes (kDa). The part of the blot showing free Ub (8.6 kDa) was cut off.