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. Author manuscript; available in PMC: 2015 Aug 1.
Published in final edited form as: FEBS J. 2014 Jul 1;281(15):3357–3381. doi: 10.1111/febs.12866

Fig. 3.

Fig. 3

In vitro Sumoylation assay revealed that Prdx6 was modified by Sumo1. The in vitro Sumoylation assay was performed according to the manufacturer’s protocol. Briefly, a combination of E1 enzyme, E2 (Ubc9) enzyme, Sumo1 wild-type (WT) protein and/or Sumo1 mutant protein and different concentrations of recombinant Prdx6 protein (TAT-HA-Prdx6) were mixed with 20μl reaction mixture containing Sumoylation buffer. Following incubation at 30°C for 3h, reaction product was incubated at 90°C with 2X SDS-gel loading buffer and was immunoblotted using anti-Sumo1 and anti-Prdx6 polyclonal antibodies. Concentration-dependent Sumoylation of recombinant wild-type Prdx6 protein was seen, as shown in figure, lanes 1 and 2 (* denotes the Sumoylation band). Mutant Sumo1 failed to conjugate to Prdx6, suggesting Prdx6 was Sumoylated in vitro.