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. Author manuscript; available in PMC: 2019 Jun 27.
Published in final edited form as: Cell Physiol Biochem. 2018 Mar 23;46(1):418–430. doi: 10.1159/000488475

Fig. 3.

Fig. 3.

Arkadia ubiquitylates polysumoylated Nrf2 through K48-linked ubiquitin chains. HepG2 cells were transfected with 2 μg of plasmid expressing empty vector (pFLAG) or FLAG-Arkadia and pRK5-HA-Ubiquitin. After 48 hr the cells were incubated with or without As2O3 (5 μM) for 4 hrs and then harvested. Whole-cell lysates were generated and immunoprecipitated with anti-Nrf2 antibody and Western blotted with (A) anti-K48-specific ubiquitin antibody or (B) anti-K63-specific ubiquitin antibody. Control IPs were blotted with anti-Nrf2 antibody (C). HepG2 cells were transfected with FLAG-Arkadia and wild-type HA-Ubiquitin or mutant plasmids expressing HA-Ub-K48R or HA-UbK48 only. Whole cell lysates were generated and immunoprecipitated with anti-Nrf2 antibody and Western blotted with anti-HA antibody. The same whole cell lysates were immunoblotted with anti-Nrf2 antibody and anti-β-actin as a loading control.