CSN5 regulates the ubiquitination of NRF2, a master regulator of expression of antioxidation genes and HO-1. (A) Immunoblot analysis of Cul3-immunoprecipitated lysates from 293 cells treated with MG132 for 6 hours and blotted with anti-CSN5, NRF2, and Keap1. (B) Immunoblot analysis of anti-Flag (NRF2)–immunoprecipitated lysates from 293 cells with the use of anti-HA (ubiquitin) antibody. 293 cells were cotransfected with the plasmids expressing HA-ubiquitin (Ub-HA), Flag-tagged NRF2 (NRF2-Flag), Myc-tagged Cul3 (Cul3-Myc), T7-tagged Keap1 (Keap1-T7), ROC1, and CSN5 siRNA (0, 100, 200, and 300nM) for 24 hours before the cells were harvested for immunoblots. (C) Immunoblot analysis of anti-NRF2 of 293 cell nuclear extracts. 293 cells were transfected with different concentrations of siRNA CSN5 (0, 100, 200, and 300nM) and expression vectors for NRF2-Flag and Cul3-Myc. Total cell lysates were subjected to immunoblot analysis with anti-Flag antibodies. (D) Immunoblot analysis of anti-NRF2 from the nuclear extracts of CSN5^fl/flLyzsM-Cre BMDMs. Data are representative of 3 independent experiments (C-D). (E) Immunoblot analysis of Cul3 in wild-type or CSN5 KO BMDMs. (F) Immunoblot analysis of NRF2-immunoprecipitated lysates of macrophages with the use of antipolyubiquitin or anti-Keap1 antibodies. (G) Immunoblot analysis of HO-1 in wild-type or CSN5 KO BMDMs. An α-tubulin blot was included to indicate lane loading. (H) Immunoblot analysis of Cul3 in 293 cells transfected with the different concentrations of siRNA CSN5 (0, 100, 200, and 300nM) for 24 hours. Data are representative of 3 experiments. An α-tubulin blot was included to indicate lane loading. Data are representative of 3 experiments (E-G).