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. Author manuscript; available in PMC: 2023 Mar 1.
Published in final edited form as: Nat Cell Biol. 2022 Sep 1;24(9):1422–1432. doi: 10.1038/s41556-022-00985-x

Figure 5. Lysine glutarylation attenuates KEAP1 binding increasing NRF2 stability.

Figure 5.

(A, B) Cycloheximide (CHX) chase analysis of half-life of endogenous NRF2 and ATF4 proteins in A375 cells transfected with indicated siRNAs. Western blot was performed on lysates of A375 cells transfected with siControl (A) or siGCDH (B) for 72 hr and then treated with 50 μM Cycloheximide (CHX) for indicated times. After quantification, signals obtained in panels A and B were used to calculate the NRF2/HSP90 and ATF4/HSP90 ratios and described the CHX treatment period as fold change relative to CHX treatment time = 0. (C) Immunoprecipitation and Western blot analysis of A375 transfected with indicated constructs. Cells were treated with the proteasomal inhibitor MG132 for 4 hr, followed by IP/Western blotting analysis with antibodies to detect K-Glu PTM, pSer/Thr PTM, KEAP1, and NRF2. (D) In vitro glutarylation assay on purified HA-NRF2 following incubation with the indicated concentration of glutaryl CoA. (E) In vitro KEAP1 binding analysis was performed using purified HA-NRF2 or K-Glu-NRF2 as bait on A375 cell lysates. Data are representative of three experiments. Statistics source data for Figure 5 can be found in Source data files - Figure 5 and uncropped scans of all blots and gels for figure 5 can be found in Source data files for blot- Figure 5.