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. Author manuscript; available in PMC: 2021 Feb 20.
Published in final edited form as: Free Radic Biol Med. 2019 Dec 26;148:70–82. doi: 10.1016/j.freeradbiomed.2019.12.037

Figure 3: Fumarate augments oxidative stress to modify Keap1 and stabilize CHOP.

Figure 3:

(A-C) Protein from mature 3T3-L1 adipocytes, control or db/db epididymal adipose tissue and control or FHKD adipocytes was immunoblotted and probed to detect heme oxygenase 1 (HOX1) protein levels. (D) Immunoblotting with anti-Keap1 and -V5 antibodies validates the successful transduction of the scrambled control or Keap1-V5 tagged lentivirus in adipocytes matured in 5 mM or 25 mM glucose. (E) Protein (600 μg) from adipocytes matured in 5 mM or 25 mM glucose was immunoprecipitated with anti-V5 agarose beads. The eluted protein was immunoblotted to detect succination (2SC) in the the total homogenate (TH) and immunoprecipitate (IP), followed by re-probing with anti-Keap1 and -V5 antibodies. Arrow indicates anticipated position of succinated Keap1 (F-G) Measurement of reactive oxygen species was performed using both Amplex Red and DCF detection methods. Control or FHKD adipocytes were cultured in 5 or 30 mM glucose, or (H) 5 mM glucose and treated with 200 pM DMF (representative experiments shown with minimum n=3/group, mean +/− SEM, *p< 0.05, **p<0.001).