Figure 6
Minocycline increases Nrf2 protein stability and suppresses Nrf2 ubiquitination.
(a) Minocycline (+Mino) has no impact on Nrf2 mRNA expression in murine podocytes compared to untreated controls (Ctrl). (b) After incubation of podocytes with the protein synthesis inhibitor cycloheximide (CHX, 10 mg/ml for 24 h) Nrf2 protein levels decline. Application of minocycline stabilizes Nrf2 levels. (c) After glucose challenge (25 mM for 24 h) protein ubiquitination (IB: Ubiquitin) is increased compared to unstressed or mannitol incubated (25 mM for 24 h) controls. Minocycline reduces glucose-induced protein ubiquitination; representative immunoblot. (d) Minocycline reduces glucose-induced Nrf2 ubiquitination. Immunoprecipitation (IP) of Nrf2 from whole cell lysates of control (5 mM glucose), glucose challenged (25 mM for 24 h), or glucose plus minocycline (10 μM, 24 h) exposed podocytes. Mean values ± SEM (a,b,d); *P < 0.05, ns: non-significant (a,b,d: Mann-Whitney-test). (a–d) Representative cropped images without further modification are shown; exemplary uncropped images are provided in Supplementary Fig. 6.
