Fig. 5
Mst1/2 downregulates Keap1-mediated ubiquitination of Nrf2. a, b RT-qPCR analysis of the mRNA levels of Nrf2 (a) and immunoblot analysis of Nrf2, HO-1, Mst1, Mst2, and GAPDH (b) in Mst1fl/flMst2fl/fl (WT) and Mst1fl/flMst2fl/fl Lyz-Cre (DKO) BMDMs treated with 10 μM antimycin A (AA) for indicated times. *P < 0.05, **P < 0.01, ***P < 0.001, compared with the control sample (Student’s t test, n = 3, experimental replicates). c Immunoblot analysis of Nrf2 ubiquitination in total lysates (bottom) and anti-IgG or anti-Nrf2 immunoprecipitates (IP, top) of WT and DKO BMDMs treated with or without antimycin A, probed with anti-ubiquitin (α-Ub) and antibodies to Nrf2, Mst1, Mst2, and GAPDH. d Immunoassay of the ubiquitination of Nrf2 in anti-Flag IP (top) and total lysates (bottom) of 293T cells expressing various combinations (above lanes) of Myc-tagged Ub, Flag-tagged Nrf2, HA-tagged Keap1, Mst2 or Mst2K/R, and Myc-tagged Ub. e Immunoblot analysis of Keap1 and Mst2 in anti-IgG or anti-Flag IP (top) and total lysates (bottom) of WT BMDM cell line stably expressing Flag-tagged Mst2 infected with E. coli (MOI: 100) for indicated times. f Immunoblot analysis of Mst1, Mst2, β-actin, and VDAC in the cytoplasmic (Cyto) and mitochondrial (Mito) fractions of WT BMDMs pretreated with or without NAC and infected with E. coli (MOI: 100) for indicated times. g SIM of the co-localization of Mst1 (red) and Keap1 (green) in WT BMDMs treated with or without NAC and infected with E. coli (blue) for 30 min; ×16 magnification of areas outlined in the main images are shown next to the main images. Scale bars, 20 μm. Data are from one experiment representative of three independent experiments with similar results
