Fig. 7

Mst1/2-mediated Keap1 phosphorylation disrupts the Keap1-Nrf2 complex. a Immunoblot analysis of Cul3 and Keap1 in total lysates (bottom) and anti-HA IP (top) of 293T cells expressing various combinations of Flag-tagged Cul3, HA-tagged Keap1^WT, Keap1^4A or Keap1^4D. b Immunoblot analysis of keap1 levels in total lysates (bottom) and anti-Flag IP (top) of 293T cells expressing various combinations of HA-tagged or Flag-tagged wild-type Keap1^WT, mutated Keap1^4A or Keap1^4D. c, d Immunoblot analysis of dimerization of Keap1 in total lysates of 293T cells expressing Flag-tagged WT or mutated Keap1 (c) or various combinations of HA-tagged WT or kinase inactivated Mst2K/R with Flag-tagged Keap1 (d) using native or regular PAGE. e SIM of HeLa cells cotransfected to express HA-tagged Keap1^4A (red) with Myc-tagged Keap1^4A, Keap1^WT or Keap1^4D (green); ×64 magnification of areas outlined in the main images are shown next to the main images. Scale bars, 20 μm. f Quantification of the percentage of foci (larger than 0.4 μM) on e.n.s. no significant, *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t test, mean and s.d. of n = 7 images). g Immunoblot analysis Nrf2 and Keap1 in total lysates (bottom) and anti-Flag IP (top) of 293T cells expressing various combinations of Flag-tagged Nrf2 and HA-tagged Keap1^WT, Keap1^4A, or Keap1^4D in present of MG132. h Immunoblot analysis of Keap1 and Nrf2 in anti-IgG or anti-Nrf2 IP (top) and total lysates (bottom) of Mst1^fl/flMst2^fl/fl (WT) and Mst1^fl/flMst2^fl/fl Lyz-Cre (DKO) BMDMs treated with antimycin A (AA) for indicated times, probed with antibodies to Keap1, Nrf2, Mst1, Mst2, and GAPDH. i Immunoassay of the ubiquitination of Nrf2 in anti-IgG or anti-Nrf2 IP (top) and total lysates (bottom) of BMDMs infected with adenovirus expressing (Ad−) Keap1^WT, Keap1^4A, Keap1^4D or control vector. j, k Immunoblot analysis of NQO1, HO-1, Nrf2, and Keap1 (j) or RT-qPCR analysis of Nqo1 and Ho-1 (k) in Raw264.7 cells expressing Flag-tagged Keap1^WT, Keap1^4A, or Keap1^4D infected with E. coli (MOI: 100). ns, not significant (P > 0.05); *P < 0.05, **P < 0.01, ***P < 0.001, compared with the control sample (Student’s t test) (mean and s.d. of n = 3, experimental replicates). Data are from one experiment representative of three independent experiments with similar results