Fig. 6

indentification of Mst1/2-mediated phosphorylation sites on Keap1. a Immunoblot analysis of Mst2, Keap1 in total lysates (bottom) and anti-Flag IP (top) of 293T cells expressing various combinations (above lanes) of Flag-tagged full length Keap1, truncated Keap1_(1–310) or Keap1_(311–624) and Myc-tagged Mst2. b Phos-tag and SDS-PAGE analysis of Flag-tagged Keap1 (1–310) expressed together with HA-tagged wild-type Mst2 or kinase-inactive Mst2K/R in 293T cells immunoprecipitated with anti-Flag conjugated beads. c Phos-tag and SDS-PAGE analysis of GST-tagged wild-type or various mutated Keap1_(1–150) expressed together with His-tagged Mst2 in 293T cells. d Mst1/2 kinases-mediated phosphorylation sites on the Keap1 showing the domains of BTB, IVR, and Kelch1-6; numbers below diagrams indicate amino acid range of each domain. e Immunoassay of the ubiquitination of Nrf2 in anti-HA IP (top) and total lysates (bottom) of 293T cells expressing various combinations (upper lanes) of Flag-tagged Cul3, Flag-tagged WT, mutated Keap1^4A or Keap1^4D, and HA-tagged Nrf2. f, g Immunoblot analysis of total lysates in 293T cells expressing various combinations (above lanes) of HA-tagged wild-type Keap1^WT, mutated Keap1^4A (f) or Keap1^4D (g) and Flag-tagged Nrf2 followed by CHX treatments with the indicated time. Data are from one experiment representative of three independent experiments with similar results