́ 3 pet28a his6 halo human keap1 gene  (Addgene inc)

Journal: Science advances

Article Title: Molecular evolution of Keap1 was essential for adaptation of vertebrates to terrestrial life.

doi: 10.1126/sciadv.adg2379

Figure Lengend Snippet: Fig. 1. Weaker interaction of Keap1 with Cul3 compared with other receptors. (A) Lysates of HEK293T cells expressing FLAG-tagged human KLHL proteins were subjected to immunoprecipitation (IP) with antibodies to FLAG, and the resulting precipitates as well as the original lysates (Input) were subjected to immunoblot analysis with the indicated antibodies (α-). GAPDH was examined as a loading control. (B) Lysates of HEK293T cells expressing FLAG-tagged human KLHL3 or Keap1 together with HA-tagged human Cul3 were subjected to immunoprecipitation with antibodies to FLAG, and the resulting precipitates as well as the original lysates were subjected to immunoblot analysis with the indicated antibodies. GAPDH was examined as a loading control. (C) Fifty-three BTB proteins previously found to interact with Cul3 in HEK293T cells (25). Z and NWD scores were obtained from CompPASS analysis (26). Cul3 interactants in the BioPlex 3.0 database (27) are shown in green, with others shown in gray. (D and E) Representative ITC titration curves for interaction between human Cul3(N) and either human KLHL3(N) (D) or human Keap1(N) (E) as well as domain organization of the protein fragments. Baseline-corrected differential power (DP) versus time as well as the normalized binding curve, with integrated changes in enthalpy (ΔH) plotted against molar ratio, are shown. Each experiment was performed independently at least twice. (F) Dissociation constants for Cullins and the indi- cated receptors. Values other than those for Keap1 and KLHL3 were obtained from previous studies (21, 23, 28, 54–62). n.d., not detected.

Article Snippet: Establishment of knock-in cells with the CRISPRCas9 system Complementary oligonucleotides containing the human Keap1 single-guide RNA (sgRNA) target sequences (table S2) were annealed and cloned into the Bbs I site of pX330 (Addgene).

Techniques: Expressing, Immunoprecipitation, Western Blot, Control, Titration, Binding Assay

Journal: Science advances

Article Title: Molecular evolution of Keap1 was essential for adaptation of vertebrates to terrestrial life.

doi: 10.1126/sciadv.adg2379

Figure Lengend Snippet: Fig. 2. C3IR of Keap1 is responsible for weak binding to Cul3. (A) Alignment of amino acid sequences for the BTB domains of human (h) KLHL3, KLHL11, SPOP, and Keap1. Information on secondary structure was obtained from PDB (4HXI for KLHL3, 4AP2 for KLHL11, 4EOZ for SPOP, and 4CXI for Keap1). The numbers of α helices and β sheets are in accordance with a previous study (30). (B) Domain organization of human Keap1, human KLHL3, and the exchange mutant proteins Keap1(N3) and Keap1(W→3) as well as a summary of their interaction with Cul3 as determined in (C). (C) HEK293T cells expressing FLAG-tagged wild-type (WT) or mutant forms of human Keap1 together with HA-tagged human Cul3 were incubated in the absence or presence of 25 μM tBHQ for 24 hours, lysed, and subjected to immunoprecipitation with antibodies to FLAG. The resulting precipitates as well as the original cell lysates were subjected to immunoblot analysis with the indicated antibodies.

Article Snippet: Establishment of knock-in cells with the CRISPRCas9 system Complementary oligonucleotides containing the human Keap1 single-guide RNA (sgRNA) target sequences (table S2) were annealed and cloned into the Bbs I site of pX330 (Addgene).

Techniques: Binding Assay, Mutagenesis, Expressing, Incubation, Immunoprecipitation, Western Blot

Journal: Science advances

Article Title: Molecular evolution of Keap1 was essential for adaptation of vertebrates to terrestrial life.

doi: 10.1126/sciadv.adg2379

Figure Lengend Snippet: Fig. 3. Evolutionary changes in C3IR of Keap1 weakened its interaction with Cul3. (A) Phylogenetic tree for the BTB domain of Keap1 proteins. The numbers as- sociated with the branches indicate bootstrap values. (B to E) Ratio of Keap1A and Keap1B mRNAs at the indicated developmental stages in O. latipes (B), D. rerio (C), X. laevis (D), and X. tropicalis (E). Data are derived from RNA-seq analysis in previous studies (50–53). (F) Alignment of amino acid sequences for the BTB domain of Keap1 proteins. Keap1A- or Keap1B-specific amino acids are highlighted in red or blue, respectively, with common amino acids shown in brown. (G) Domain organization of zKeap1A, zKeap1B, and the C3IR mutants zKeap1A(A→B) and zKeap1B(B→A) as well as a summary of their interaction with zCul3 as determined in (H) and (I). (H and I) Lysates of HEK293T cells expressing FLAG-tagged zKeap1A, zKeap1B, zKeap1A(A→B), or zKeap1B(B→A) together with HA-tagged zCul3A (H) or zCul3B (I) were subjected to immunoprecipitation with antibodies to FLAG. (J) Domain organization of human Keap1 and the C3IR mutants hKeap1(W→3), hKeap1(W→A), and hKeap1(W→B) as well as a summary of their interaction with human Cul3 as determined in (K). (K) Lysates of HEK293T cells expressing FLAG-tagged hKeap1, hKeap1(W→3), hKeap1(W→A), or hKeap1(W→B) together with HA-tagged hCul3 were subjected to immunoprecipitation with antibodies to FLAG. (L and M) Lysates of HEK293T cells expressing HA- tagged hCul3 together with FLAG-tagged hKeap1 or its exchange mutants containing C3IR of Keap1 proteins from the indicated species were subjected to immuno- precipitation with antibodies to FLAG (L). The relative extent of HA-hCul3 binding to the hKeap1 mutants was also quantitated by densitometry (M). Quantitative data are means from three independent experiments.

Article Snippet: Establishment of knock-in cells with the CRISPRCas9 system Complementary oligonucleotides containing the human Keap1 single-guide RNA (sgRNA) target sequences (table S2) were annealed and cloned into the Bbs I site of pX330 (Addgene).

Techniques: Derivative Assay, RNA Sequencing, Expressing, Immunoprecipitation, Binding Assay

Journal: Science advances

Article Title: Molecular evolution of Keap1 was essential for adaptation of vertebrates to terrestrial life.

doi: 10.1126/sciadv.adg2379

Figure Lengend Snippet: Fig. 4. Evolutionary changes in C3IR of Keap1 attenuate ubiquitylation activity and promote Nrf2-dependent responses to oxidative stress. (A) HEK293T cells expressing Myc-Keap1 or its indicated C3IR mutants together with HA-ubiquitin (Ub) and FLAG-Nrf2 were treated with 10 μM MG132 for 6 hours, lysed, and subjected to immunoprecipitation with antibodies to FLAG. Asterisks indicate nonubiquitylated forms of FLAG-Nrf2. (B) Median fluorescence intensity (MFI) determined by flow cy- tometry for EGFP-Nrf2 expressed in HEK293T cells together with FLAG-Keap1 or its C3IR mutants. Data are means; n = 4. ****P < 0.0001 versus WT. (C) MFI of EGFP-Nrf2 expressed together with FLAG-Keap1 or its C3IR mutants that were treated with 1 μM MLN4924 for 24 hours before washout of MLN4924 and incubation of the cells with cycloheximide (CHX; 50 μg/ml) for the indicated times. Data are expressed relative to the value for time 0. (D to F) MFI of EGFP-Nrf2 expressed together with FLAG-Keap1 or its C3IR mutants during exposure to 25 μM tBHQ (D), 100 μM genistein (E), or 400 nM doxorubicin (F). (G) WT or Keap1 C3IR mutation knock-in HEK293T cells were exposed to 25 μM tBHQ, lysed, and subjected to immunoblot analysis with antibodies to Nrf2 (left). The relative abundance of endogenous Nrf2 was also quantitated (right). Data are means ± SEM; n = 5. (H and I) RT and real-time PCR analysis of HO1 (H) and NQO1 (I) mRNA abundance in WT or Keap1 C3IR mutation knock-in cells at 8 hours after treatment with 25 μM tBHQ. Data are means; n = 5. NS, not significant. (J and K) Flow cytometric analysis of WT or Keap1 C3IR mutation knock-in cells that were negative for annexin V and propidium iodide (PI) staining after treatment with 0.003% H2O2 (J) or 5 μM doxorubicin (K) for 24 hours. Data are means; n = 5.

Article Snippet: Establishment of knock-in cells with the CRISPRCas9 system Complementary oligonucleotides containing the human Keap1 single-guide RNA (sgRNA) target sequences (table S2) were annealed and cloned into the Bbs I site of pX330 (Addgene).

Techniques: Activity Assay, Expressing, Ubiquitin Proteomics, Immunoprecipitation, Fluorescence, Incubation, Mutagenesis, Knock-In, Western Blot, Real-time Polymerase Chain Reaction, Staining

Journal: Science advances

Article Title: Molecular evolution of Keap1 was essential for adaptation of vertebrates to terrestrial life.

doi: 10.1126/sciadv.adg2379

Figure Lengend Snippet: Fig. 5. Keap1W→A/W→A knock-in mice are more sensitive to APAP- induced oxidative stress. (A) Sche- matic representation of exon 2 of the WT mouse Keap1 allele, the ssODN, and the W→A (replacement of C3IR of the mouse protein with that of zKeap1A) mutant allele after homol- ogous recombination. The sgRNA and its protospacer adjacent motif are in- dicated by continuous and dotted overlines, respectively. The 5′ un- translated region and open reading frame of Keap1 are represented by the light and dark gray boxes, respec- tively. The mutation is shown in red. (B) GSEA plot performed for a gene set consisting of Nrf2 target genes with RNA-seq data obtained from the liver of 8-week-old Keap1W→A/W→A

Article Snippet: Establishment of knock-in cells with the CRISPRCas9 system Complementary oligonucleotides containing the human Keap1 single-guide RNA (sgRNA) target sequences (table S2) were annealed and cloned into the Bbs I site of pX330 (Addgene).

Techniques: Knock-In, Mutagenesis, RNA Sequencing