Journal: The Journal of Biological Chemistry

Article Title: Transforming Growth Factor-β Induces Transcription Factors MafK and Bach1 to Suppress Expression of the Heme Oxygenase-1 Gene *

doi: 10.1074/jbc.M113.450478

Figure Lengend Snippet: TGF-β induces MafK and Bach1. A , NMuMG cells were treated with TGF-β (5 ng/ml) for 1 h. mRNA for the small Maf family of transcription factors (MafF, MafG, and MafK), Bach1, Smad7, and β-actin was detected by semiquantitative RT-PCR. B , NMuMG cells were treated with TGF-β (5 ng/ml) for 4 h. Immunoblot analysis was performed using anti-MafK, -Bach1, -phospho-Smad2 ( P-Smad2 ), -Smad2, and -β-actin antibodies as indicated. Bach1 was detected after immunoprecipitation ( IP ) with anti-Bach1 antibody to increase the sensitivity. C , NMuMG cells were treated with a TGF-β type I receptor kinase inhibitor, SD-208 (0, 0.03, 0.1, and 0.3 μ m ) for 30 min before treatment with TGF-β (5 ng/ml) for 1 h. MafK, Bach1, Smad7, and β-actin mRNAs were detected by semiquantitative RT-RCR.

Article Snippet: Soluble chromatin was diluted with 9 volumes of dilution buffer for immunoprecipitation (16.7 m m Tris-HCl, pH 8.1, 1.2 m m EDTA, 167 m m NaCl, 0.01% SDS, 1.1% Triton X-100, 20,000 KIU/ml aprotinin, 1 μg/ml leupeptin) and incubated with normal rabbit IgG, anti-Nrf2 (H-300, Santa Cruz Biotechnology), anti-MafK , anti-Bach1 , or anti-Smad2/3 (clone 18, BD Transduction Laboratories) antibody with end-over-end rotation at 4 °C overnight followed by incubation with 25 μl of Dynabeads ® Protein A (Invitrogen) at 4 °C for 1 h. DNA was extracted from the Dynabeads by means of phenol-chloroform extraction.

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Transforming Growth Factor-β Induces Transcription Factors MafK and Bach1 to Suppress Expression of the Heme Oxygenase-1 Gene *

doi: 10.1074/jbc.M113.450478

Figure Lengend Snippet: MafK and MafG, but not MafF, suppress transcriptional activity of HO-1 . A , pHO1-luc reporter activities were activated by overexpression of Nrf2, and the effects of MafF, MafG, and MafK were examined. Error bars represent S.D. B , interaction between Nrf2 and small Mafs was examined by coprecipitation assays in 293T cells. MafF, MafG, and MafK all coprecipitated Nrf2. C , binding of small Mafs to AREs from HO-1 . HA-tagged MafF, MafG, and MafK were expressed in 293T cells, and the cell lysates were incubated with biotinylated double-stranded DNA fragments and precipitated with avidin beads. Coprecipitated proteins were detected with anti-HA antibody. D , chromatin immunoprecipitation analysis using anti-FLAG antibody detected binding of MafK and MafG to the HO-1 promoter region including ARE (E2) in NMuMG-MafK ( K ) and NMuMG-MafG ( G ) cells, respectively. DNAP , DNA affinity precipitation; IP , immunoprecipitation.

Article Snippet: Soluble chromatin was diluted with 9 volumes of dilution buffer for immunoprecipitation (16.7 m m Tris-HCl, pH 8.1, 1.2 m m EDTA, 167 m m NaCl, 0.01% SDS, 1.1% Triton X-100, 20,000 KIU/ml aprotinin, 1 μg/ml leupeptin) and incubated with normal rabbit IgG, anti-Nrf2 (H-300, Santa Cruz Biotechnology), anti-MafK , anti-Bach1 , or anti-Smad2/3 (clone 18, BD Transduction Laboratories) antibody with end-over-end rotation at 4 °C overnight followed by incubation with 25 μl of Dynabeads ® Protein A (Invitrogen) at 4 °C for 1 h. DNA was extracted from the Dynabeads by means of phenol-chloroform extraction.

Techniques: Activity Assay, Over Expression, Binding Assay, Incubation, Avidin-Biotin Assay, Chromatin Immunoprecipitation, Affinity Precipitation, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Transforming Growth Factor-β Induces Transcription Factors MafK and Bach1 to Suppress Expression of the Heme Oxygenase-1 Gene *

doi: 10.1074/jbc.M113.450478

Figure Lengend Snippet: Cooperative functions of TGF-β/Smad signaling with MafK and Bach1. A and B , binding of Smad3 to MafK ( A ) and Bach1 ( B ). FLAG-MafK, FLAG-Bach1, Myc-Smad3, and a constitutively active TGF-β type I receptor (ALK5TD-V5) were transfected to 293T cells as indicated, and binding of MafK or Bach1 and Smad3 was examined by immunoprecipitation ( IP ) with anti-Myc antibody followed by immunoblotting with anti-FLAG antibody. Arrow , Myc-Smad3; *, IgG. C and D , chromatin immunoprecipitation analyses using anti-Smad2/3 antibody detected binding of Smad2/3 to the HO-1 promoter region including ARE (E2) in NMuMG cells ( C ). Binding of Smad2/3 to the Smad7 promoter region was used as a positive control ( D ). Error bars represent S.D.

Article Snippet: Soluble chromatin was diluted with 9 volumes of dilution buffer for immunoprecipitation (16.7 m m Tris-HCl, pH 8.1, 1.2 m m EDTA, 167 m m NaCl, 0.01% SDS, 1.1% Triton X-100, 20,000 KIU/ml aprotinin, 1 μg/ml leupeptin) and incubated with normal rabbit IgG, anti-Nrf2 (H-300, Santa Cruz Biotechnology), anti-MafK , anti-Bach1 , or anti-Smad2/3 (clone 18, BD Transduction Laboratories) antibody with end-over-end rotation at 4 °C overnight followed by incubation with 25 μl of Dynabeads ® Protein A (Invitrogen) at 4 °C for 1 h. DNA was extracted from the Dynabeads by means of phenol-chloroform extraction.

Techniques: Binding Assay, Transfection, Immunoprecipitation, Western Blot, Chromatin Immunoprecipitation, Positive Control