topflash fopflash reporter vector  (Millipore)

Journal: EBioMedicine

Article Title: Bach1-induced suppression of angiogenesis is dependent on the BTB domain

doi: 10.1016/j.ebiom.2019.102617

Figure Lengend Snippet: The BTB domain of Bach1 is required for the Bach1-induced suppression of Wnt/β-catenin signaling and Bach1-TCF4 binding. ( a ) AdGFP, AdBach1, and AdBach1-ΔBTB HUVECs were transfected with a TOPflash or FOPflash reporter, and luciferase activity was quantified in the presence (+) and absence (–) of Wnt3a (200 ng/mL) stimulation; results were normalized to measurements in AdGFP-transfected cells cultured without Wnt3a ( n = 3, * P < 0.05, ** P < 0.01, one-way analysis of variance). ( b ) VEGF-A mRNA levels in AdGFP, AdBach1, and AdBach1-ΔBTB HUVECs were measured via real-time PCR and normalized to measurements in AdGFP-transfected cells ( n = 3, ** P < 0.01, one-way analysis of variance). ( c ) AdGFP, AdBach1, and AdBach1-ΔBTB HUVECs were transfected with a reporter construct coding for luciferase production from the VEGF promoter; then, luciferase activity was quantified and normalized to measurements in AdGFP-transfected cells ( n = 3, * P < 0.05, one-way analysis of variance). ( d ) HEK293T cells that had been transfected with an empty vector or with a Bach1-Flag or Bach1-ΔBTB-Flag and MARES reporter for 24 h and incubated with or without Wnt3a for 12 h, luciferase activity was quantified and normalized to measurements in control vector-transfected cells ( n = 3, * P < 0.05, ** P < 0.01, one-way analysis of variance). (e) Bacterially expressed GST-tagged versions of the full Bach1 protein (Bach1-Full-GST) or with mutated versions of Bach1 that lacked either the N-terminal BTB domain or the other Bach1 deletion mutants was incubated with Flag-tagged TCF4; then, the reaction products were precipitated with glutathione-Sepharose 4B beads, and TCF4 was detected in the precipitate via Western blot with anti-Flag antibodies. ( f ) HEK293T cells were transfected with 2 vectors, one coding for TCF4-HA, and the other coding for Flag-tagged versions of the full Bach1 sequence and Bach1 sequences lacking amino acid residues 81–89 (Bach1-Δ(81–89)), the cells were lysed; then, Bach1 and Bach1-Δ(81–89) were immunoprecipitated from the lysate with an anti-Flag antibody, and TCF4 was detected in the precipitate with an anti-HA antibody. ( g ) HEK293T cells were transfected with Flag-tagged Bach1 and lysed; then, the lysate was incubated with GST or GST-tagged versions of TCF4 or the indicated TCF4-deletion mutants, the GST-bound proteins were eluted, and the presence of Bach1 was evaluated via Western blot with an anti-Flag antibody.

Article Snippet: The TOPflash and FOPflash reporter vector were purchased from Millipore (Billerica, MA).

Techniques: Binding Assay, Transfection, Luciferase, Activity Assay, Cell Culture, Real-time Polymerase Chain Reaction, Construct, Plasmid Preparation, Incubation, Western Blot, Sequencing, Immunoprecipitation