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. Author manuscript; available in PMC: 2021 Jun 22.
Published in final edited form as: J Immunol. 2020 Jan 31;204(6):1641–1649. doi: 10.4049/jimmunol.1900094

FIGURE 4.

FIGURE 4.

TGF-β induced GATA2/Smad4 complex formation and enhanced Mcpt1 transactivation through the GATA-Smad region. (A) The mRNA levels of GATA1 and GATA2 in TGF-β–treated BMMCs. The data are shown as the mean ± SD of three independent experiments performed with triplicate samples. (B) Association between exogenous GATA2 and Smad4. HEK293T cells cotransfected with expression plasmids for Flag–GATA2 and Myc–Smad4 were stimulated by TGF-β at 24 h after transfection and were harvested after 2 h incubation in the presence or absence of TGF-β. The cell lysates were subjected to immunoprecipitation (IP) and immunoblotting (IB). Aliquot lysates were loaded to independent gels; one for staining by anti-Myc and another for anti-Flag. A typical result of three independent experiments was shown. (C) Western blotting profiles of immunoprecipitated samples (left) and band intensity determined in three independent experiments (right). HEK293T cells were cotransfected with Flag-tagged GATA2 and Myc-tagged Smad2 and HA-tagged Smad4 expression plasmids. After 24 h, cells were treated with TGF-β for an additional 2 h and then subjected to IP and Western blotting assays using the indicated Abs. A single transferred membrane was reprobed. Band densities are shown as ratio to that of control IgG without TGF-β stimulation (n = 3; three independent experiments performed with a single sample). (D) Transcriptional activity driven by the promoter (−299/+32) or the GATA–Smad (−3420/−3218) + promoter (−299/+32) of the Mcpt1 gene was determined by a luciferase assay. TGF-β (10 ng/ml) was added to the culture medium of 293T transfectants at 4 h after transfection, and the cells were harvested after an additional 24 h incubation. (E) The effects of wild-type and 28 bp-deleted GATA-Smad regions on GATA2 and TGF-β signaling-mediated transcriptional activity. After 4 h incubation in culture media without FCS, transfectants were stimulated by 10 ng/ml TGF-β (E and F). (F) Transcriptional activity of tandem repeats of the minimal elements and its mutants. Luciferase activity was normalized to that of β-galactocidase (D–F). The data are expressed as mean ± SD of triplicate samples and are shown as fold change relative to promoterless, untreated cells (D–F). Similar results were obtained in two independent experiments (D–F). *p < 0.05.