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Blood, Vol. 113, Issue 10, 2191-2201, March 5, 2009
SCL and associated proteins distinguish active from repressive GATA transcription factor complexes
Blood Tripic et al.
113: 2191
Supplemental materials for: Tripic et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 87.1 KB)
- Table S1. DNA segments (PDF, 23.9 KB)
- Figure S1. CBP levels at GATA-1 regulated genes (JPG, 68.8 KB)
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ChIP analysis with CBP antibodies or isotype-matched control antibodies (IgG) of the indicated sites at the β-globin (Hbb-b1) locus (top panel), the Kit (middle panel), and Gata2 (bottom panel) genes. The region 1 kb upstream region of the Hbb-b1 promoter (–1 kb) served as a negative control. ChIP experiments were performed in G1E cells and G1E-ER4 cells after E2 treatment for 21–24h. The data shown are the averages of two independent experiments. Error bars represent standard deviations. Note that while CBP is recruited by GATA-1 to active genes such as the β-globin locus as previously observed 24 it is also found at the GATA-1 repressed genes Kit and Gata2.
- Figure S2. Co-occupancy of Ldb-1, LMO2, and E2A at GATA-1–activated genes (JPG, 77.5 KB)
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ChIP analysis using Ldb-1, LMO2, and E2A antibodies. Experiments were performed in G1E cells and G1E-ER4 cells after E2 treatment for 21–24h. The data are the averages of three or more independent experiments. Error bars represent standard deviations.
- Figure S3. Representative Western blots with indicated antibodies of nuclear extracts from G1E cells and G1E-ER4 cells after E2 treatment for 24 h (JPG, 50.8 KB)
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- Figure S4. Time course ChIP experiments using anti–GATA-1 and anti-SCL antibodies were performed in G1E-ER4 cells at indicated time points following treatment with estradiol (JPG, 46 KB)
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Primer pairs were directed against the Hbb-b1 promoter or as control a region 1 kb upstream. The data shown are the averages of two independent experiments. Error bars represent standard deviations.
- Figure S5. Association of the SCL complex with the active and inactive α-globin (Hba-a1) locus (JPG, 82.5 KB)
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ChIP analysis using GATA-1, GATA-2, SCL, Ldb-1, LMO2, and E2A antibodies or isotype-matched control antibodies. ChIP experiments were done in G1E cells and G1E-ER4 cells after E2 treatment for 21–24h. The data shown are the averages of three or more independent experiments. Error bars represent standard deviations.
- Figure S6. SCL is recruited to GATA-1 target genes in primary erythroid cells (JPG, 83.2 KB)
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ChIP analysis of indicated genes in fetal liver cells from E14.5 mice using GATA-1, SCL, Ldb-1, and LMO2 antibodies or isotype-matched control antibodies. Two independent ChIP experiments were performed using separate mice and the data sets were averaged. Error bars represent standard deviations.
- Figure S7. Reduced occupancy of Ldb-1, LMO2, and E2A at GATA-1–repressed Kit gene (JPG, 61.3 KB)
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ChIP analysis using Ldb-1, LMO2, E2A, or isotype matched control antibodies and primer sets for Kit locus . ChIP experiments were performed in G1E cells and G1E-ER4 cells after E2 treatment for 24 hours. The data are the averages of three or more independent experiments. Error bars represent standard deviations.
- Figure S8. LMO2 is required for activation but not repression of GATA-1–regulated genes (JPG, 54 KB)
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Bcl-XL–expressing G1E-ER4 cells expressing an shRNA against LMO2 or empty vector were treated with estradiol (E2) for 24 hours and mRNA levels of indicated genes were determined by real-time RT-PCR. Results were normalized to Gapdh mRNA. The data are the averages of 4 independent experiments. Error bars represent standard deviation.
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