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Status |
Public on May 08, 2019 |
Title |
Stat3 at 400nM concentration |
Sample type |
protein |
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Source name |
Mouse Stat3 (DBD, phosphorylated)
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Organism |
Mus musculus |
Characteristics |
transcription factor: Stat3 protein concentration: Protein concentration: 400nM
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Growth protocol |
GATA1, TCF3: Gateway-compatible clones containing the full-length genes were purchased from GeneCopoeia and the genes were transferred into pDEST15 using the LR Clonase reaction (Life Technologies). This vector enables the production of N-terminally GST-tagged proteins. Cells were grown in LB broth to an OD600 of 0.4 to 0.6 and then expression was induced with IPTG at 30° to 37° C. Pelleted cells were frozen, stored at -20 C, and thawed cells were lysed with lysozyme. The DBD of E2F7 was a kind gift from the Nevins lab (Duke University), where it was expressed and purified as described in Liu et al. 2003 (DOI: 10.1074/jbc.M113.467506). The DBD of EGR1 was a kind gift from the Iwahara lab (University of Texas Medical Branch), where it was expressed and purified as described in Chattopadhyay et al. 2016 (DOI: 10.1021/acs.biochem.6b00757). FOXO3 was a kind gift from the Nevins lab (Duke University), where it was expressed in human 293 cells, as described in Shats et al. 2013 (DOI: 10.1158/0008-5472.CAN-13-0453). MITF was a kind gift from the Young lab (MIT), where it was expressed and purified as described in Lin et al. 2013 (DOI: 10.1016/j.cell.2012.08.026). The DBD of mouse Stat3 was a kind gift from the Arbely lab (Ben Gurion University) where it was expressed and purified as described in Belo et al. 2018 (DOI: https://doi.org/10.1101/537696).
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Extracted molecule |
protein |
Extraction protocol |
GST-tagged GATA1 and TCF3 were purified from the soluble portion of the lysate using GST resin (GE Healthcare) according to manufacturer’s instructions. The DBD of E2F7 (from the Nevins lab) was purified as described in Liu et al. 2003 (DOI: 10.1074/jbc.M113.467506). The DBD of EGR1 (from the Iwahara lab) was purified as described in Chattopadhyay et al. 2016 (DOI: 10.1021/acs.biochem.6b00757. FOXO3 (cell lysates from the Nevins lab) was purified by immunoprecipitation using anti-Flag M2-agarose affinity gels (Sigma) according to manufacturer's instructions. MITF (from the Young lab) was purified as described in Lin et al. 2013 (DOI: 10.1016/j.cell.2012.08.026). The DBD of mouse Stat3 (from the Arbely lab) was purified as described in Belo et al. 2018 (DOI: https://doi.org/10.1101/537696).
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Label |
Alexa 488
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Label protocol |
Proteins were tagged with N-terminal GST (EGR1, GATA1, TCF3), N-terminal 6xHis (E2F7, Stat3, MITF), or C-terminal Flag (FOXO3) by cloning. Protein-bound arrays were incubated with Alexa-488-conjugated antibody (to GST: Invitrogen A11131, to 6xHis: Qiagen 35310, to Flag: Fisher 50-193-003).
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Hybridization protocol |
Double-stranded microarrays were first pre-moistened in PBS / 0.01% Triton X-100 for 5 min and blocked with PBS / 2% (wt/vol) nonfat dried milk (Sigma) for 1 h. Microarrays were then washed once with PBS / 0.1% (vol/vol) Tween-20 for 5 min and once with PBS / 0.01% Triton X-100 for 2 min. Proteins were diluted to 200 or 100 nM in a 175-μl protein binding reaction containing PBS / 2% (wt/vol) milk / 51.3 ng/μl salmon testes DNA (Sigma) / 0.2 μg/μl bovine serum albumin (New England Biolabs). Preincubated protein binding mixtures were applied to individual chambers of a four-chamber gasket cover slip in a steel hybridization chamber (Agilent), and the assembled microarrays were incubated for 1 h at room temperature. Microarrays were again washed once with PBS / 0.5% (vol/vol) Tween-20 for 3 min, and then once with PBS / 0.01% Triton X-100 for 2 min. Alexa-488-conjugated antibody was diluted to 50 μg/ml in PBS / 2% milk and applied to a single-chamber gasket cover slip (Agilent), and the assembled microarrays were again incubated for 1 h at 20°C. Finally, microarrays were washed twice with PBS / 0.05% (vol/vol) Tween-20 for 3 min each, and once in PBS for 2 min. After each hour-long incubation step, microarrays and cover slips were disassembled in a staining dish filled with 500 ml of the first wash solution. All washes were performed in Coplin jars on an orbital shaker at 125 r.p.m. Immediately following each series of washes, microarrays were rinsed in PBS (slowly removed over approximately 10 seconds) to ensure removal of detergent and uniform drying.
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Scan protocol |
Protein-bound microarrays were scanned to detect Alexa-488-conjugated antibody (488 nm ex, 522 nm em) using at least two different laser power settings to best capture a broad range of signal intensities and ensure signal intensities below saturation for all spots. Microarray TIF images were analyzed using GenePix Pro version 6.0 software (Molecular Devices), bad spots were manually flagged and removed, and data from multiple Alexa 488 scans of the same slide were combined using masliner (MicroArray LINEar Regression) software.
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Data processing |
To correct for any possible non-uniformities in protein binding, we adjusted the Alexa 488 signals according to their positions on the microarray. We calculated the median normalized intensity of the 15 x 15 block centered on each spot and divided the spot's signal by the ratio of the median within the block to the median over the entire chamber. Next, the Seed-and-wobble algorithm was applied to compute enrichment scores (E-scores) and median intensities for all possible 8-mers.
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Submission date |
May 07, 2019 |
Last update date |
May 08, 2019 |
Contact name |
Raluca Gordan |
E-mail(s) |
raluca.gordan@duke.edu
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Organization name |
Duke University
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Department |
Center for Genomic and Computational Biology
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Street address |
101 Science Dr, CIEMAS 2179
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City |
Durham |
State/province |
NC |
ZIP/Postal code |
27708 |
Country |
USA |
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Platform ID |
GPL23935 |
Series (2) |
GSE130831 |
QBiC-Pred: Quantitative Predictions of Transcription Factor Binding Changes Due to Sequence Variants I |
GSE130837 |
QBiC-Pred: Quantitative Predictions of Transcription Factor Binding Changes Due to Sequence Variants |
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