MassIVE MSV000092739

Complete Public PXD044816

PML::RARA and GATA2 proteins interact via DNA templates to induce aberrant self-renewal in hematopoietic cells

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Description

The global protein interactions of the oncofusion protein PML::RARA were identified using proximity labeling followed by mass spectrometry. Briefly, Lineage-depleted bone marrow cells from C57Bl/6 mice were transduced with MSCV-IRES-GFP retroviruses containing an enhanced biotin ligase (TurboID) fused to either the N-terminus or C-terminus of PML::RARAWT (TurboID-PML::RARAWT and PML::RARAWT-TurboID respectively), TurboID-PML::RARAC88A (mutation in the DNA binding domain of RARA), or a TurboID cDNA alone. A flexible Glycine/Serine linker placed between TurboID and PML::RARA allows TurboID to freely rotate, and biotinylate proteins within 10 angstroms of the TurboID-PML::RARA fusion protein. Four days following the first round of transduction, cells were flow sorted on GFP+ cells. Two days following the sort, 1-5 million cells were treated with 50 uM Biotin for four hours at 37 degrees C. Cell lysates were prepared, sonicated, and incubated with streptavidin agarose beads overnight. Beads were washed, peptides were eluted and digested, and then run on a timsTOF Pro 2. [doi:10.25345/C5ZP3W98W] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: PML::RARA ; TurboID ; Proximity labeling ; Biotin ligase ; mass spectrometry

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Principal Investigators:
(in alphabetical order) 		Timothy J. Ley, Washington University in St. Louis, United States of America
Submitting User: jimmalone
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