Protein interaction AP-MS data for TAP-CSAG1 and TAP-CSAG2 from HEK293 cells
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LTQ Orbitrap Elite
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Kidney
SUBMITTER: 
Patrick Potts
LAB HEAD: Patrick Ryan Potts
PROVIDER: PXD020231 | Pride | 2020-09-01
REPOSITORIES: Pride
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| DatasetEV1.xlsx | Xlsx | |||
| Pottsgrp_060817_cta_VectorControl.raw | Raw | |||
| Pottsgrp_060817_cta_csag1.raw | Raw | |||
| Pottsgrp_060817_cta_csag2.raw | Raw | |||
| checksum.txt | Txt |
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Publications
CSAG2 is a cancer-specific activator of SIRT1.
Yang Xu X Potts Patrick Ryan PR
EMBO reports 20200805 9
SIRT1 is a NAD<sup>+</sup> -dependent deacetylase that controls key metabolic and signaling pathways, including inactivating the p53 tumor suppressor. However, the mechanisms controlling SIRT1 enzymatic activity in the context of cancer are unclear. Here, we show that the previously undescribed CSAG2 protein is a direct activator of SIRT1. CSAG2 is normally restricted to expression in the male germline but is frequently re-activated in cancers. CSAG2 is necessary for cancer cell proliferation an ...[more]
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Project description:Purpose: Investigation of genome-wide changes in R-loop levels after knockdown of DDX41 U2OS cells in comparison to a control knockdown. Method: Concanavalin A-coated beads were activated in Binding Buffer (20 mM Hepes-KOH pH 7.9, 10 mM KCl, 1mM CaCl2, 1 mM MnCl2). 5*105 U2OS cells were washed twice with Wash Buffer (Hepes-NaOH pH7.5, 150 mM NaCl, 0.5 mM Spermidine, 1 mM protease inhibitor) at room temperature and afterwards immobilized on the activated beads in 50 µl Wash Buffer containing 0.05% Digitonin. Either pA-MNase or RHΔ-MNase were added to the cells overnight at 4°C on a rotating wheel. After three washes with Wash Buffer containing 0.05% Digitonin, samples resuspended in 100 µl Dig-Wash-Buffer were equilibrated on ice. Activity of the MNase was triggered by adding 2mM CaCl2 to the samples for 30 minutes. 2x Stop Buffer (68 µl 5M NaCl, 40 µl 0.5 M EDTA, 20 µl 0.2 M EGTA, 10 µl 5% digitonin, 5 µl 10mg/ml RnaseA, 20 pg/ml spike-in Drosophila DNA (Active Motif)) was mixed with the samples to stop the reaction. Chromatin fragments were released by incubating the samples for 20 minutes at 37°C and centrifugation at 16.000×g for 10 minutes at 4°C. Supernatants were incubated at 70°C in the presence of 0.1% SDS and 5 µg proteinase K. Before library preparation, the DNA was recovered by phenol-chloroform extraction Results: We were able to map R-loop dynamics genome-wide in U2OS cells in biological triplicates. MapR signal was significantly increased around the transcription start site in the absence of DDX41 and decreased after treatment with ActinomycinD.
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