Proteomics

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Protein interaction AP-MS data for TAP-CSAG1 and TAP-CSAG2 from HEK293 cells


ABSTRACT: Ten 15 cm2 plates of HEK293 cells stably expressing TAP-vector, TAP-CSAG1, or TAP-CSAG2 were lysed with TAP lysis buffer (10% (v/v) glycerol, 50 mM HEPES-KOH pH 7.5, 100 mM KCl, 2 mM EDTA, 0.1% (v/v) NP-40, 10 mM NaF, 0.25 mM Na3VO4, 50 mM β-glyerolphosphate, 2 mM DTT, and 1X protease inhibitor cocktail (Sigma)) and cleared supernatants were bound to IgG-Sepharose beads (GE Amersham) and then washed in lysis buffer and TEV buffer (10 mM HEPES-KOH pH 8.0, 150 mM NaCl, 0.1% (v/v) NP-40, 0.5 mM EDTA, 1 mM DTT, and 1X protease inhibitor cocktail). Protein complexes were cleaved off the beads by TEV protease (Sigma) and incubated with calmodulin-Sepharose beads (GE Amersham) in calmodulin binding buffer (10 mM HEPES-KOH pH 8.0, 150 mM NaCl, 1 mM Mg acetate, 1 mM imidazole, 0.1% (v/v) NP-40, 6 mM CaCl2, 10 mM 2-mercaptoethanol) and then washed in calmodulin rinse buffer (50 mM Ammonium bicarbonate pH 8.0, 75 mM NaCl, 1 mM Mg Acetate, 1 mM Imidazole, 2 mM CaCl2) before eluted with SDS sample buffer, subjected to SDS-PAGE, and stained with GelCode Blue stain (Thermo Fisher Scientific) before protein identification by LC-MS/MS.

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

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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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