Computational protocol: RBPJ/CBF1 interacts with L3MBTL3/MBT1 to promote repression of Notch signaling via histone demethylase KDM1A/LSD1

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Protocol publication

[…] U87‐MG cells transfected with pcDNA3‐HA‐DEST encoding RBPJ, L3MBTL3, or EGFP control were collected, washed with ice‐cold PBS, and lysed in ice‐cold lysis buffer [50 mM Tris pH 7.8, 150 mM NaCl, 0.5% NP‐40, 10% glycerol, 2 mM NaF, 2 mM Na3VO4, and Complete® protease inhibitor (1× final, Roche®, 05 056 489 001)]. HA‐tagged proteins were affinity‐purified with 50 μl of α‐HA agarose beads (Sigma®, A2095) at 4°C for 2 h with rotation. Beads were washed four times with lysis buffer, three times with washing buffer (50 mM Tris pH 7.8, 100 mM NaCl, 0.1% NP‐40), and three times with 50 mM NH4HCO3. Proteins were eluted twice with 50 μl of 1% ammonia (NH4OH; Sigma®, 338818), dried, and resuspended in 20 μl Laemmli sample buffer. Proteins were resolved via SDS–PAGE and the whole gel lanes were cut into five pieces that were individually subjected to in‐gel tryptic digestion, as previously described (Shevchenko et al, ). Peptides were dried and analyzed via LC‐MS/MS system, as follows.Peptides were resolved on a nano‐capillary reverse phase column (PicoFrit column, New Objective®) using a 5–50% acetonitrile gradient at 300 nl/min and directly introduced into an ion‐trap mass spectrometer (LTQ XL, Thermo Fisher®). Data‐dependent MS/MS spectra on the five most intense ions from each full MS scan were collected (relative collision energy ~35%). Proteins were identified by searching the data against Swiss‐Prot human database (January 9th 2013) appended with decoy (reverse) sequences using the X!Tandem/Trans‐Proteomic Pipeline software suite (Pedrioli, ). All peptides and proteins with a PeptideProphet (Keller et al, ) and ProteinProphet (Nesvizhskii et al, ) probability score of >0.8 (false discovery rate < 2% estimated using a target‐decoy strategy) were considered positive identifications. Proteins were considered potential RBPJ interactors if they were identified with two or more mass spectra in both duplicate RBPJ AP‐MS experiments but not in the EGFP‐negative AP‐MS control experiments. Proteins identified in > 10% of the AP‐MS experiments available in the CRAPome database version 1.1, a contaminant repository for AP‐MS data (Mellacheruvu et al, ), were considered background contaminants and removed from the analysis. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (Vizcaino et al, ) partner repository with the dataset identifier PXD004196. […]

Pipeline specifications

Software tools TPP, PeptideProphet, ProteinProphet
Databases UniProt ProteomeXchange CRAPome
Application MS-based untargeted proteomics
Organisms Drosophila melanogaster, Caenorhabditis elegans