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General Information
Name
TF_Interaction_Network-2
Species
D. melanogaster
Result type
FlyBase ID
FBlc0000679
Project
Data Provider
Title
624 high-confidence co-complex membership interactions (2% FDR) involving 647 proteins, 229 of which are transcription factors.
Status
Current
Accessions
    Biosample Source
    Overview
    Strain
    Stage
    Sex
    Tissue isolated
    Other tissues studied
    Cell component
    Key genes
    Sample preparation

    Open reading frames were transferred from the BDGP Drosophila melanogaster expression-ready clone set (FBrf0213191) to the pMK33-C-FLAG-HA acceptor vector (see DPiM_cDNA_bait collection report). Each clone was transiently transfected into a 54 ml culture of Drosophila S2R+ cells. Protein expression was induced with 0.35 mM CuSO4 for 24 hours and nuclear extracts were prepared (PMID: 6828386) then diluted 1:1 with dialysis buffer (20mM HEPES [pH 7.6], 20% glycerol, 100 mM KCl, 2mM MgCl2, 0.1 mM EDTA, 1 mM DTT, 0.25 mM PMSF and Roche Complete protease inhibitor).

    Biosamples analyzed by this result (1)
    Biosample
    Type
    Title
    D. melanogaster, S2R+ cell line, transiently transfected with bait protein, source for TFIN affinity purifications.
    Data Analyzed
    Assay methods
    Key genes
    Protocol

    Each clarified lysate was bound to 40 ul of crosslinked HA immunoaffinity resin (Sigma) for 3 hr at 4oC. Unbound proteins were washed off with dialysis buffer. Bound proteins were eluted using IgG elution buffer (Thermo Scientific Pierce), and neutralized with 1 M Tris [pH 8.0].

    Mode of Assay

    The copurified proteins were precipitated using trichloroacetic acid, washed with TCA and acetone, dried, digested overnight with trypsin, and analyzed by LC-MS/MS. MS/MS spectra were searched with SEQUEST (PMID: 18774840) against FlyBase Release 5.41.

    Raw Data Analyzed (1)
    Assay / Reagent collection
    Type
    Title
    Coaffinity purification of proteins interacting with transcription-related factors, identification by mass spectrometry, S2R+ cells.
    Processed Data Analyzed (0)
    Result
    Type
    Title
    Analysis
    Methods
    Reference Genome
    Reference Annotation
    Data analysis

    Following processing and filtering, a high-confidence TF interaction map was generated using the HGSCore method to distinguish specific interactions as described previously (FBrf0216491), but with indirect prey-prey interactions filtered out to focus the network on the TF-interacting subspace. To draw the cutoff for interaction specificity and determine the FDR, we ran HGSCore on 40 simulated data sets randomly sampled from the real data set, until convergence on a cutoff score, resulting in a 2% FDR.

    Comments
    Associated Data
    Size
    Associated interactions
    618 Interactions
    Files
    Additional Information
    Synonyms and Secondary IDs (4)
    Reported As
    Symbol Synonym
    High-Confidence TF Interaction Network
    High-Confidence TF Network Interactions
    TF_Interaction_Network-2
    Name Synonyms
    624 high-confidence co-complex membership interactions (2% FDR) involving 647 proteins, 229 of which are transcription factors.
    Secondary FlyBase IDs
      References (2)