IndraLab

Statements

Flag binds MYSM1. 20 / 20
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"NIH/3T3 cells were co-transfected with Myc-STAT1, Flag-MYSM1 and HA-Ub or its mutants (HA-K48/K63)."
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"As an internal quality control, successful and specific co-IP of fusion proteins by anti-Flag compared with control IgG were affirmed in lysates from MYSM1-Flag transfected 293T cells vs. GFP-Flag controls, respectively, prior to sample analysis by mass spectrometry ( xref C)."
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"The results demonstrated that exogenous Flag-MYSM1 and Myc-STAT1 were also efficiently co-immunoprecipitated (Figure xref H)."
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"In addition, as proof of principle of the co-IP approach, MYSM1 interaction with ubiquitinated histone H2A (H2Aub), a known substrate of the H2A deubiquitinase, was detectable in the anti-Flag pull-down from MYSM1-Flag transfectants, as expected ( xref C, lower panel)."
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"As presented inFigure xref C-D, the degradation rate of STAT1 was reduced in Flag-MYSM1 overexpressing NIH/3T3 cells under CHX treatment."
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"To identify new candidate MYSM1 interaction partners and further investigate the molecular function of MYSM1 in DNA damage responses (DDR), we subsequently performed co-immunoprecipitation (co-IP) experiments in 293T cells transiently overexpressing MYSM1-Flag or GFP-Flag upon treatment with etoposide, followed by an SCX-HPLC MS/MS mass spectrometry proteomics approach [ xref , xref ]."
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"NIH/3T3 cells were co-transfected with Myc-TRIM21, Flag-MYSM1 (MYSM1-WT) or its mutants lacking SANT domain (△SANT, aa 113–164), SWIRM domain (△SWIRM, aa 363–464), or MPN domain (△MPN aa 563–673) (Fig.  xref A)."
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"Identification of MYSM1 Interaction Partners by Co-Immunoprecipitation-coupled Mass Spectrometry in MYSM1-Flag 293T Cells."
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"Analogous to the loss-of-function assessment of MYSM1, the gain-of-function of MYSM1 was evaluated by transfecting HL-1 cells with Flag-MYSM1 plasmid (oeMYSM1, Fig. xref D)."
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"A total of 256 modified proteins showed significantly reduced ubiquitination levels in cells overexpressing Flag-MYSM1 (Figure xref A below)."
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"To explore the potential substrates of MYSM1, we transfected Flag-MYSM1 or Flag-EV plasmid into HL-1 cells before DOX treatment and then screened for specific proteins interacting with MYSM1 through Co-IP combined with LC-MS/MS (Fig.  xref A)."
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"Subsequently, the IP eluates from MYSM1-Flag and GFP-Flag transfected 293T cells, processed according to the workflow in xref A, were subjected to SCX-HPLC MS/MS mass spectrometry in order to identify novel MYSM1 interactions in DDR."
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"The Co-IP of Flag-MYSM1 was verified by immunoblotting (Fig.  xref B)."
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"At first, robust expression of GFP-Flag and MYSM1-Flag fusion proteins, as well as DNA damage induction by etoposide, were confirmed in comparison to endogenous MYSM1 levels in parental and DMSO-treated 293 T cells by Western blot ( xref B) and by IF analyses (not shown)."
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"Then Flag-MYSM1 and Myc-TRIM21 plasmids were co-transfected into NIH/3T3 cells, confirming the combination of exogenous MYSM1 and TRIM21 (Fig.  xref G)."
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"We also performed a Co-IP assay in NIH/3T3 co-transfecting with Flag-MYSM1 and Myc-STAT1."
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"Specific potential MYSM1 interactors were classified as proteins with at least > 16-fold (log2(16) = 4) differential enrichment in MYSM1-Flag anti-Flag vs. IgG eluates, but not in the GFP-Flag eluates."
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"To determine the domain required for MYSM1-STAT1 interaction, we co-transfected NIH/3T3 cells with Myc-STAT1, Flag-MYSM1 (MYSM1-WT) or its mutants lacking SANT domain (△SANT), SWIRM domain (△SWIRM), or MPN domain (△MPN) (Figure xref F)."
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"In differential display analyses, relevant DNA damage-dependent MYSM1 candidate interaction partners were defined as proteins differentially enriched by at least 4-fold (log2(4) = 2) in MYSM1-Flag eluates upon etoposide vs. DMSO treatment by anti-Flag vs. IgG IP, but not present in GFP-Flag eluates ( xref A)."
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"CUT&Tag using antibodies against Flag and analysis with deepTools revealed a gradual enhanced enrichment of Flag peaks in NSCs that stably expressed Mysm1-Flag with increasing induction time (Fig. xref )."
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