function generator 182 Search Results


fancd2  (Novus Biologicals)
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Novus Biologicals fancd2
Workflow for MS-based quantification of CFS associated proteins. ( A ) Experimental workflow for SILAC-based quantitative MS identification of CFS associated proteins. ( B ) Flow cytometry analysis of cell cycle distribution of SILAC labeled cells used for enrichment of CFSs as illustrated in (A). ( C ) IF of <t>FANCD2</t> and EdU incorporation to assess formation of FANCD2 foci at late replicating regions with and without APH treatments. Cells were synchronized as in (A) and (B).
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Workflow for MS-based quantification of CFS associated proteins. ( A ) Experimental workflow for SILAC-based quantitative MS identification of CFS associated proteins. ( B ) Flow cytometry analysis of cell cycle distribution of SILAC labeled cells used for enrichment of CFSs as illustrated in (A). ( C ) IF of FANCD2 and EdU incorporation to assess formation of FANCD2 foci at late replicating regions with and without APH treatments. Cells were synchronized as in (A) and (B).

Journal: Nucleic Acids Research

Article Title: Proteomic characterization of chromosomal common fragile site (CFS)-associated proteins uncovers ATRX as a regulator of CFS stability

doi: 10.1093/nar/gkz510

Figure Lengend Snippet: Workflow for MS-based quantification of CFS associated proteins. ( A ) Experimental workflow for SILAC-based quantitative MS identification of CFS associated proteins. ( B ) Flow cytometry analysis of cell cycle distribution of SILAC labeled cells used for enrichment of CFSs as illustrated in (A). ( C ) IF of FANCD2 and EdU incorporation to assess formation of FANCD2 foci at late replicating regions with and without APH treatments. Cells were synchronized as in (A) and (B).

Article Snippet: The primary antibodies used were ATRX (Santa Cruz, sc-55584), FANCD2 (Novus Biologicals, NB100-182), 53BP1 (Novus Biologicals, 100-304A2), Cyclin A (Abcam, ab16726) and DAXX (Bethyl Laboratories, A301-353A).

Techniques: Multiplex sample analysis, Flow Cytometry, Labeling

Overview and analysis of MS-based identification of CFS associated proteins. ( A ) Results and filtering of identifications from MS analysis. ( B ) GO term enrichment analysis for biological process and cellular localization within the 226 proteins that were more abundant in the FANCD2 pull-down compared to IgG. Analysis was performed using the DAVID tool with the human genome as reference. ( C ) Functional network analysis of all 226 FANCD2 enriched proteins. The color of the nodes indicates the level on enrichment in the FANCD2 pull-down compared to IgG. Diamond shaped nodes indicate those that were enriched in the FANCD2 pull-down from APH treated cells compared to vehicle treated. Proteins with well-defined relevant functions have been grouped. The network was generated using the STRING database in Cytoscape.

Journal: Nucleic Acids Research

Article Title: Proteomic characterization of chromosomal common fragile site (CFS)-associated proteins uncovers ATRX as a regulator of CFS stability

doi: 10.1093/nar/gkz510

Figure Lengend Snippet: Overview and analysis of MS-based identification of CFS associated proteins. ( A ) Results and filtering of identifications from MS analysis. ( B ) GO term enrichment analysis for biological process and cellular localization within the 226 proteins that were more abundant in the FANCD2 pull-down compared to IgG. Analysis was performed using the DAVID tool with the human genome as reference. ( C ) Functional network analysis of all 226 FANCD2 enriched proteins. The color of the nodes indicates the level on enrichment in the FANCD2 pull-down compared to IgG. Diamond shaped nodes indicate those that were enriched in the FANCD2 pull-down from APH treated cells compared to vehicle treated. Proteins with well-defined relevant functions have been grouped. The network was generated using the STRING database in Cytoscape.

Article Snippet: The primary antibodies used were ATRX (Santa Cruz, sc-55584), FANCD2 (Novus Biologicals, NB100-182), 53BP1 (Novus Biologicals, 100-304A2), Cyclin A (Abcam, ab16726) and DAXX (Bethyl Laboratories, A301-353A).

Techniques: Functional Assay, Generated

ATRX associates with chromatin under conditions that induce CFS expression. ( A ) IF analysis of ATRX (green) in G2 cells. HeLa cells were treated with DMSO or APH (0.2 μM) for 20 h, and G2 cells were selected based on DAPI profile. Representative images are shown. ( B ) Analysis of the number of ATRX and FANCD2 foci across the cell cycle. HeLa cells were incubated with DMSO or APH (0.2 μM) for 20 h and EdU (20 μM) was incorporated for the last 30 min. Analysis was performed using quantitative image-based cytometry (QIBC). DAPI and EdU profiles were used to determine cell cycle stages (G1, early, mid and late S and G2). Z-stack images were acquired to determine the number of foci per cell. The average number of ATRX foci per cell in each stage is illustrated in the box plots for vehicle (blue) or after APH treatment (green). Data shown corresponds to the average of >700 cells per condition. Means, SDs, maximum and minimum values are indicated. Significance was evaluated with unpaired t -test **** P ≤ 0.0001. ( C ) IF analysis for colocalization of ATRX (green) and FANCD2 (red) in G2 cells. HeLa cells were treated with DMSO or APH (0.2 μM) for 20 h. Two different colocalization patterns are indicated: complete foci overlap (middle row and indicated by white arrows) and adjacent foci (bottom row). Representative images are shown. Selected regions are magnified. ( D ) Quantification of FANCD2 and ATRX colocalizing foci per G2 cell. Data shown correspond to biological duplicates of 300 cells per condition. Means and SDs are indicated. Significance of the total number of colocalizing foci was evaluated with unpaired t -test **** P ≤ 0.0001. ( E ) Quantification of the percentage of FRA16D loci colocalizing with ATRX foci. HeLa cells were treated with DMSO or APH (0.2 μM) for 20 h and arrested at G2. Data shown corresponds to biological triplicates of 300 cells per condition. Means and SDs are indicated. Significance was evaluated with unpaired t -test. * P ≤ 0.05. ( F ) Example of ATRX IF (red) colocalizing with FRA16D (green) in G2 cells. Selected regions are magnified.

Journal: Nucleic Acids Research

Article Title: Proteomic characterization of chromosomal common fragile site (CFS)-associated proteins uncovers ATRX as a regulator of CFS stability

doi: 10.1093/nar/gkz510

Figure Lengend Snippet: ATRX associates with chromatin under conditions that induce CFS expression. ( A ) IF analysis of ATRX (green) in G2 cells. HeLa cells were treated with DMSO or APH (0.2 μM) for 20 h, and G2 cells were selected based on DAPI profile. Representative images are shown. ( B ) Analysis of the number of ATRX and FANCD2 foci across the cell cycle. HeLa cells were incubated with DMSO or APH (0.2 μM) for 20 h and EdU (20 μM) was incorporated for the last 30 min. Analysis was performed using quantitative image-based cytometry (QIBC). DAPI and EdU profiles were used to determine cell cycle stages (G1, early, mid and late S and G2). Z-stack images were acquired to determine the number of foci per cell. The average number of ATRX foci per cell in each stage is illustrated in the box plots for vehicle (blue) or after APH treatment (green). Data shown corresponds to the average of >700 cells per condition. Means, SDs, maximum and minimum values are indicated. Significance was evaluated with unpaired t -test **** P ≤ 0.0001. ( C ) IF analysis for colocalization of ATRX (green) and FANCD2 (red) in G2 cells. HeLa cells were treated with DMSO or APH (0.2 μM) for 20 h. Two different colocalization patterns are indicated: complete foci overlap (middle row and indicated by white arrows) and adjacent foci (bottom row). Representative images are shown. Selected regions are magnified. ( D ) Quantification of FANCD2 and ATRX colocalizing foci per G2 cell. Data shown correspond to biological duplicates of 300 cells per condition. Means and SDs are indicated. Significance of the total number of colocalizing foci was evaluated with unpaired t -test **** P ≤ 0.0001. ( E ) Quantification of the percentage of FRA16D loci colocalizing with ATRX foci. HeLa cells were treated with DMSO or APH (0.2 μM) for 20 h and arrested at G2. Data shown corresponds to biological triplicates of 300 cells per condition. Means and SDs are indicated. Significance was evaluated with unpaired t -test. * P ≤ 0.05. ( F ) Example of ATRX IF (red) colocalizing with FRA16D (green) in G2 cells. Selected regions are magnified.

Article Snippet: The primary antibodies used were ATRX (Santa Cruz, sc-55584), FANCD2 (Novus Biologicals, NB100-182), 53BP1 (Novus Biologicals, 100-304A2), Cyclin A (Abcam, ab16726) and DAXX (Bethyl Laboratories, A301-353A).

Techniques: Expressing, Incubation, Cytometry

ATRX regulates CFS stability in a DAXX-dependent manner. ( A ) Quantification of 53BP1 N.B. in HeLa cells transfected with the indicated siRNAs. Two days post-transfection, cells were treated with DMSO or APH (0.2 μM) for 20 h. Cyclin A negative cells were selected as G1 cells. Data correspond to three biological replicates of >600 cells per condition. Means and SDs are indicated. Significance was assessed by unpaired t -test. n.s. >0.05;*** P ≤ 0.001;**** P ≤ 0.0001. ( B ) IF analysis of ATRX (green) and DAXX (red) in G2 cells. HeLa cells were transfected with the indicated siRNAs and two days post-transfection, cells were treated with APH (0.2 μM) for 20 h. Representative images are shown. (C) Quantification of 53BP1 N.B. in HeLa cells transfected with the indicated siRNAs. Two days post-transfection, cells were treated with DMSO or APH (0.2 μM) for 20 h. Cyclin A negative cells were selected as G1 cells. Data correspond to biological triplicates of >600 cells per condition. Means and SDs are indicated. Significance was assessed by unpaired t -test. n.s. >0.05;**** P ≤ 0.0001. ATRX, FANCD2 and DAXX depletion was verified by IF in all the experiments performed.

Journal: Nucleic Acids Research

Article Title: Proteomic characterization of chromosomal common fragile site (CFS)-associated proteins uncovers ATRX as a regulator of CFS stability

doi: 10.1093/nar/gkz510

Figure Lengend Snippet: ATRX regulates CFS stability in a DAXX-dependent manner. ( A ) Quantification of 53BP1 N.B. in HeLa cells transfected with the indicated siRNAs. Two days post-transfection, cells were treated with DMSO or APH (0.2 μM) for 20 h. Cyclin A negative cells were selected as G1 cells. Data correspond to three biological replicates of >600 cells per condition. Means and SDs are indicated. Significance was assessed by unpaired t -test. n.s. >0.05;*** P ≤ 0.001;**** P ≤ 0.0001. ( B ) IF analysis of ATRX (green) and DAXX (red) in G2 cells. HeLa cells were transfected with the indicated siRNAs and two days post-transfection, cells were treated with APH (0.2 μM) for 20 h. Representative images are shown. (C) Quantification of 53BP1 N.B. in HeLa cells transfected with the indicated siRNAs. Two days post-transfection, cells were treated with DMSO or APH (0.2 μM) for 20 h. Cyclin A negative cells were selected as G1 cells. Data correspond to biological triplicates of >600 cells per condition. Means and SDs are indicated. Significance was assessed by unpaired t -test. n.s. >0.05;**** P ≤ 0.0001. ATRX, FANCD2 and DAXX depletion was verified by IF in all the experiments performed.

Article Snippet: The primary antibodies used were ATRX (Santa Cruz, sc-55584), FANCD2 (Novus Biologicals, NB100-182), 53BP1 (Novus Biologicals, 100-304A2), Cyclin A (Abcam, ab16726) and DAXX (Bethyl Laboratories, A301-353A).

Techniques: Transfection