mdc1  (Bioss)

Journal: Cancers

Article Title: Low-Dose Pesticides Alter Primary Human Bone Marrow Mesenchymal Stem/Stromal Cells through ALDH2 Inhibition

doi: 10.3390/cancers13225699

Figure Lengend Snippet: Pesticide exposure for 21 days induces DNA damage in normal BM-MSCs. ( A ) DNA damage with double strand breaks, quantified by ℗-S139-γH2AX immunoreactivity by flow cytometry, increases in BM-MSCs after pesticide exposure for 21 days (left panel, n = 6), as shown in a representative experiment (middle panel). Comet assays reveal increased nucleus DNA migration after exposure of BM-MSCs to pesticides for 21 days, as shown in a representative experiment (right panel). ( B ) ℗-S139 γH2AX, 53BP1, MDC1 triple immunostaining in BM-MSCs indicates a higher number of double positive 53BP1/℗-S139 γH2AX signals in the nucleus of exposed cells (FOCI) and a loss of MDC1 staining. Data are expressed as percent of the control ± SEM; *, p < 0.05; **, p < 0.01; Friedman followed by post hoc Dunn’s multiple comparisons test. Scalebar: 50 µm ( A ) and 10 µm ( B ).

Article Snippet: After 21 days, cells were fixed with 4% paraformaldehyde, permeabilized (PBS/Triton X-100 0.1%, 10 min, room temperature), blocked with gelatin from cold water fish skin 5%, Triton X-100 0.1% (Sigma-Aldrich) in PBS, and were incubated overnight at 4 °C with anti-phospho-S139 γH2AX monoclonal mouse antibody (Abcam) at 1/1000, anti-MDC1 polyclonal rabbit antibody (Novus Biologicals, Centennial, CO, USA) at 1/500 and anti-53BP1 polyclonal goat antibody (R&D systems) at 1/1000, followed by a secondary anti-mouse antibody labeled with TRITC (Jackson Immunoresearch, Ely, UK), Alexa Fluor ® 647 conjugated anti-goat (Jackson Immunoresearch) and FITC-conjugated anti-rabbit (Jackson Immunoresearch) at 1/2000 each at room temperature for 2 h. Fluorescence images were acquired with a Leica TCS SP8 (Leica, Wetzlar, Germany) confocal microscope and analyzed using ImageJ software.

Techniques: Flow Cytometry, Migration, Pesticides, Triple Immunostaining, Staining, Control

Journal: eLife

Article Title: Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response

doi: 10.7554/eLife.61407

Figure Lengend Snippet: ( A–E ) Immunofluorescence microscopy in combination with PLA for protein-protein interactions (red dots) within single cells of stably expressing ( A ) MCF7-GFP, ( B ) MCF7-cavin1-GFP, ( C ) MCF7-cavin2-GFP, ( D ) MCF7-cavin3-GFP, and ( E ) MCF7-CAV1-GFP using monoclonal GFP and polyclonal BRCA1 antibodies. DNA was counterstained with DAPI (blue). Scale bars represent 10 μm. ( F ). Number of red dots/PLA signals in 40–50 cells for each MCF7-GFP-expressing cell line was quantified from three independent experiments using a nested ANOVA. Each biological replicate is color-coded, and the mean ± SEM is presented as a black bar. **p<0.05, **p<0.01.

Article Snippet: Antibody , MDC1 rabbit polyclonal , Novus , Novus Cat#NB10056657, RRID: AB_838567 , WB 1:100.

Techniques: Immunofluorescence, Microscopy, Protein-Protein interactions, Stable Transfection, Expressing

Journal: eLife

Article Title: Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response

doi: 10.7554/eLife.61407

Figure Lengend Snippet: ( A ) Immunofluorescence microscopy in combination with PLA to detect and visualize protein-protein interactions (red dots) within single cells of stably expressing MCF7-GFP and ( B ) MCF7-cavin3-GFP cells using monoclonal BRCA1 (MS 110) and polyclonal GFP antibodies. DNA was counterstained with DAPI (blue). Scale bars represent 10 μm. ( C ) Number of red dots/PLA signals in 40–50 cells for each MCF7-GFP-expressing cell line quantified from three independent experiments using a nested ANOVA. Each biological replicate is color-coded with the mean ± SEM presented as a black bar. **p<0.01.

Article Snippet: Antibody , MDC1 rabbit polyclonal , Novus , Novus Cat#NB10056657, RRID: AB_838567 , WB 1:100.

Techniques: Immunofluorescence, Microscopy, Protein-Protein interactions, Stable Transfection, Expressing

Journal: eLife

Article Title: Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response

doi: 10.7554/eLife.61407

Figure Lengend Snippet: A431 cells treated with control (Con) or siRNAs specific to cavin3 or BRCA1 (oligo 1 and 2). Cells were left untreated or subjected to UV treatment. Cells were subject to immunofluorescence microscopy in combination with PLA using monoclonal mouse BRCA1 and polyclonal rabbit cavin3 antibodies. The number of PLA signals in 40–50 cells was quantified from three independent experiments using a nested ANOVA. Each biological replicate is color-coded with the mean ± SEM presented as a black bar. *p<0.05, **p<0.001.

Article Snippet: Antibody , MDC1 rabbit polyclonal , Novus , Novus Cat#NB10056657, RRID: AB_838567 , WB 1:100.

Techniques: Control, Immunofluorescence, Microscopy

Journal: eLife

Article Title: Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response

doi: 10.7554/eLife.61407

Figure Lengend Snippet: ( A ) Z-score for HeLa WT and cavin3 KO cells (replicates Rep. 1–3) showing upregulated proteins (red) and downregulated proteins (blue). ( B ) Volcano plot showing proteins (red dots) identified by Gene Ontology Biological Process (GOBP) involved in DNA repair. ( C ) Volcano plot showing DNA repair proteins upregulated in cavin3 KO cells. ( D ) Volcano plot showing proteins of the BRCA1 A-complex, BRCA1, BRCC3, MDC1, and UBE4A downregulated in cavin3 HeLa KO cells and upregulation of 53BP1 with a heatmap analysis of the expression of each of these proteins in replicate (Rep. 1–3) HeLa WT and cavin3 KO cells.

Article Snippet: Antibody , MDC1 rabbit polyclonal , Novus , Novus Cat#NB10056657, RRID: AB_838567 , WB 1:100.

Techniques: Expressing

Journal: eLife

Article Title: Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response

doi: 10.7554/eLife.61407

Figure Lengend Snippet: ( A ) Representative western blot analysis of WT and cavin3 KO cells UV time course for cavin3, BRCA1, CAV1, Rad51, and Tubulin. ( B ) Protein components of the BRCA1 A-complex. Blue-colored circles: proteins downregulated in the label-free quantitative (LFQ) proteomics; yellow-colored circles: proteins not detected in the LFQ proteomics of cavin3 KO cells. ( C ) Representative western blot analysis of cavin3, BRCA1, pH2AX, UIM1C/Rap80, BARD1, Rad51, MDC1, RNF168, BRCC36, Merit40, BRCA2, CAV, PKM, PGK1, and Actin in WT and cavin3 KO HeLa cells untreated (-) or UV treated (UV) followed by a 4 hr chase. Quantitation of protein levels from three independent experiments is presented in . ( D ) Representative immunofluorescence images of BRCA1 foci after UV treatment in WT HeLa cells. ( E ) Percentage of cells with more than five BRCA1 foci, Rap80 foci, and γH2AX foci in WT and cavin3 KO cells following UV treatment and a 30 min chase. The results are presented as mean ± SD using a one-way ANOVA and Bonferroni’s multiple comparisons test from three independent experiments. ( F ) WT and cavin3 KO cells untreated or treated with the PARP inhibitor (AZD2461, PARPi) 5 nM for 6 days were subjected to comet assays. The results are presented as the mean ± SEM using a one-way ANOVA and Bonferroni’s multiple comparisons test from three independent experiments. Each biological replicate is color-coded. Extent Tail Moment was calculated as described in Materials and methods. NS: not significant; **p<0.01, ***p<0.001; ****p<0.0001. Figure 8—source data 1. Raw western data for HeLa WT and cavin3 KO cells time course after UV treatment with molecular weight markers for . Western blot analysis of ( A ) anti-rabbit cavin3, ( B ) anti-rabbit CAV1, ( C ) anti-rabbit BRCA1, ( D ) anti-rabbit RAD51, and ( E ) anti-mouse Tubulin antibodies in (1) WT control, (2) WT UV 30 min chase, (3) WT UV 60 min chase, (4) WT UV 120 min chase, (5) WT UV 240 min chase, (6) cavin3 KO control, and (7) cavin3 KO UV 30 min chase, cavin3 KO UV 60 min chase, cavin3 KO UV 120 min chase, and cavin3 KO 240 min chase. Figure 8—source data 2. Raw western data for HeLa WT and cavin3 KO cells untreated or UV treatment for 4 hr with molecular weight markers for . ( A ) Western blot analysis of anti-rabbit cavin3, ( B ) anti-rabbit BRCA1, ( C ) anti-rabbit Rad51 and γH2AX, ( D ) anti-rabbit BRCC36, ( E ) anti-sheep Merit40, ( F ) anti-rabbit BRCA2, ( G ) anti-rabbit CAV1, ( H ) anti-rabbit PKM, ( I ) anti-rabbit PGK1, and ( J ) anti-actin antibodies in (1) WT untreated cells, (2) WT + UV treatment and a 4 hr chase, (3) cavin3 KO cells, and (4) cavin3 KO + UV treatment and a chase 4 hr chase time.

Article Snippet: Antibody , MDC1 rabbit polyclonal , Novus , Novus Cat#NB10056657, RRID: AB_838567 , WB 1:100.

Techniques: Western Blot, Quantitation Assay, Immunofluorescence, Molecular Weight, Control

Journal: eLife

Article Title: Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response

doi: 10.7554/eLife.61407

Figure Lengend Snippet: ( A ) mRNA analysis of cavin3 in WT and cavin3 KO cells from three independent experiments performed in triplicate samples as mean ± SD using Student's t-test, **p<0.01. ( B ) Representative western blot analysis of equally loaded lysates for cavin3, DDX21, ACLY, gamma-catenin, alpha-catenin, ACCA, EGFR, CAV1, and Actin in WT and cavin3 KO HeLa cell. ( C ) Western blot analysis of equally loaded lysates from WT, cavin3 KO, and cavin3 KO transfected with cavin3 for cavin3, GFP, CAV1, Caldesmon, DDX21, and Tubulin as the loading control. Western blots are representative of three independent experiments. ( D ) Representative western blot analysis of lysates from WT, and cavin3 KO cells were western blotted for cavin3, RAP80/UIM1C, BRCA1 Santa Cruz (SC), BRCA1 Proteintech (PT), BARD1, Merit40, MDC1, BRCC3, Rad51, BRCC45, 53BP1, FANCD2, HLTF, and Actin as the loading control. Figure 1—figure supplement 1—source data 1. Raw western data for HeLa WT and cavin3 KO cells with molecular weight markers for . ( A ) Western blot analysis of anti-rabbit cavin3, ( B ) anti-rabbit CAV1, ( C ) anti-rabbit ACLY, ( D ) anti-mouse alpha-catenin, ( E ) anti-rabbit ACCA, and ( F ) anti-rabbit EGFR antibodies in (1) WT HeLa cells and (2) cavin3 KO cells. Figure 1—figure supplement 1—source data 2. Raw western data for HeLa WT, cavin3 KO, and cavin3 KO with cavin3-GFP cells with molecular weight markers for . ( A ) Western blot analysis of anti-rabbit cavin3, ( B ) anti-rabbit CAV1, ( C ) anti-mouse GFP, ( D ) anti-rabbit DDX21, ( E ) anti-rabbit Caldesmon and ( F ) anti-Tubulin antibodies in (1) HeLa WT, (2) cavin3 KO cells, and (3) cavin3KO + cavin3 GFP-expressing cells. Figure 1—figure supplement 1—source data 3. Raw western data for HeLa WT, cavin3 KO, and cavin3 KO with cavin3-GFP cells with molecular weight markers for . ( A ) Western blot analysis of anti-rabbit cavin3, ( B ) anti-rabbit CAV1, ( C ) anti-mouse GFP, ( D ) anti-rabbit DDX21, ( E ) anti-rabbit Caldesmon, and ( F ) anti-Tubulin antibodies in (1) HeLa WT, (2) cavin3 KO cells, and (3) cavin3KO + cavin3 GFP-expressing cells.

Article Snippet: Antibody , MDC1 rabbit polyclonal , Novus , Novus Cat#NB10056657, RRID: AB_838567 , WB 1:100.

Techniques: Western Blot, Transfection, Control, Molecular Weight, Expressing

Journal: eLife

Article Title: Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response

doi: 10.7554/eLife.61407

Figure Lengend Snippet: Densitometry analysis was performed of the protein levels of cavin3, BRCA1, P139 γH2AX, RAP80, BARD1, RAD51, MDC1, RNF168, BRCC36, Merit40, BRCA2, CAV1, PKM, PGK1, and Actin in in WT and cavin3 KO cells subjected to UV treatment and a 4 hr chase from 2 to 3 independent experiments presented as mean ± SD using a one-way ANOVA and Bonferroni’s multiple comparisons test. NS: not significant; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Antibody , MDC1 rabbit polyclonal , Novus , Novus Cat#NB10056657, RRID: AB_838567 , WB 1:100.

Techniques:

Journal: eLife

Article Title: Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response

doi: 10.7554/eLife.61407

Figure Lengend Snippet:

Article Snippet: Antibody , MDC1 rabbit polyclonal , Novus , Novus Cat#NB10056657, RRID: AB_838567 , WB 1:100.

Techniques: Sequencing, In Situ, CRISPR, Protease Inhibitor, Software

Journal: Molecular Cell

Article Title: MDC1 Interacts with TOPBP1 to Maintain Chromosomal Stability during Mitosis

doi: 10.1016/j.molcel.2019.02.014

Figure Lengend Snippet: A Conserved Acidic Sequence Motif near the N Terminus of MDC1 Binds to TOPBP1 (A) Schematic showing the layout of conserved domains and motifs in MDC1. Names of the known MDC1 binding partners, NBS1, RNF8, and H2AX, are shown below the motifs with which they interact. The FHA domain promotes MDC1 dimerization; hence, its binding partner is MDC1. Key phosphorylated residues are highlighted in bold. (B) Identification of TOPBP1 as a specific interactor for the MDC1-S196 phosphopeptide by LC-MS/MS and label-free quantification. Scatterplot depicts log 2 fold enrichment of MDC1-S196 versus MDC1-pS196 peptide-binding proteins from 2 independent experiments. See for raw MS data. (C) Peptide pull-downs from HeLa nuclear extracts using biotinylated peptides corresponding to residues surrounding MDC1-S196, either native (S196) or phosphorylated (pS196). (D) HA immunoprecipitations from 293FT cells transfected with the indicated HA-tagged MDC1 variants.

Article Snippet: MDC1 pS196 affinity purified polyclonal antibody was custom made by 21st Century Biochemicals (Marlboro), using the synthetic peptide sequence SVIVPE[pS]DEEGHSP for immunization.

Techniques: Sequencing, Binding Assay, Phospho-proteomics, Liquid Chromatography with Mass Spectroscopy, Quantitative Proteomics, Transfection

Journal: Molecular Cell

Article Title: MDC1 Interacts with TOPBP1 to Maintain Chromosomal Stability during Mitosis

doi: 10.1016/j.molcel.2019.02.014

Figure Lengend Snippet: BRCT Domains 1 and 2 of TOPBP1 Interact with MDC1 via Two Phosphorylated Residues, Ser168 and Ser196 (A) Schematic showing the layout of conserved domains and motifs in TOPBP1. Numbered boxes represent BRCT domains, with phosphopeptide-binding domains in green and domains lacking phosphopeptide-binding activity in gray. Names of known TOPBP1 binding partners are shown below the domains they interact with. AAD = ATR-activation domain. (B) GFP pull-downs from 293FT cells transfected with the indicated GFP-tagged TOPBP1 constructs. (C) Sequence alignment showing the conservation of Ser168, Ser196, and surrounding residues in MDC1 in vertebrates. Key phospho-serines are highlighted in bold. (D) HA-immunoprecipitations from 293FT cells transfected with the indicated HA-tagged MDC1 variants. (E) Fluorescence polarization with recombinant TOPBP1 BRCT domains 0–2 and MDC1-pS168 phosphopeptide. K155E is a mutation in TOPBP1 BRCT domain 1; K250E is a mutation in BRCT domain 2. ND = not determined. Dotted line indicates threshold for specific protein-protein interactions. (F) Fluorescence polarization with recombinant TOPBP1 BRCT domains 0–2 and MDC1-pS196 phosphopeptide. See also Figure S1 .

Article Snippet: MDC1 pS196 affinity purified polyclonal antibody was custom made by 21st Century Biochemicals (Marlboro), using the synthetic peptide sequence SVIVPE[pS]DEEGHSP for immunization.

Techniques: Phospho-proteomics, Binding Assay, Activity Assay, Activation Assay, Transfection, Construct, Sequencing, Fluorescence, Recombinant, Mutagenesis, Protein-Protein interactions

Journal: Molecular Cell

Article Title: MDC1 Interacts with TOPBP1 to Maintain Chromosomal Stability during Mitosis

doi: 10.1016/j.molcel.2019.02.014

Figure Lengend Snippet: MDC1 Phosphorylation and TOPBP1 Binding Are Mediated by CK2 (A) HA-immunoprecipitations from 293FT cells transfected with the indicated constructs. (B) 293FT cells treated with 10 μM CK2 inhibitor CX-4945 were harvested for western blotting with the indicated antibodies. AKT-pS129 is a positive control as AKT is a known CK2 substrate ( Siddiqui-Jain et al., 2010 ). (C) GFP pull-downs from 293FT cells transfected with the indicated GFP-tagged TOPBP1 constructs and treated with 10 μM CK2 inhibitor CX-4945 or DMSO vehicle control. (D) Western blots of an in vitro kinase assay with recombinant CK2 as the kinase, and GST-tagged MDC1 fragment (encompassing residues 109–330) or GST alone as substrates. (E) GST pull-downs from HeLa nuclear extracts with WT and mutant versions of GST-tagged MDC1 fragment (encompassing residues 109–330), preincubated or not with recombinant CK2 and ATP. See also Figure S2 .

Article Snippet: MDC1 pS196 affinity purified polyclonal antibody was custom made by 21st Century Biochemicals (Marlboro), using the synthetic peptide sequence SVIVPE[pS]DEEGHSP for immunization.

Techniques: Phospho-proteomics, Binding Assay, Transfection, Construct, Western Blot, Positive Control, Control, In Vitro, Kinase Assay, Recombinant, Mutagenesis

Journal: Molecular Cell

Article Title: MDC1 Interacts with TOPBP1 to Maintain Chromosomal Stability during Mitosis

doi: 10.1016/j.molcel.2019.02.014

Figure Lengend Snippet: MDC1 Is Required for TOPBP1 Recruitment to Sites of DSBs in G1 Phase Cells (A) Schematic representation of the human MDC1 gene locus, illustrating the hybridization site of the gRNA selected for the generation of Δ MDC1 cell line used in this study (gRNA sequence in the ). (B) Western blots of total cell extract of irradiated WT U2OS cells and Δ MDC1 cells showing that the ATM and ATR pathways are normally activated in the knock-out cell line in response to IR (3 Gy). (C) Immunofluorescence experiment of irradiated WT U2OS cells and Δ MDC1 cells stained with NBS1 and 53BP1 antibodies. Cells were co-stained with MDC1 antibodies to show lack of MDC1 expression in the knock-out cell line. (D) Quantitative assessment of γH2AX foci in irradiated WT U2OS cells and Δ MDC1 cells (box and whiskers represent minimum to maximum and individual data points are also shown; t test, α = 0.05, at least 130 cells per condition). (E) Western blot showing no signal with the MDC1-pS168 and -pS196 phospho-specific antibodies in Δ MDC1 cells. NBS1 is a loading control. (F) Immunofluorescence experiment of WT U2OS cells, Δ MDC1 cells, and Δ 53BP1 cells stained with TOPBP1 antibodies 3 h after IR (3 Gy). Cells were co-stained with Cyclin A antibodies to distinguish G1 phase from S/G2 phase cells. (G) Immunofluorescence experiment of irradiated WT U2OS cells and Δ 53BP1 G1 cells stained with MDC1 and TOPBP1 antibodies 3 h after IR (3 Gy). All scale bars represent 10 μm. See also Figure S3 .

Article Snippet: MDC1 pS196 affinity purified polyclonal antibody was custom made by 21st Century Biochemicals (Marlboro), using the synthetic peptide sequence SVIVPE[pS]DEEGHSP for immunization.

Techniques: Hybridization, Sequencing, Western Blot, Irradiation, Knock-Out, Immunofluorescence, Staining, Expressing, Control

Journal: Molecular Cell

Article Title: MDC1 Interacts with TOPBP1 to Maintain Chromosomal Stability during Mitosis

doi: 10.1016/j.molcel.2019.02.014

Figure Lengend Snippet: Direct Interaction with MDC1 Is Essential for TOPBP1 Recruitment to DSBs in Mitotic Cells (A) Confocal microscopy of U2OS cells expressing GFP-tagged MDC1 WT and mutants 3 h after treatment with 3 Gy IR. (B) Quantitative analysis of GFP-MDC1 and TOPBP1 colocalization by SQUASSH. Upper graph: object number colocalization (fraction of objects in each channel that overlap ≥50%). Lower graph: object size colocalization (area of object overlap divided by total object area). Each data point represents one cell (n = 10); bars represent the mean. One-way ANOVA and Dunnett’s multiple comparison test were performed to test for difference of WT versus mutants. All mutant cell lines are significantly different from WT (p ≤ 0.0006). (C) Confocal microscopy of U2OS cells arrested in mitosis by nocodazole (100 ng/mL) and treated with 0.5 Gy IR. (D) Confocal microscopy of U2OS Δ MDC1 and Δ 53BP1 cells arrested in mitosis by nocodazole (100 ng/mL) and treated with 0.5 Gy IR. (E) Quantitative analysis of TOPBP1-γH2AX colocalization in Δ MDC1 and Δ 53BP1 cells by SQUASSH. Each data point represents one cell (n = 8); bars represent the mean. (F) Confocal microscopy of U2OS cells expressing GFP-tagged MDC1 WT and mutants, arrested in mitosis by nocodazole (100 ng/mL) 1 h after treatment with 0.5 Gy IR. (G) Quantitative analysis of GFP-MDC1 and TOPBP1 colocalization by SQUASSH. Upper graph: object number colocalization. Lower graph: object size colocalization. Each data point represents one cell (n = 10); bars represent the mean. All scale bars represent 10 μm. See also Figure S3 .

Article Snippet: MDC1 pS196 affinity purified polyclonal antibody was custom made by 21st Century Biochemicals (Marlboro), using the synthetic peptide sequence SVIVPE[pS]DEEGHSP for immunization.

Techniques: Confocal Microscopy, Expressing, Comparison, Mutagenesis

Journal: Molecular Cell

Article Title: MDC1 Interacts with TOPBP1 to Maintain Chromosomal Stability during Mitosis

doi: 10.1016/j.molcel.2019.02.014

Figure Lengend Snippet: The MDC1-TOPBP1 Interaction Promotes Genome Stability during Mitosis (A) Clonogenic survival assay of mitotic and interphase U2OS, Δ MDC1 , and Δ MDC1 cells stably transfected with MDC1 WT and TOPBP1 binding mutants (mean of 3 independent experiments; error bars: ±SD). (B) Quantification of background γH2AX (−IR) and residual γH2AX foci 20 min, 6 h, and 24 h after irradiation of mitotic cells and subsequent release from mitotic arrest (red bars represent the mean; n = 3). One-way ANOVA and Dunnett’s multiple comparison test were performed for the 24 h time point to test for difference of the mean of Δ MDC1 versus complemented cell lines. (C) Quantification of micronuclei formation 4 h after irradiation of mitotic cells and subsequent release from mitotic arrest (bars represent mean ± SD, n = 3, unpaired t test, α = 0.05, at least 1,000 cells per condition). (D) Quantification of micronuclei formation in untreated U2OS cell lines after staining of micronuclei for CENPA (stacked bars represent means of two independent experiments of at least 700 cells assessed per condition). All scale bars represent 10 μm. See also Figure S4 .

Article Snippet: MDC1 pS196 affinity purified polyclonal antibody was custom made by 21st Century Biochemicals (Marlboro), using the synthetic peptide sequence SVIVPE[pS]DEEGHSP for immunization.

Techniques: Clonogenic Cell Survival Assay, Stable Transfection, Transfection, Binding Assay, Irradiation, Comparison, Staining

Journal: Molecular Cell

Article Title: MDC1 Interacts with TOPBP1 to Maintain Chromosomal Stability during Mitosis

doi: 10.1016/j.molcel.2019.02.014

Figure Lengend Snippet: TOPBP1 Can Bridge MDC1-Bound DSBs Acquired during Mitosis (A) Examples of chromosomal aberrations in metaphase spreads derived from U2OS Δ MDC1 cells and Δ MDC1 cells stably transfected with TOPBP1 binding mutants, hybridized with a telomere Cy3-labeled PNA probe. Scale bar represents 10 μm. (B) Quantification of chromosomal aberrations: chromosome breaks, chromatid breaks, fragments, and chromosome fusions were scored (bars represent mean ± SD; n = 18, unpaired t test, α = 0.05). (C) Airyscan high-resolution confocal single slice images of GFP-TOPBP1 and MDC1 foci in mitosis 1 h after 0.5 Gy IR. “Intra” indicates an intrachromosomal TOPBP1 filament; “inter” indicates an interchromosomal TOPBP1 filament. (D) Quantification of TOPBP1 foci structures induced by IR in mitotic cells (100 foci scored). Foci structures were determined manually by inspecting images slice by slice in ZEN. Filaments were defined by >1 diffraction-limited spot connected within a TOPBP1 focus. See also and .

Article Snippet: MDC1 pS196 affinity purified polyclonal antibody was custom made by 21st Century Biochemicals (Marlboro), using the synthetic peptide sequence SVIVPE[pS]DEEGHSP for immunization.

Techniques: Derivative Assay, Stable Transfection, Transfection, Binding Assay, Labeling

Journal: Molecular Cell

Article Title: MDC1 Interacts with TOPBP1 to Maintain Chromosomal Stability during Mitosis

doi: 10.1016/j.molcel.2019.02.014

Figure Lengend Snippet:

Article Snippet: MDC1 pS196 affinity purified polyclonal antibody was custom made by 21st Century Biochemicals (Marlboro), using the synthetic peptide sequence SVIVPE[pS]DEEGHSP for immunization.

Techniques: Recombinant, Protease Inhibitor, Plasmid Preparation, Staining, Mutagenesis, Mass Spectrometry, Software, Sequencing