polyclonal antibody against mdc1  (Novus Biologicals)

Journal: Journal of Biological Chemistry

Article Title: Disassembly of MDC1 Foci Is Controlled by Ubiquitin-Proteasome-dependent Degradation

doi: 10.1074/jbc.m801082200

Figure Lengend Snippet: FIGURE 1. Degradation of MDC1 is dependent on the ubiquitin-protea- some system. A, MDC1 is a ubiquitylated protein. MCF7 cells were trans- fectedwithaH6-Myc-Ubexpressionvectororacontrolvector.After48h,cells were exposed to PIs for 4 h. Cell lysates were immunoprecipitated with an anti-MDC1 antibody. The MDC1 immunocomplexes were separated on a SDS-5% polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. The membrane was cut vertically and Western-blotted with mixed antibodies including mouse anti-Myc and rabbit anti-MDC1 (lanes 1, 2, and 3) or MDC1 antibody alone (lanes 4 and 5). The left and right membrane halves were lined up by using protein size markers. B, conjugated ubiquitin moieties in MDC1 are dependent on lysine 48 of ubiquitin. MCF7 cells were transfected with a H6-Myc-Ub vector, a vector with a 48R substitution. The ubiquitylation of MDC1 was analyzed as described in A. C, treatment with PIs induces the accumulation of MDC1 protein levels. Exponentially growing MCF7 cells were treated with PIs for 4 h. MDC1 protein expression was iden- tified by immunoblotting using anti-MDC1 antibody (Novus). -Actin is shown as a protein loading control. D, MDC1 protein was identified by immu- nofluorescence staining. MCF7 cells were fixed with 4% formaldehyde and the MDC1 protein was identified by the anti-MDC1 antibody and a Alex- aFluor488-labeled anti-rabbit IgG secondary antibody. The cell nuclei were stained with DAPI (blue).

Article Snippet: Immunoprecipitationswere performed overnight with polyclonal antibodies against MDC1 (Bethyl, rabbit anti-MDC1 A300–053A).

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Membrane, Western Blot, Transfection, Plasmid Preparation, Expressing, Control, Staining, Labeling

Journal: Journal of Biological Chemistry

Article Title: Disassembly of MDC1 Foci Is Controlled by Ubiquitin-Proteasome-dependent Degradation

doi: 10.1074/jbc.m801082200

Figure Lengend Snippet: FIGURE 2. Increased ubiquitylation of MDC1 in response to DSBs. A, ubiq- uitylated MDC1 associates with chromatin before and after exposure to ion- izing radiation (IR). MCF7 cells were transfected with the H6-Myc-Ub and exposed to PIs after 48 h. Four hours after IR treatment, chromatin fractions were prepared as described under “Experimental Procedures.” The chroma- tin-bound proteins were analyzed by immunoprecipitation using the anti- MDC1 antibody. Anti-MDC1 immunoprecipitates were separated by SDS-5% polyacrylamide gel. Immunoblotting was performed as described in Fig. 1. BandC,decreasedMDC1proteinlevelsinresponsetoIR.Exponentiallygrow- ing MCF7 cells were exposed to 8 Gy of IR. After 4 h, the cell lysates were fractionated to separate chromatin. Samples from the whole cell lysate (WCL) or the chromatin fraction (Chro) were analyzed by immunoblotting with anti- MDC1 antibody. The loading control was monitored by being probed with indicated antibodies.

Article Snippet: Immunoprecipitationswere performed overnight with polyclonal antibodies against MDC1 (Bethyl, rabbit anti-MDC1 A300–053A).

Techniques: Transfection, Immunoprecipitation, Western Blot, Control

Journal: Journal of Biological Chemistry

Article Title: Disassembly of MDC1 Foci Is Controlled by Ubiquitin-Proteasome-dependent Degradation

doi: 10.1074/jbc.m801082200

Figure Lengend Snippet: FIGURE3.MDC1regulatesconjugatedubiquitinfociinresponsetoDNAdamage.AandB,knockdownofRNF8 by siRNA. Exponentially growing MCF7 cells were transfected with two rounds of RNF8 (siRNF8) or control siRNA (sicon),separatedbya24-hourinterval.Experimentswereperformed72hafterfirstsiRNAtransfection.RNF8knock- down was confirmed by Western blot and immunostaining. RNF8 knockdown by immunofluorescence staining analogous to A. C, residual FK2 foci in cells with RNF8 knockdown. MCF7 cells transfected with siRNA twice were irradiatedwith8Gy,andimmunostainingwasperformed4hafterIR.Brieflythecellsweredetergent-extractedwith 0.5% Triton X-100 CSK buffer and fixed with 4% formaldehyde. The fixed cells were then permeabilized using 0.1% Triton X-100, which was followed by blocking and incubation with FK2 and anti-RNF8 antibodies. Foci were visual- ized by AlexaFluor594-labeled anti-mouse IgG secondary antibody (red) or AlexaFluor488-labeled anti-rabbit anti- body (green). Cell nuclei were stained with DAPI. D, MDC1 foci co-localize with conjugated ubiquitin in both untreated and IR-treated cells. Exponentially growing MCF7 cells were mock-treated or irradiated with 8 Gy and fixedattheindicatedtimepoints.ThefixedcellswithandwithoutIRtreatmentwereco-stainedwithanti-MDC1and anti-ubiquitin (FK2) antibodies. This was followed by incubation with the secondary antibodies. Co-localization is indicated by a yellow signal. E–G, MDC1 knockdown resulted in inhibition of conjugated ubiquitin foci. Exponen- tiallygrowingMCF7cellsweretransfectedwithtworoundsofMDC1orcontrolsiRNAasdescribedinAandB.MDC1 knockdown was monitored by Western blot (E) and immunostaining (F).

Article Snippet: Immunoprecipitationswere performed overnight with polyclonal antibodies against MDC1 (Bethyl, rabbit anti-MDC1 A300–053A).

Techniques: Transfection, Control, Western Blot, Immunostaining, Knockdown, Immunofluorescence, Staining, Blocking Assay, Incubation, Labeling, Ubiquitin Proteomics, Irradiation, Inhibition

Journal: Journal of Biological Chemistry

Article Title: Disassembly of MDC1 Foci Is Controlled by Ubiquitin-Proteasome-dependent Degradation

doi: 10.1074/jbc.m801082200

Figure Lengend Snippet: FIGURE 4. Kinetics of MDC1 and -H2AX focus formation without and with PI treatment. A, representative MDC1 foci in cells with and without PI treatment at 4 h after irradiation with 8 Gy. B, dynamics of MDC1 focus formation after irradiation of MCF7 cells with 8 Gy. The percentage of cells with more than five nuclear foci was calculated. In each experiment, 200–300 nuclei were counted per data point. Error bars indicate S.E. from three independent experiments. C, representative -H2AX foci in cells with and without PI treatment 4 h after irradiation with 8 Gy. D, dynamics of -H2AX focus formation after irradiation of MCF7 cells with 8 Gy. -H2AX foci kinetics was determined as in B.

Article Snippet: Immunoprecipitationswere performed overnight with polyclonal antibodies against MDC1 (Bethyl, rabbit anti-MDC1 A300–053A).

Techniques: Irradiation

Journal: Journal of Biological Chemistry

Article Title: Disassembly of MDC1 Foci Is Controlled by Ubiquitin-Proteasome-dependent Degradation

doi: 10.1074/jbc.m801082200

Figure Lengend Snippet: FIGURE5.DetectionoftheenhancedMDC1proteinaccumulationatchro- mosomalDSBsitesinthepresenceofPIsasdeterminedbyChIP.A,induc- tion of site-directed DSBs, as reflected by subsequent homology-mediated repair, was measured by dual-color flow cytometric detection of GFP-positive cells. Representative flow cytometric analyses of H1299/DR-GFP cells treated with and without infection of the adenoviral I-SceI expression construct (Ad- SceI-NG) are shown. Left panel show cells without DSB induction, with few GFP-positive cells detected. The right panel shows cells 24 h after infection with Ad-Sce-ING. B, expression of HA-tagged I-SceI endonuclease was moni- tored by Western blot using anti-HA antibody. H1299/DR-GFP cells were infected with and without Ad-SceI-NG, and whole cell lysate was prepared at theindicatedtimepoints.C,schematicrepresentationofthepositionofprim- ers used for real-time PCR quantification of ChIPs respective to the DSB cre- ated by I-SceI endonuclease in vivo. Real-time PCR on ChIP samples were car- ried out at 94–378 nucleotides from the break (52). D, ChIP of MDC1 protein on DSB after Ad-SceI-NG. -Fold enrichment represents the enrichment of MDC1 proteins compared with an IgG control and normalized to a PCR inter- nal control for a chromosomal locus distinct from the DSB site (52). Data points represent an average of four independent repeated experiments. E, ChIP analysis of MDC1 in the presence and absence of PIs (upper panel). Samples were prepared in parallel to monitor the amount of pulled-down MDC1 proteins using immunoprecipitation. The anti-MDC1 immunoprecipi- tate was probed with anti-MDC1 antibody (bottom panel). Shown is one rep- resentative result from three independent repeated experiments.

Article Snippet: Immunoprecipitationswere performed overnight with polyclonal antibodies against MDC1 (Bethyl, rabbit anti-MDC1 A300–053A).

Techniques: Infection, Expressing, Construct, Western Blot, Real-time Polymerase Chain Reaction, In Vivo, Control, Immunoprecipitation

Journal: Journal of Biological Chemistry

Article Title: Disassembly of MDC1 Foci Is Controlled by Ubiquitin-Proteasome-dependent Degradation

doi: 10.1074/jbc.m801082200

Figure Lengend Snippet: FIGURE 6. Kinetics of BRCA1 focus formation with and without PI treat- ment. A, illustration of MDC1 foci and BRCA1 foci at the 4-hour time point. Cells lose MDC1 foci as BRCA1 foci are being formed (upper row). After treat- ment, MDC1 foci persist while BRCA1 foci fail to form (lower row). B, Western blot developed using an anti-BRCA1 antibody (Oncogene, Ab-1) on whole cellextractsfromMCF7cellswithorwithoutPItreatmentfor4–6h.C,dynam- ics of BRCA1 focus formation after irradiation of MCF7 cells with 8 Gy. The percentage of cells with more than five nuclear foci was calculated. In each experiment, 200–300 nuclei were counted per data point. Error bars indicate S.E. from three independent repeated experiments with and without PI treatment.

Article Snippet: Immunoprecipitationswere performed overnight with polyclonal antibodies against MDC1 (Bethyl, rabbit anti-MDC1 A300–053A).

Techniques: Western Blot, Irradiation

Journal: Journal of Biological Chemistry

Article Title: Disassembly of MDC1 Foci Is Controlled by Ubiquitin-Proteasome-dependent Degradation

doi: 10.1074/jbc.m801082200

Figure Lengend Snippet: FIGURE 7. Importance of MDC1 ubiquitylation and RNF8 for BRCA1 foci recruitment. A, BRCA1 focus formation at the 4 h after IR in control siRNA, RNF8 siRNA, or MDC1 siRNA-transfected cells. The proportion of cells with IR-induced BRCA1 foci is shown. Bars represent the mean with S.E. based on 3–4 independent experiments. B, effect of persisting MDC1 foci on recruitment of BRCA1 in the presence of PIs is independent of RNF8. Exponentially growing MCF7 cells were transfected twice with either control (sicon) or RNF8 siRNA (siRNF8) as described in Fig. 3. Cells were then treated with DMSO or PIs for 1–2 h and exposed to 8 Gy IR. Focus formation was analyzed 4 h after IR as in Fig. 3. One representative result from three independent experiments is shown. C and D, stability of BRCA1 protein expression in siRNA-transfected cells. Lysate protein was resolved by SDS-PAGE and visualized using an anti-BRCA1 antibody (Oncogene, Ab-1) 72 h after the first siRNA transfection.

Article Snippet: Immunoprecipitationswere performed overnight with polyclonal antibodies against MDC1 (Bethyl, rabbit anti-MDC1 A300–053A).

Techniques: Control, Transfection, Expressing, SDS Page

Journal: Virology

Article Title: The cellular Mre11 protein interferes with adenovirus E4 mutant DNA replication.

doi: 10.1016/j.virol.2007.03.049

Figure Lengend Snippet: Fig. 4. Mre11 inhibits DNA replication in E4 mutant infected cells. HeLa cells were transfected with control siRNA or Mre11 siRNA prior to infection with Ad5 or H5dl1007 at 3 FFU/cell for 24 h. (A) Mre11 knockdown was monitored by immunofluorescence (scale bar 10 μm) and (B) western blotting of 75 μg of total protein prepared 96 h after mock (−) or Mre11-specific siRNA (+) transfection, using goat polyclonal antibody against Mre11. Additional controls demonstrating the specificity of Mre11 expression knockdown included no treatment (UT), non-targeting siRNA (non-targeting), siRNA against GAPD, and treatment with the transfection reagent alone (D1). (C) Panels a–c represent host Mre11 and viral E2-72kDa distribution in untransfected cells infected with H5dl1007 at 24 hpi. The distribution patterns of Mre11 and 72 kDa in siRNA transfected HeLa cells infected for 24 h with H5dl1007 are shown in panels d–f. (D) Levels of viral DNA synthesis in Ad5 and H5dl1007 infected HeLa, MO59J, and MO59K cells with and without Mre11 siRNA transfection were quantified by Southern analysis of 10 μg of EcoRI-digested total DNA prepared at 24 hpi. The C fragment from the DNA digestion was used for comparison between Ad5 and H5dl1007.

Article Snippet: Rabbit polyclonal antibody against Mdc1 (Bethyl Laboratories) was diluted to 1:2500 for use.

Techniques: Mutagenesis, Infection, Transfection, Control, Knockdown, Immunofluorescence, Western Blot, Expressing, DNA Synthesis, Comparison

Journal: Virology

Article Title: The cellular Mre11 protein interferes with adenovirus E4 mutant DNA replication.

doi: 10.1016/j.virol.2007.03.049

Figure Lengend Snippet: Fig. 5. The host Mdc1 protein is relocalized in response to Ad infection. HeLa cells were infected with Ad5 and E4 mutant H5dl1007 at 3 FFU/cell for 4, 6, and 10 h. (A) Uninfected (UI) cells were stained with antibodies specific for host Mdc1 (panel a) and viral E2-72kDa proteins (panel b) and analyzed by confocal microscopy. (B) Confocal microscopy was used to analyze the distribution of host Mdc1 (panels d, g, and j and panels m, p, and s) and viral 72 kDa (panels e, h, and k and panels n, q, and t) in Ad5 (panels d–l) and H5dl1007 (panels m–u) infections.

Article Snippet: Rabbit polyclonal antibody against Mdc1 (Bethyl Laboratories) was diluted to 1:2500 for use.

Techniques: Infection, Mutagenesis, Staining, Confocal Microscopy

Journal: Virology

Article Title: The cellular Mre11 protein interferes with adenovirus E4 mutant DNA replication.

doi: 10.1016/j.virol.2007.03.049

Figure Lengend Snippet: Fig. 6. Mdc1 is not required to recruit Mre11 to E4 mutant DNA replication foci. HeLa cells were transfected with control siRNA or Mdc1 siRNA before being infected with Ad5 or H5dl1007 at 3 FFU/cell for 24 h. (A) Mdc1 knockdown was monitored by immunofluorescence (scale bar 10 μm) and (B) western blotting of 75 μg of total protein prepared 96 h after mock (−) or Mdc1-specific siRNA (+) transfection, using rabbit polyclonal antibody against Mdc1. Additional controls demonstrating the specificity of Mdc1 expression knockdown included no treatment (UT), non-targeting siRNA (non-targeting), siRNA against GAPD (GAPD), and treatment with the transfection reagent alone (D1). (C) Mre11 (panels a, d, g, and j) and Mdc1 (panels b, e, h, and k) distribution in untransfected cells (panels a–f) early (top) and late (bottom) in H5dl1007 infection. The distribution of Mre11 and Mdc1 in siRNA transfected HeLa cells infected with H5dl1007 is shown in panels g–l. (D) Levels of viral DNA synthesis in cells with and without Mdc1 siRNA transfection were quantified by Southern analysis with 10 μg of EcoRI-digested viral DNA. The C fragment from the digestion was used for comparison between Ad5 and H5dl1007.

Article Snippet: Rabbit polyclonal antibody against Mdc1 (Bethyl Laboratories) was diluted to 1:2500 for use.

Techniques: Mutagenesis, Transfection, Control, Infection, Knockdown, Immunofluorescence, Western Blot, Expressing, DNA Synthesis, Comparison