Journal: Nucleic Acids Research

Article Title: And-1 is required for homologous recombination repair by regulating DNA end resection

doi: 10.1093/nar/gkw1241

Figure Lengend Snippet: And-1 forms complexes with HR repair proteins and is required for HR repair. ( A ) Identification of CtIP-associated proteins that are involved in HR repair by mass spectrometry. ( B ) CtIP interacts with And-1. Co-immunoprecipitation (co-IP) assays were performed using U2OS cell lines. Cell lysates were immunoprecipitated with control IgG, anti-CtIP or anti-And-1 antibodies and the IPs were then resolved by SDS-PAGE and immunoblotted for the indicated proteins. Left panel, immunoprecipitation with anti-CtIP antibody. Right panel, immunoprecipitation with anti-And-1 antibody. ( C ) And-1 depletion impairs HR repair. U2OS-DR-GFP cells treated with the indicated siRNAs were transfected with pCBASce plasmids 48 h post siRNA transfection. The percentage of GFP-positive cells was determined by flow cytometry 48 h after plasmid transfection. The data were normalized to those obtained from cells transfected with control siGl2 (set as 1.0). Data represent means ± SD from three independent experiments. * P ≤ 0.05. ( D ) U2OS cell survival after exposing cells transfected with indicated siRNAs to the indicated doses of camptothecin. Data represent means ± SD from three independent experiments. * P ≤ 0.05; ** P ≤ 0.01.

Article Snippet: Recombinant CtIP proteins (H00005932-P01) were purchased from Novus Biologicals.

Techniques: Mass Spectrometry, Immunoprecipitation, Co-Immunoprecipitation Assay, Control, SDS Page, Transfection, Flow Cytometry, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: And-1 is required for homologous recombination repair by regulating DNA end resection

doi: 10.1093/nar/gkw1241

Figure Lengend Snippet: And-1 is recruited to DSB sites. ( A ) And-1 co-localizes with γ-H2AX at DSB sites induced by laser micro-irradiation. U2OS cells were micro-irradiated with laser and fixed for immunostaining using the indicated antibodies 10 min after irradiation. For each experiment, >50 cells were counted and the percentage of cells exhibiting γ-H2AX strips or both γ-H2AX and And-1 strips was determined. Data represent means ± SD from three independent experiments. *** P ≤ 0.001. ( B ) And-1 co-localizes with γ-H2AX at DSB sites induced by UVC light. U2OS cells labeled with or without 10 μM BrdU for 72 h were covered with a 5 μm polycarbonate isopore membrane filter and subjected to UVC irradiation (30J/m 2 ). 30 min after irradiation, cells were harvested and immunostained for the indicated proteins. For each experiment, >100 cells were counted and the percentage of cells exhibiting And-1 foci was determined. Data represent means ± SD from three independent experiments. *** P ≤ 0.001. ( C ) And-1 recruitment to laser-induced DSB sites at the indicated time points. U2OS cells were micro-irradiated with laser and then processed at indicated time points for immunostaining for indicated proteins. ( D ) CtIP recruitment to laser-induced DSB sites at indicated time points. U2OS cells were micro-irradiated with laser and then processed at the indicated time points for immunostaining for indicated proteins.

Article Snippet: Recombinant CtIP proteins (H00005932-P01) were purchased from Novus Biologicals.

Techniques: Irradiation, Immunostaining, Labeling, Membrane

Journal: Nucleic Acids Research

Article Title: And-1 is required for homologous recombination repair by regulating DNA end resection

doi: 10.1093/nar/gkw1241

Figure Lengend Snippet: And-1 is required for efficient recruitment of CtIP to DSB sites. ( A ) And-1 depletion impairs CtIP recruitment to DSB sites. U2OS cells treated with siGl2 or two independent siAnd-1s (siA-1 or siA-2) were micro-irradiated by laser and co-immunostained for γ-H2AX and CtIP 15 min post irradiation. For each experiment, >50 cells were counted and the percentage of γ-H2AX cells exhibiting CtIP strips was determined. Data represent means ± SD from three independent experiments. N.S., not significant; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001. ( B ) Restoration of CtIP recruitment to DSB sites in cells expressing siRNA resistant And-1. U2OS cells expressing the mutant And-1 (330–1129) were transfected with the indicated siRNAs and subjected to treatment as described in Figure . For each experiment, >50 cells were counted and the percentage of γ-H2AX cells exhibiting CtIP strips was determined. Data represent means ± SD from three independent experiments. N.S., not significant; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001.

Article Snippet: Recombinant CtIP proteins (H00005932-P01) were purchased from Novus Biologicals.

Techniques: Irradiation, Expressing, Mutagenesis, Transfection

Journal: Nucleic Acids Research

Article Title: And-1 is required for homologous recombination repair by regulating DNA end resection

doi: 10.1093/nar/gkw1241

Figure Lengend Snippet: And-1 depletion impairs DNA damage response induced by end resection. ( A and B ) Depletion of And-1 impairs Chk1 and RPA phophorylation but not Chk2 phosphorylation after camptothecin exposure. U2OS cells transfected with the indicated siRNAs were treated with camptothecin (1 μM) for 1 h. Cells were then harvested and immunoblotted for indicated proteins. Asterisks in A and B: hyper-phosphorylated RPA32. ( C ) Depletion of And-1 impairs Chk1 phosphorylation at both early and late time-points after continuous camptothecin exposure. U2OS cells transfected with the indicated siRNAs were treated with camptothecin (1 μM) and harvested at the indicated time-points and immunoblotted for the indicated proteins. ( D ) U2OS cells transfected with indicated siRNAs were synchronized by using a double-thymidine (2 mM) block and then released for 6h. Cells enriched in S/G2 phase were harvested for IP. CtIP or IgG IPs were immunoblotted for the indicated proteins.

Article Snippet: Recombinant CtIP proteins (H00005932-P01) were purchased from Novus Biologicals.

Techniques: Phospho-proteomics, Transfection, Blocking Assay

Journal: Nucleic Acids Research

Article Title: And-1 is required for homologous recombination repair by regulating DNA end resection

doi: 10.1093/nar/gkw1241

Figure Lengend Snippet: The C-terminus of And-1 is required for its interaction with CtIP and localization to DSB sites. ( A ) And-1 directly interacts with CtIP. Purified recombinant CtIP proteins (400 ng) were mixed with recombinant And-1protiens (200 ng) or BSA (200 ng) as described in Method. CtIP was immunoprecipitated with anti-CtIP antibody and IPs were then resolved by SDS-PAGE and immunoblotted for the indicated proteins. ( B ) The associations of And-1 or its mutants with CtIP. FLAG-And-1 and its mutants were expressed in 293T cells. FLAG-IPs were resolved by SDS-PAGE and immunoblotted for the indicated proteins. ( C ) The localization of And-1 and its mutants at DSB sites. U2OS cells expressing the indicated And-1 or it mutants were subjected to the treatment as in Figure . ( D ) The associations of CtIP or its mutants with And-1. FLAG-CtIP and its mutants were expressed in 293T cells. Left panel, schematic of the CtIP protein domains and deletion mutants used for protein–protein interactions; Right panel, FLAG-IPs were resolved on SDS-PAGE and immunoblotted for the indicated proteins.

Article Snippet: Recombinant CtIP proteins (H00005932-P01) were purchased from Novus Biologicals.

Techniques: Purification, Recombinant, Immunoprecipitation, SDS Page, Expressing, Protein-Protein interactions