rad50 upstate  (Cell Signaling Technology Inc)

Journal: Nucleic Acids Research

Article Title: ATM-dependent phosphorylation of MRE11 controls extent of resection during homology directed repair by signalling through Exonuclease 1

doi: 10.1093/nar/gkv754

Figure Lengend Snippet: Investigation of MRE11 phosphorylation kinetics at MRE11S676S678. ( A ) MRE11pS676pS678 antibody immunoprecipitate the phosphorylated form of MRE11 after ionizing radiation (IR). Western blot (MRE11) of immunoprecipitated MRE11 using an antibody against MRE11S676S678 (nonP) site and the corresponding phosphorylated MRE11pS676pS678 (pSpS) sites from control and ATLD2 lymphoblastoid cells treated with 0 or 10 Gy IR. Unbound sample from each immunoprecipitation was run in parallel and also immunoblotted for total MRE11 (GeneTex). ( B ) Dose-dependent increase of MRE11 phosphorylation following IR. Western blot of immunoprecipitated MRE11 using an antibody against MRE11pS676pS678 (pSpSMRE11) sites from control and A-T lymphoblastoid cells treated with 0, 2, 5 or 10 Gy IR. ATLD2 cells were also run as a negative control (0 and 10 Gy). Western blot for the MRN complex proteins, MRE11, RAD50 and NBS1. ( C ) Increasing MRE11 phosphorylation over 2 h post 5 Gy. Western blot of immunoprecipitated MRE11 using an antibody against MRE11pS676pS678 (pSpSMRE11) sites from control lymphoblastoid cells either untreated (−) or harvested at 0.6, 2, 4 or 10 h post 5 Gy IR. ATLD2 lymphoblastoid cells were either untreated (−) or harvested at 2 and 10 h post 5 Gy IR and run as a negative control. ( D ) MRE11 phosphorylation in response to DNA double strand break inducing agents. Western blot of immunoprecipitated MRE11 using an antibody against MRE11pS676pS678 (pSpSMRE11) sites from control cells treated with either IR, camptothecin (CPT), hydrogen peroxide (H2O2), etoposide (ETOP), cisplatin (CISP) or methlymethanesulfonate (MMS).

Article Snippet: Whole cell extracts or immune complexes were separated by electrophoresis on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and proteins transferred to nitrocellulose membranes using Towbin's buffer (20% methanol, 50 mM Tris, 40 mM glycine and 0.02% SDS) at 100 V for 1 h. Blots were incubated with antibodies against MRE11 (12D7; GeneTex), Phospho-SQ/TQ (Cell Signaling Technologies), RAD50 (Upstate), NBS1 (Novus Biologicals), ATM (2C1; GeneTex), ATM pS1981 (GeneTex), SMC1 and SMC1 pS957 (GeneTex), Kap1 and Kap1 p824 (Novus Biologicals), GAPDH (GeneTex) and GFP (Abcam).

Techniques: Phospho-proteomics, Western Blot, Immunoprecipitation, Control, Negative Control

Journal: Nucleic Acids Research

Article Title: ATM-dependent phosphorylation of MRE11 controls extent of resection during homology directed repair by signalling through Exonuclease 1

doi: 10.1093/nar/gkv754

Figure Lengend Snippet: Investigation of ATM signalling, cell survival and chromosomal aberrations in the cell line expressing non-phosphorylatable MRE11S676AS678A. ( A ) Stable MRN complex in WT and non-phosphorylatable mutant MRE11 corrected cell lines. Western blot of immunoprecipitated MRE11 from control (MCR5) and ATLDMRE11 (WT), ATLDS676AS678A (MUT) and ATLDVEC (VEC) cell lines. Also immunoblotted for NBS1 and RAD50. ( B ) ATM signalling in the non-phosphorylatable mutant MRE11 cell line is comparable to the WT corrected cell line. Total cell extracts from ATLDMRE11 (WT), ATLDS676AS678A (MUT) and ATLDVEC (VEC) cell lines were extracted 30 min post 5 Gy (+) or left as unirradiated controls (−) and western blotted for ATM S1981 autophosphorylation and total ATM, as well as the ATM kinase substrates, SMC1 and KAP1. Tubulin was immunoblotted as loading control. ( C ) The non-phosphorylatable mutant MRE11 cell line displays increased cellular sensitivity to IR. Plot of percent survival after 0, 1, 2, 3, 4 and 5 Gy IR. NFF (control), ATLDMRE11 (WT), ATLDS676AS678A (MUT), ATLDVEC (VEC), A-T, ATLDS676A (S676A) and ATLDS678A (S678A) cell lines were treated with increasing doses of IR and percent survival assessed by clonogenic cell survival assay. The mean ± standard deviation is plotted from 3 independent experiments. ( D ) Non-phosphorylatable mutant MRE11 cell line is unable to correct radiation induced chromosomal aberrations. Aberrations (chromatid breaks, chromosome breaks and interchanges) were scored from Giemsa stained metaphases in 2 Gy irradiated NFF (control), A-T, ATLDMRE11 (WT), ATLDS676AS678A (MUT), ATLDVEC (VEC), ATLDS676A (S676A) and ATLDS678A (S678A) cell lines. Induced chromosomal aberrations (ICA) were totalled and divided by number of metaphases as indicated in parentheses.

Article Snippet: Whole cell extracts or immune complexes were separated by electrophoresis on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and proteins transferred to nitrocellulose membranes using Towbin's buffer (20% methanol, 50 mM Tris, 40 mM glycine and 0.02% SDS) at 100 V for 1 h. Blots were incubated with antibodies against MRE11 (12D7; GeneTex), Phospho-SQ/TQ (Cell Signaling Technologies), RAD50 (Upstate), NBS1 (Novus Biologicals), ATM (2C1; GeneTex), ATM pS1981 (GeneTex), SMC1 and SMC1 pS957 (GeneTex), Kap1 and Kap1 p824 (Novus Biologicals), GAPDH (GeneTex) and GFP (Abcam).

Techniques: Expressing, Mutagenesis, Western Blot, Immunoprecipitation, Control, Clonogenic Cell Survival Assay, Standard Deviation, Staining, Irradiation