Journal: Cell reports

Article Title: Competition between NBS1 and ATMIN controls ATM signaling pathway choice.

doi: 10.1016/j.celrep.2012.11.002

Figure Lengend Snippet: Figure 4. Antagonism and Redundancy of ATMIN and NBS1 for ATM Signaling and Function (A)H&E stainingonrepresentativeintestinesofmice with the indicated genotypes, 5 days after Villin- creERT-mediated deletion of Atmin and Nbs1. (B) High-magnification representation of H&E- stained crypts. (C)RepresentativeMCM6 IHCon intestines of mice with the indicated genotypes, 5 days after Villin- creERT-mediated deletion of Atmin and Nbs1. (D) Quantification of (C); **p = 0.0093. (E) Cells were treated with 20 Gy irradiation and fixed at the indicated time points post treatment. The percentage of subG1 cells was determined by flow cytometry analysis. In (D) and (E), error bars show the SD of at least three independent experiments, and Student’s t test was used for statistical analysis. (F) Four days after Villin-creERT-mediated dele- tion of Atmin and Nbs1, mice were treated with the indicated doses of irradiation (n = 5 mice per genotype per dose). (G) H&E staining on representative intestines of mice with the indicated genotypes, 24 hr after treatment with 14 Gy irradiation. (H) Representative 53BP1 IHC in intestines of mice with the indicated genotypes, 24 hr after treatment with 14 Gy irradiation. Arrowheads in- dicate cells with 53BP1 foci. (I) Mice were culled 2 hr after 10 Gy irradiation, and proteinlysateswere prepared from intestinal tissue. Lysates were resolved on a 6% SDS-PAGE gel and the membrane was probed for pS1987-ATM, pS957-SMC1, SMC, pS15-p53, p53, and actin. See also Figure S4.

Article Snippet: The following antibodies were used: pS1981-ATM (10H11.E12; Cell Signaling), ATM (2C1; Santa Cruz), pS824-Kap1 (Bethyl Laboratories), Kap1 (Bethyl Laboratories), b-actin (A5060; Sigma), pS957-SMC1 (5D11G5; Millipore), SMC1 (AB3908; Millipore), pS966-SMC1 (A300-050A; Bethyl Laboratories), p53 (2524; Cell Signaling), NBS1 (NB100-143, Novus Biologicals), DHX9 (gift from Professor F. Grosse), pS15-p53 (9284 and 9286; Cell Signaling), pATM (2152-1, Epitomics), horseradish-peroxidase-conjugated goat anti-mouse/rabbit immunoglobulin G (Jackson).

Techniques: Staining, Irradiation, Cytometry, SDS Page, Membrane

Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: Human Rad9 is required for the activation of S-phase checkpoint and the maintenance of chromosomal stability.

doi: 10.1111/j.1365-2443.2005.00840.x

Figure Lengend Snippet: Figure 2 Rad9 is required for the phosphorylation of Smc1 and Chk1 as an early response induced by replication block. (A) Cells transfected with vector or Rad9 RNAi construct were incubated in the presence of 5 µg/mL APH for 15 min, 30 min, 60 min or 240 min before harvest. Extracts of these cells were analyzed by immunoblotting with indicated antibodies: anti-pS966-Smac1, ant-total Smac1, anti-pS345Chk1 and anti-total Chk1. (B) Vector or Rad9 RNAi transfected cells were left untreated, or treated with 10 µm MNNG for 1 h (and harvested at indicated time), or 5 µg/mL aphidicolin (APH) for 1 h before harvest, followed by immunoblotting with anti-pS966-Smc1 and anti-total Smc1.

Article Snippet: Smc1-pS966 (BL311) and Smc1 (BL308) antibodies were from Bethyl Laboratories (Montgomery, TX, USA).

Techniques: Phospho-proteomics, Blocking Assay, Transfection, Plasmid Preparation, Construct, Incubation, Western Blot