Journal: The Journal of Cell Biology

Article Title: Loss of ATM kinase activity leads to embryonic lethality in mice

doi: 10.1083/jcb.201204035

Figure Lengend Snippet: ATM D2899A and Q2740P mutations display severely impaired kinase activity in vitro and cause embryonic lethality in mice. (A) Pairwise local alignments of human ( Homo sapiens ) and mouse ( Mus musculus ) ATM protein sequences surrounding sites of mutagenesis. (B) Kinase assay with 0.2 nM WT, D2889A, or Q2730P mutant dimeric human ATM, 3.6 nM MRN, 50 nM GST-p53 substrate, and linear DNA probed with an antibody to p53 S15 phosphorylation. A titration of ATM is shown for each sample. (C) Sequence chromatograms showing the amino acid mutations generated in mouse BAC RP24-122F10. (D and E) Thymocytes were harvested for lysate preparation and Western blot analysis. Molecular mass markers are in kilodaltons. (F and G) Tables showing the expected and observed genotypes of 3-wk-old pups from breeding Atm Tg Atm +/− with Atm +/− mice. The boxes highlight the comparison between the total number of ATM kinase-inactive and Atm -null live-born pups.

Article Snippet: In brief, 40 μg protein was loaded on 4–12% Bis-Tris gels (NuPAGE; Life Technologies) and probed with the following antibodies using HRP-conjugated secondary antibodies and ECL Western blotting detection reagents (GE Healthcare): ATM (clone 5C2; 1:400; Novus Biologicals), α-Tubulin (clone B512; 1:30,000; Sigma-Aldrich), KAP1 S824p (1:700; Bethyl Laboratories, Inc.), CHK1 S317p (1:500, Bethyl Laboratories, Inc.), and SMC1 (1:8,000; Novus Biologicals).

Techniques: Activity Assay, In Vitro, Mutagenesis, Kinase Assay, Phospho-proteomics, Titration, Sequencing, Generated, Western Blot, Comparison

Journal: The Journal of Cell Biology

Article Title: Loss of ATM kinase activity leads to embryonic lethality in mice

doi: 10.1083/jcb.201204035

Figure Lengend Snippet: Conditional ATM D2899A mice display defects mildly more severe than ATM deficiency in B cell development, genome stability, and sensitivity to PARP inhibitor. (A) Western blot analysis of splenic B cells stimulated with LPS and RP105 for 72 h, FACS sorted for YFP expression, and harvested for lysate preparation after a 10-Gy irradiation. Shown for comparison are sorted cells from two individual Atm TgD2899A Atm flox/− mice. In addition to the labeled Atm genotype, note that mice are also Cd19 cre/+ Rosa26 YFP/+ , for which YFP expression (as a marker of Cre recombinase expression) is found in pro-B cells and becomes progressively higher through B cell development to the mature B cell stage. Molecular mass markers are in kilodaltons. (B) YFP + frequency from freshly isolated B cells of the following subsets of bone marrow and spleen harvested and stained with antibodies to B220, CD43, and IgM for flow cytometric analysis. Pro-B (B220 + CD43 + IgM − ), pre-B (B220 + CD43 − IgM − ), immature B (B220 + CD43 − IgM int ), mature B (B220 + IgM + ), and splenic mature B (B220 + IgM + ) are shown. *, P < 0.00001. (C) YFP + absolute cell numbers from each subset in bone marrow and spleen. Legend same as in B. *, P < 0.05. (D) YFP + fraction of live B220 + splenic B cells stimulated with LPS as a function of days in culture. Each data point represents the YFP + frequency measured by flow cytometry, relative to day 0. For days 5 and 6, P < 0.0001 for Atm TgD2899A Atm flox/− versus Atm flox/+ , and P < 0.05 for Atm TgD2899A Atm flox/− versus Atm flox/− . (E) Splenic YFP + B cells stimulated with LPS, IL4, and RP105 for 72 h were harvested for metaphase chromosome preparation. FISH was performed on slides with a probe for telomeres and counterstained with DAPI. Frequency of aberrations per metaphase is shown for chromatid breaks (CTB), chromosome breaks (CSB), radials, and translocations ( Atm flox/+ : n = 105 from 1 mouse; Atm TgD2899A Atm flox/− : n = 313 from 3 mice; Atm flox/− : n = 200 from 2 mice). (F) YFP + fraction of live B220 + splenic B cells stimulated with LPS and 1 µM PARP inhibitor. Each data point represents the YFP + frequency measured by flow cytometry relative to no treatment of the same genotype. Legend same as in D. On days 3 and 4, P < 0.05 for Atm TgD2899A Atm flox/− versus Atm flox/− . Error bars represent standard deviation. PARPi, PARP inhibitor.

Article Snippet: In brief, 40 μg protein was loaded on 4–12% Bis-Tris gels (NuPAGE; Life Technologies) and probed with the following antibodies using HRP-conjugated secondary antibodies and ECL Western blotting detection reagents (GE Healthcare): ATM (clone 5C2; 1:400; Novus Biologicals), α-Tubulin (clone B512; 1:30,000; Sigma-Aldrich), KAP1 S824p (1:700; Bethyl Laboratories, Inc.), CHK1 S317p (1:500, Bethyl Laboratories, Inc.), and SMC1 (1:8,000; Novus Biologicals).

Techniques: Western Blot, Expressing, Irradiation, Comparison, Labeling, Marker, Isolation, Staining, Flow Cytometry, Standard Deviation