atr antibody  (Bio-Techne corporation)

Journal: Nature communications

Article Title: ATR regulates neuronal activity by modulating presynaptic firing.

doi: 10.1038/s41467-021-24217-2

Figure Lengend Snippet: Fig. 1 Deletion of ATR in Purkinje cells leads to locomotor dysfunction and learning defects in mice. a Sagittal brain sections of the control (Ctr) and ATR-PCΔ (PCΔ) brains were stained with cresyl violet (Nissl staining). Upper panel shows the complete cerebellum. The magnified view of the frames in the upper and mid panels are shown in the lower panels, respectively. The images are representative from 3 to 4 mice of each genotype analyzed. DCN Deep Cerebellar Nuclei, ML Molecular Layer, PCL Purkinje Cell Layer, GCL Granular Cell Layer. b Sagittal sections of 3-month-old control and ATR-PCΔ mice were stained with DAPI, Calbindin and GFAP antibodies to label nuclei, Purkinje cells and Bergmann glia, respectively. ML Molecular Layer, PCL Purkinje Cell Layer, GCL Granular Cell Layer. c Quantification of total Purkinje cell number compared to control at the indicated age. The data is represented as fold changes compared to the control group. The number of mice is indicated within the bar. Error bars indicate SD. Student’s t-test (unpaired, two- tailed) is performed for the statistical analysis. The p values are indicated in the graphs. d Quantification of the thickness of the molecular layer of the cerebellum of mice at the indicated age. The number of mice is indicated within the bar. Student’s t-test (unpaired, two-tailed) is performed for the statistical analysis. The p values are indicated in the graphs. e The rotarod performance of 4–9-month-old mice on five consecutive days. Error bars indicate SEM. P values: day 2, p = 0.014; day 3, p = 0.002; day 5, p = 0.028; group comparison p = 0.034. f The rotarod performance of 18–20-month-old mice on five consecutive days. Error bars indicate SEM. P values: day 1 p = 0.025; day 5, p = 0.024, group comparison p = 0.041. g The beam walking performance was tested in the 18–20-month-old group. The latencies on 1 cm width beam platform on three consecutive days are shown. Error bars indicate SEM. p = 0.072. h The number of hindlimb slips during the beam walking performance test. Error bars indicate SEM. P values: day 2, p = 0.015; group comparison, p = 0.017. Two-way repeated measures ANOVA with Sidak’s multiple comparisons test or MWU within individual days was performed for the statistical analysis in e–h. *p < 0.05, **p < 0.01. The number of mice (n) tested are indicated within the graph legend. Source data are provided as a Source Data file.

Article Snippet: The following antibodies and dilutions were used in the study: ATR 1:250 (SantaCruz, #sc-515173), GAPDH 1:5000 (Sigma-Aldrich, #G8795), ATM 1:1000 (Novus Biologicals, # NB100-309), PAR 1:1000 (Trevigen, # 4336-BPC-100), PARP1 1:1000 (SantaCruz, #sc-1561), Chk1 1:1000 (Bethyl Laboratories, #A300162A), phospho-Chk1 1:1000 (Bethyl Laboratories, #A300-163A), γH2AX S139 1:1000 (Upstate, #05-636), Syt1 1:1000 (Synaptic Systems, #105011), Syt2 1:1000 (Synaptic Systems, #105123), Slc6a7 1:400 (GeneTex, #GTX65943), NR2B 1:1000 (Millipore, #06-600), GluR1 1:1000 (SantaCruz, #sc-13152), Kv1.1 1:200 (Alomone, #APC-009), GAD67 1:1000 (Abcam, #ab26116), phospho-(S/T) ATM/ATR substrate 1:1000 (Cell Signaling, #2851 S), tdTomato 1:1000 (SICGEN, #AB8181200), H3 1:1000 (Abcam #ab1791).

Techniques: Control, Staining, Two Tailed Test, Comparison

Journal: Nature communications

Article Title: ATR regulates neuronal activity by modulating presynaptic firing.

doi: 10.1038/s41467-021-24217-2

Figure Lengend Snippet: Fig. 3 ATR deletion in excitatory neurons of the mouse forebrain (ATR-FBΔ) leads to developments of epileptic signs. a Dorsal views of the brains from 3-month-old control (Ctr) and ATR-FBΔ (FBΔ) mice. Ob Olfactory Bulb, Ctx Cortex, Cb Cerebellum. b Coronal brain sections of 3-month-old control and ATR-FBΔ brains were stained with Nissl. The upper panel shows the complete half hemisphere and the lower panel displays the magnified images of the hippocampal regions. The images are representative from 3 to 4 mice per genotype analyzed. Ctx Cortex, HC Hippocampus, Amy. Amygdala, DG Dentate Gyrus, CA Cornu Ammonis, H Hilus. c Hippocampal astrogliosis in ATR-FBΔ mice. Coronal sections from control and ATR-FBΔ littermates at 10–12 months old after epileptic seizures were stained with DAPI, GFAP and NeuN to label nuclei, astrocytes and post-mitotic neurons, respectively. Right panels show magnifications of the dentate gyrus. Neurons are leaving the dentate gyrus (arrows) and expression of GFAP is markedly increased. CA Cornu Ammonis, DG Dentate Gyrus, H Hilus, GL Granular Layer. The graph shows the quantification of GFAP-positive cells within the hippocampal area from at least 2–3 sections per animal. The number of mice used is indicated within the bar. Data are presented as mean values of ± SD. Student’s t-test (unpaired, two- tailed). p = 0.013. *p < 0.05. d Mossy fiber sprouting in the hippocampus of 10-month-old epileptic ATR-FBΔ mice. Coronal sections of brains were stained with DAPI, ZnT-3 and SMI312 antibodies to label nuclei, mossy fibers and axonal neurofilaments, respectively. White arrows indicate the mossy fiber sprouting in IML. Neurofilaments follow the same pattern of ZnT-3 staining. The magnified images of the white rectangles on the DG and CA3 areas are shown in the right panels. The images are representative from 3 mice per genotype analyzed. CA Cornu Ammonis, DG Dentate Gyrus, SPMF Suprapyramidal Mossy Fibers, IPMF Infrapyramidal Mossy Fibers, H Hilus, GL Granular Layer, ML Molecular Layer, IML Inner Molecular Layer. Source data are provided as a Source Data file.

Article Snippet: The following antibodies and dilutions were used in the study: ATR 1:250 (SantaCruz, #sc-515173), GAPDH 1:5000 (Sigma-Aldrich, #G8795), ATM 1:1000 (Novus Biologicals, # NB100-309), PAR 1:1000 (Trevigen, # 4336-BPC-100), PARP1 1:1000 (SantaCruz, #sc-1561), Chk1 1:1000 (Bethyl Laboratories, #A300162A), phospho-Chk1 1:1000 (Bethyl Laboratories, #A300-163A), γH2AX S139 1:1000 (Upstate, #05-636), Syt1 1:1000 (Synaptic Systems, #105011), Syt2 1:1000 (Synaptic Systems, #105123), Slc6a7 1:400 (GeneTex, #GTX65943), NR2B 1:1000 (Millipore, #06-600), GluR1 1:1000 (SantaCruz, #sc-13152), Kv1.1 1:200 (Alomone, #APC-009), GAD67 1:1000 (Abcam, #ab26116), phospho-(S/T) ATM/ATR substrate 1:1000 (Cell Signaling, #2851 S), tdTomato 1:1000 (SICGEN, #AB8181200), H3 1:1000 (Abcam #ab1791).

Techniques: Control, Staining, Expressing, Two Tailed Test

Journal: Nature communications

Article Title: ATR regulates neuronal activity by modulating presynaptic firing.

doi: 10.1038/s41467-021-24217-2

Figure Lengend Snippet: Fig. 4 The CA3 region is susceptible to epileptiform activity in the ATR-deleted hippocampus. a Deletion of ATR increases the amplitude of the preictal epileptiform discharges (PEDs). Example 30-second sections of 40-minute extracellular field potential recordings of PEDs in CA3. Zoom-in representative PEDs (0.5–5000 Hz) in population field activity band (PFA, 1–100 Hz) and fast ripple activity band (FRA, 200–500 Hz). b PEDs show higher instantaneous power in ATR-FBΔ (FBΔ) CA3 slices compared to controls (Ctr). Color-coded raster plots of the PED timing in all recorded slices. Colors represent the maximum instantaneous power of each PED in 1–100 Hz band. The slices are arranged in descending order, by the first slice having the highest number of detected PEDs. The slices containing PEDs with a power-level beyond 0.4 µV2 (high-activity PEDs) are designated with a black square next to them. The ratio of slices with high-activity versus those with low-activity PED instantaneous power is increased after ATR deletion (right panel); Chi-square test (one- tailed), p = 0.012. c The instantaneous power of the whole field potential signal in 1–100 Hz band, computed within non-overlapping 1-min bins. The mean (solid line) ± SEM (shaded area). d Same as (c), but only for isolated PEDs (peak values) within 5-min bins. A one-tailed permutation test method of Cohen is used to compare the results of the two groups at each bin, with p < 0.05 (see Methods). e ATR-deletion leads to an increase in the power spectrum of PEDs over 1–100 Hz band. Power spectral density (PSD) of the PED events (solid lines), together with that of the baseline (i.e. non-PED epochs; dotted lines). f The summed baseline-normalized power presents an increase in the power of PEDs over 1–100 Hz shown in (e). Data presented as mean ± SEM. g ATR-deletion induces stronger fast ripples within PEDs. Same as (f), but for the fast ripple band (200–500 Hz). h Same as (f), but for the gamma band (30–90 Hz). i ATR-deletion increases the rate of spikes within PEDs, as detected in the multiple unit activity signal (MUA, > 500 Hz). Data are obtained from 16 slices of 4 control and 18 slices of 4 ATR-FBΔ mice and presented as mean ± SEM. Two-tailed t-test for (e–i): p = 0.026 for (e) and (f), p = 0.023 for (g), p = 0.027 for (h), and p = 0.016 for (i). *p < 0.05. Source data are provided as a Source Data file.

Article Snippet: The following antibodies and dilutions were used in the study: ATR 1:250 (SantaCruz, #sc-515173), GAPDH 1:5000 (Sigma-Aldrich, #G8795), ATM 1:1000 (Novus Biologicals, # NB100-309), PAR 1:1000 (Trevigen, # 4336-BPC-100), PARP1 1:1000 (SantaCruz, #sc-1561), Chk1 1:1000 (Bethyl Laboratories, #A300162A), phospho-Chk1 1:1000 (Bethyl Laboratories, #A300-163A), γH2AX S139 1:1000 (Upstate, #05-636), Syt1 1:1000 (Synaptic Systems, #105011), Syt2 1:1000 (Synaptic Systems, #105123), Slc6a7 1:400 (GeneTex, #GTX65943), NR2B 1:1000 (Millipore, #06-600), GluR1 1:1000 (SantaCruz, #sc-13152), Kv1.1 1:200 (Alomone, #APC-009), GAD67 1:1000 (Abcam, #ab26116), phospho-(S/T) ATM/ATR substrate 1:1000 (Cell Signaling, #2851 S), tdTomato 1:1000 (SICGEN, #AB8181200), H3 1:1000 (Abcam #ab1791).

Techniques: Activity Assay, One-tailed Test, Isolation, Control, Two Tailed Test

Journal: Nature communications

Article Title: ATR regulates neuronal activity by modulating presynaptic firing.

doi: 10.1038/s41467-021-24217-2

Figure Lengend Snippet: Fig. 7 ATR deletion in neurons does not cause activation of the DDR. a Western blot analysis of the hippocampus of 3-month-old control (Ctr) and ATR- FBΔ (FBΔ) mice for indicated proteins. The DDR activation is controlled by HCT116 cells without (−) or with (+) tBHP for 15 min. GAPDH serves as loading control. Four mice (numbers on the top of lanes) of the indicated genotypes were analyzed. b Immunostaining of ATR-PCΔ cerebellum with antibodies against Calbindin (for PCs) and γH2AX (DNA damage marker, white arrows). The γH2AX signal is controlled by the cortical sections of mice after 4 Gy ionizing irradiation (IR). The images are representative from 3 to 4 mice per genotype analyzed. Source data are provided as a Source Data file.

Article Snippet: The following antibodies and dilutions were used in the study: ATR 1:250 (SantaCruz, #sc-515173), GAPDH 1:5000 (Sigma-Aldrich, #G8795), ATM 1:1000 (Novus Biologicals, # NB100-309), PAR 1:1000 (Trevigen, # 4336-BPC-100), PARP1 1:1000 (SantaCruz, #sc-1561), Chk1 1:1000 (Bethyl Laboratories, #A300162A), phospho-Chk1 1:1000 (Bethyl Laboratories, #A300-163A), γH2AX S139 1:1000 (Upstate, #05-636), Syt1 1:1000 (Synaptic Systems, #105011), Syt2 1:1000 (Synaptic Systems, #105123), Slc6a7 1:400 (GeneTex, #GTX65943), NR2B 1:1000 (Millipore, #06-600), GluR1 1:1000 (SantaCruz, #sc-13152), Kv1.1 1:200 (Alomone, #APC-009), GAD67 1:1000 (Abcam, #ab26116), phospho-(S/T) ATM/ATR substrate 1:1000 (Cell Signaling, #2851 S), tdTomato 1:1000 (SICGEN, #AB8181200), H3 1:1000 (Abcam #ab1791).

Techniques: Activation Assay, Western Blot, Control, Immunostaining, Marker, Irradiation

Journal: Nature communications

Article Title: ATR regulates neuronal activity by modulating presynaptic firing.

doi: 10.1038/s41467-021-24217-2

Figure Lengend Snippet: Fig. 8 ATR deletion upregulates presynaptic proteins SYT2 and PROT. a Western blot analysis of the indicated proteins in whole-cell extract (S1) and synaptosome (P2) fractions of 3-month-old hippocampus of control (Ctr) and ATR-FBΔ (FBΔ) mice. GAPDH controls loading. Note, GAD67 expression is controlled by a separate control GAPDH below. The right panel shows the quantification of the indicated proteins after normalization with GAPDH. The data is represented as relative fold change in protein expression in P2 fraction, compared to control mice. N = 3 mice for each genotype. The vertical dashed line in the graph indicates control mice. Data are mean values ± SD. Student’s t-test (unpaired, one-tailed for ATR, SYT2 and PROT, two-tailed for others). P value: ATR, p = 0.025; SYT, p = 0.001; PROT, p = 0.029. *p < 0.05, **p < 0.01. b ATR interacts with SYT2 and PROT in vitro. Murine neuroblastoma cells (N2a) were transfected with Tomato-SYT2 or Tomato-PROT treated with or without with 2 µM ATR inhibitor VE-821. SYT2 and PROT were immunoprecipitated with Tomato antibody and blotted by indicated antibodies. The experiment was repeated four times. c Immunoprecipitation of protein extract from hippocampi and cerebella using antibodies as indicated. IP against IgG serves as control. Histone H3 serves as loading control for the inputs. IgG light chain was used to control the amount of antibodies used for IP. Note, an overloading of IgG in IP-IgG lanes correlates with unspecific binding signals in these samples. The number indicates individual mice. The experiment was repeated twice. Source data are provided as a Source Data file.

Article Snippet: The following antibodies and dilutions were used in the study: ATR 1:250 (SantaCruz, #sc-515173), GAPDH 1:5000 (Sigma-Aldrich, #G8795), ATM 1:1000 (Novus Biologicals, # NB100-309), PAR 1:1000 (Trevigen, # 4336-BPC-100), PARP1 1:1000 (SantaCruz, #sc-1561), Chk1 1:1000 (Bethyl Laboratories, #A300162A), phospho-Chk1 1:1000 (Bethyl Laboratories, #A300-163A), γH2AX S139 1:1000 (Upstate, #05-636), Syt1 1:1000 (Synaptic Systems, #105011), Syt2 1:1000 (Synaptic Systems, #105123), Slc6a7 1:400 (GeneTex, #GTX65943), NR2B 1:1000 (Millipore, #06-600), GluR1 1:1000 (SantaCruz, #sc-13152), Kv1.1 1:200 (Alomone, #APC-009), GAD67 1:1000 (Abcam, #ab26116), phospho-(S/T) ATM/ATR substrate 1:1000 (Cell Signaling, #2851 S), tdTomato 1:1000 (SICGEN, #AB8181200), H3 1:1000 (Abcam #ab1791).

Techniques: Western Blot, Control, Expressing, One-tailed Test, Two Tailed Test, In Vitro, Transfection, Immunoprecipitation, Binding Assay

Journal: Molecular cell

Article Title: MRI is a DNA Damage Response Adaptor during Classical Non-Homologous End Joining

doi: 10.1016/j.molcel.2018.06.018

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit anti-ATR , Novus Biologicals , Cat#NB100-323.

Techniques: Blocking Assay, Virus, Recombinant, Cell Isolation, Magnetic Beads, Mass Spectrometry, Sequencing, Magnetic Resonance Imaging, Plasmid Preparation, Software, High Content Screening, Flow Cytometry, Inverted Microscopy, Laser-Scanning Microscopy, Spectrophotometry, Irradiation