Journal: Neuron

Article Title: Reversible Conformational Conversion of α-Synuclein into Toxic Assemblies by Glucosylceramide

doi: 10.1016/j.neuron.2017.12.012

Figure Lengend Snippet: Key Resources Table

Article Snippet: The following anti-α-Syn primary antibodies were used: Name epitope (residues) Company Cat # dilution F-11 2-25 Santa Cruz sc-514908 1:500 LS-C352877 15-55 LifeSpan Biosciences LS-C352877 1:500 5C2 61-95 Novus Biologicals NBP1-04321 1:200 syn-1 91-99 BD Biosciences 610787 1:500 syn211 121-125 Sigma-Aldrich 36-008-25UL 1:500 S9500 ∼130 Biomol GmbH S9500-01E.100 1:500 Primary antibodies were detected with fluorescent-conjugated secondary antibodies as described under western blot analysis and quantified with Li-Cor Image Studio software.

Techniques: Virus, Recombinant, DNA Extraction, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, CRISPR, Sequencing, Plasmid Preparation, shRNA, Software

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

Journal: iScience

Article Title: Dorsal raphe nucleus to pre-Bötzinger complex serotonergic neural circuit is involved in seizure-induced respiratory arrest

doi: 10.1016/j.isci.2022.105228

Figure Lengend Snippet: Optogenetic activation of TPH2-ChETA neurons in DR-mediated reduction of PTZ-induced S-IRA was significantly reversed by the injection of KET into the PBC (A) Schematic illustration of the pAAV-TPH2 PRO-ChETA-EYFP-WPRES-PAS delivery into the DR of DBA/1 mice and implantation of the optic fiber into the DR and cannula into the bilateral PBC to receive PS of the DR. (B) Compared with that in the control group treated with PTZ without PS with a microinjection of 400 nL 25% DMSO into the bilateral PBC, the incidence of PTZ-induced S-IRA was significantly reduced in the group injected PTZ with PS in the DR with microinjection of 400 nL 25% DMSO into the bilateral PBC (n = 7 and n = 6, respectively; p < 0.01). Compared with the group with PTZ with PS in the DR with microinjection of 400 nL 25% DMSO into the bilateral PBC, the incidence of PTZ-induce S-IRA was significantly reduced in the group with PTZ that received PS and microinjection of 400 nL KET into the bilateral PBC (n = 6 and n = 7, respectively; p < 0.01). (C) No obvious differences were observed between treatment groups in the GSz latency (s) (p > 0.05). (D) Duration of wild running and clonic seizure in the group with 25% DMSO + NO PS was significantly higher than the group with 25% DMSO + PS and the group with KET 400 nL + PS (p < 0.001 and p < 0.001, respectively). (E and F) The seizure score or duration of tonic seizures in the group with 25% DMSO + PS was significantly reduced compared to the group with 25% DMSO + NO PS and the group with KET 400 nL + PS (p < 0.01 and p < 0.001, respectively). (G–J) Representative images of the delivery of pAAV-TPH2 PRO-ChETA-EYFP-WPRES-PAS into the DR and injection of KET into the bilateral PBC. (H) Staining of ChETA, TPH2, and DAPI in the DR. (I) All implantation devices in a DBA/1 mouse. (J) The tracks for KET injection into the bilateral PBC and staining for NK1R, 5-HT2AR, and DAPI in the bilateral PBC. No PS, no photostimulation; PS, photostimulation.

Article Snippet: For microinjection of KET in the bilateral PBC, guide cannulas (O.D.0.48 × I.D.0.34 mm/M3.5,62033, RWD Life Science Inc.) were implanted on both sides.

Techniques: Activation Assay, Injection, Control, Microinjection, Staining

Journal: iScience

Article Title: Dorsal raphe nucleus to pre-Bötzinger complex serotonergic neural circuit is involved in seizure-induced respiratory arrest

doi: 10.1016/j.isci.2022.105228

Figure Lengend Snippet: Activation of ChETA-TPH2 neurons in the PBC could have an effect on S-IRA but did not significantly reduce the incidence of S-IRA in DBA/1 mice or prevent death (A) Schematic illustration of the pAAV-TPH2-PRO-ChETA-EYFP-WPRES-PAS delivery into the bilateral PBC of DBA/1 mice and implantation of the optic fiber and cannula to receive PS of the PBC and observe changes in the incidence of PTZ-induced S-IRA upon the microinjection of KET in the PBC. (B–F) The tracks of optic fibers with cannula implanted into each side of the bilateral PBC and staining for ChETA, TPH2, NK1R, 5-HT2AR, and DAPI expression in the bilateral PBC. (G) Compared with that in the control group treated with PTZ without PS, the incidence of PTZ-induce S-IRA was not significantly reduced by PS of the bilateral PBC (p > 0.05). Furthermore, no significant differences were observed between the group with PS and microinjection of vehicle into the bilateral PBC and the group with PS and microinjection of KET into the bilateral PBC (p > 0.05). (H–K) No obvious differences between treatment groups were observed in the seizure score, duration of wild running and clonic seizure, AGSz latency, duration of tonic seizure, or seizure score (p > 0.05). No PS, no photostimulation; PS, photostimulation.

Article Snippet: For microinjection of KET in the bilateral PBC, guide cannulas (O.D.0.48 × I.D.0.34 mm/M3.5,62033, RWD Life Science Inc.) were implanted on both sides.

Techniques: Activation Assay, Microinjection, Staining, Expressing, Control

Journal: iScience

Article Title: Dorsal raphe nucleus to pre-Bötzinger complex serotonergic neural circuit is involved in seizure-induced respiratory arrest

doi: 10.1016/j.isci.2022.105228

Figure Lengend Snippet: Mechanism by which 5-HT neurons in the brain modulate S-IRA and SUDEP via the neural circuit between DR and PBC SUDEP, sudden unexpected death in epilepsy; S-IRA, seizure-induced respiratory arrest; KET, ketanserin; PBC, pre-Bötzinger complex; DR, dorsal raphe nucleus; 5-HT2AR, 5-HT2A receptor.

Article Snippet: For microinjection of KET in the bilateral PBC, guide cannulas (O.D.0.48 × I.D.0.34 mm/M3.5,62033, RWD Life Science Inc.) were implanted on both sides.

Techniques:

Journal:

Article Title: Identification of the Tumor Cells in Peripheral T-Cell Lymphomas by Combined Polymerase Chain Reaction-Based T-Cell Receptor ? Spectrotyping and Immunohistological Detection with T-Cell Receptor ? Chain Variable Region Segment-Specific Antibodies

doi:

Figure Lengend Snippet: Antibodies Used for Single and Double Stains

Article Snippet: All cases had been diagnosed according to the World Health Organization classification and were characterized by conventional immunohistochemistry with antibodies against CD3, CD5, CD4, CD8, TIA-1, GranzymeB, Perforin and TCRβ chain, TCRδ chain, and CD94 , thus identifying the tumor cells morphologically. table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Antibody Clone Company Dilution Conjugation Detection Vβ 2 MPB2D5 BC 1:200 Donkey anti-mouse CY3 Vβ 5.1 IMMU157 BC 1:400 Donkey anti-mouse CY3 Vβ 5.2 36213 BC 1:100 Donkey anti-mouse CY3 Vβ 5.3 3D11 BC 1: 50 Donkey anti-mouse CY3 Vβ 8.1 and Vβ 8.2 56C5 BC 1:200 Donkey anti-mouse CY3 Vβ 9 FIN9 BC 1:100 Donkey anti-mouse CY3 Vβ14 CAS1.1.3 BC 1: 50 Donkey anti-mouse CY3 Vβ17 E17.5F3 BC 1: 50 Donkey anti-mouse CY3 Vβ 18 BA62 BC 1:20 Donkey anti-mouse CY3 Vβ 22 IMMU546 BC 1:20 Donkey anti-mouse CY3 CD 3 Polyclonal Dako 1: 2 Donkey anti-rabbit CY2 CD 4 MT310 Dako 1: 10 FITC CD 4 BL4 BC 1:100 Avidin-biotin POX CD 5 BL1A BC 1: 10 FITC CD 5 BL1A BC 1:400 Avidin-biotin POX CD 8 B9.11 BC 1: 10 FITC CD 8 5C2–2H2 Dianova 1:20 Donkey anti-mouse IgM Cy5 CD 8 C8/144B Dako 1:1600 Avidin-biotin POX TIA-1 2G9 BC 1:200 Donkey anti-mouse CY3 TIA-1 2G9 BC 1:200 Avidin-biotin POX GranzymeB GrB-7 Monosan 1:100 Donkey anti-mouse CY3 GranzymeB GrB-7 Monosan 1:20 Avidin-biotin POX Perforin 5B10 Novocastra 1:20 Donkey anti-mouse CY3 Perforin 5B10 Novocastra 1:10 Avidin-biotin POX CD94 HP-3D9 Dako 1:100 Avidin-biotin POX PanTCRaβ 8A3 Serotec 1:100 Avidin-biotin POX PanTCRγδ 5A6.E9 BC 1:100 Avidin-biotin POX Open in a separate window Detection with fluorescent-labeled antibodies (1:100) (Dianova) or Zenon mouse IgG Labeling kits (Molecular Probes, Leiden, The Netherlands).

Techniques: Conjugation Assay, Avidin-Biotin Assay