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:

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

Journal: PLoS ONE

Article Title: Vesicular Location and Transport of S100A8 and S100A9 Proteins in Monocytoid Cells

doi: 10.1371/journal.pone.0145217

Figure Lengend Snippet: (A) S100A9 in the biotinylated plasma membrane fraction of THP-1 cells: non-biotinylated membrane fraction (lane a) and biotinylated cell surface fraction (lane b). (B) S100A8 expression in biotinylated THP-1 cells (same sample fractions as in (A). (C) Expression of S100A9 (left) and S100A8 (right) in the non-biotinylated membrane fraction (lane a) and biotinylated cell surface fraction (lane b) of human monocytes. Antibodies used were; α-S100A9 (NOVUS clone 1C10) and α-S100A8 (BMA; T1030; clone 8-5C2). 4 ng recombinant hS100A9 (rA9) was loaded.

Article Snippet: Then antibodies raised against human S100A8 (mAb 8-5C2 from BMA Biomedicals, Augst, Switzerland), S100A9 (mAb 43/8; produced at Active Biotech AB), S100A8/S100A9 (mAb 27E10; BMA Biomedicals, Augst, Switzerland), RAGE/Fc (MAB11451; R&D Systems Minneapolis, MN, USA) or TLR4 (MAB14781; R&D Systems, Minneapolis, MN, USA) were injected for 2–3 min in 10 mM Hepes, 0.15 M NaCl and 0.005% Surfactant P20 (HBS-P), pH 7.4, containing 1 mM Ca ++ and 10 μM Zn ++ .

Techniques: Clinical Proteomics, Membrane, Expressing, Recombinant

Journal: PLoS ONE

Article Title: Vesicular Location and Transport of S100A8 and S100A9 Proteins in Monocytoid Cells

doi: 10.1371/journal.pone.0145217

Figure Lengend Snippet: (A) Binding of anti-S100A8 (8-5C2), S100A9 (43/8) and S100A8/S100A9 (27E10) to SA captured biotinylated plasma membranes. Antibodies were injected at 6.25–50 nM for 2 min at 30 μL/min in assay buffer (HBS-P with 1 mM Ca ++ + 20 μM Zn ++ ). A 15 μL pulse of 10 mM glycine-HCl was used for regeneration. Capture level ~ 2.1 kRU. Kinetic evaluation was made using a 1:1 model for mAb 43/8 at 25 and 50 nM. An affinity of 7.0 nM ( k on 1.9 × 10 5 1/Ms; k off 1.3 × 10 −3 1/s) was calculated with maximal binding of 46 RU. (B) Injection of non-biotinylated THP-1 lysate over immobilized mAb 8-5C2, 43/8 and 27E10 (density ~ 3 kRU). Lysate, 0.45 mg/mL, diluted 5 to 40 times in assay buffer, injected for 3 min at 20 μL/min. Distinct binding was obtained when the THP-1 lysate was injected over the 27E10 surface whereas no binding was observed when lysate was passed over 8-5C2 and 43/8. (C) Binding of ten-fold diluted non-biotinylated THP-1 lysate to mAb 27E10 is inhibited after pre-incubation with 50 nM 27E10 but not with 8-5C2 or 43/8. Binding was expressed as % of response without antibody. (D) Sensorgrams obtained after injection of 50 nM mAb 8-5C2, 43/8 and 27E10 over SA captured (level ~ 1.1 kRU) solubilized membranes from surface biotinylated human monocytes. An affinity of ~ 6.1 nM (( k on 2.7 × 10 5 1/Ms; k off 1.6 × 10 −3 1/s) and maximal binding at ~ 12 RU was obtained after fit of sensorgram obtained with the 43/8 antibody to a 1:1 model. (E) Interaction of non-biotinylated lysate from human monocytes with 8-5C2, 43/8 and 27E10. Serially diluted lysate (5- to 40-fold in assay buffer) was injected over immobilized antibodies as in . Binding at high levels was demonstrated to mAb 27E10 (reaching steady state levels at ~ 1,000 RU) and was completely blocked when lysate (diluted 5-fold) was pre-incubated with 50 nM 27E10 but not with 8-5C2 or 43/8 (Fig 2C). Specific (i.e. displaceable by pre-incubation with anti-S100A9; data not shown) binding at much lower levels was also shown to 43/8 whereas non-specific binding was demonstrated to 8-5C2 (Fig 2E).

Article Snippet: Then antibodies raised against human S100A8 (mAb 8-5C2 from BMA Biomedicals, Augst, Switzerland), S100A9 (mAb 43/8; produced at Active Biotech AB), S100A8/S100A9 (mAb 27E10; BMA Biomedicals, Augst, Switzerland), RAGE/Fc (MAB11451; R&D Systems Minneapolis, MN, USA) or TLR4 (MAB14781; R&D Systems, Minneapolis, MN, USA) were injected for 2–3 min in 10 mM Hepes, 0.15 M NaCl and 0.005% Surfactant P20 (HBS-P), pH 7.4, containing 1 mM Ca ++ and 10 μM Zn ++ .

Techniques: Binding Assay, Clinical Proteomics, Injection, Incubation

Journal: PLoS ONE

Article Title: Vesicular Location and Transport of S100A8 and S100A9 Proteins in Monocytoid Cells

doi: 10.1371/journal.pone.0145217

Figure Lengend Snippet: THP1 cells were cultured for 48h in presence or absence of 10ng/ml TNFα and stained with antibodies against S100A9 (purple) (A). THP-1 cells were treated with either TNFα (B) or TNFα + IL10 (C); stained with anti-S100A8 (red), anti-S100A9 (purple) and anti-S100A8/S100A9 (green) antibodies. Arrows indicate the vesicles containing only hS100A9 or hS100A8. (D) Distribution of hS100A8 and hS100A9 proteins in THP-1 cells by Transmission Electron Microscopy. Black and white boxes represent magnification of areas where S100A8 and S100A9 hetero- or homodimers can be observed respectively. Further, black arrows highlight cytoplasmic vesicles or plasma membrane patches where hS100A9 (big dots) and hS100A8 (small dots) proteins co-localize, while red arrows point to vesicles containing only hS100A9 or hS100A8 homodimers.

Article Snippet: Then antibodies raised against human S100A8 (mAb 8-5C2 from BMA Biomedicals, Augst, Switzerland), S100A9 (mAb 43/8; produced at Active Biotech AB), S100A8/S100A9 (mAb 27E10; BMA Biomedicals, Augst, Switzerland), RAGE/Fc (MAB11451; R&D Systems Minneapolis, MN, USA) or TLR4 (MAB14781; R&D Systems, Minneapolis, MN, USA) were injected for 2–3 min in 10 mM Hepes, 0.15 M NaCl and 0.005% Surfactant P20 (HBS-P), pH 7.4, containing 1 mM Ca ++ and 10 μM Zn ++ .

Techniques: Cell Culture, Staining, Transmission Assay, Electron Microscopy, Clinical Proteomics, Membrane

Journal: PLoS ONE

Article Title: Vesicular Location and Transport of S100A8 and S100A9 Proteins in Monocytoid Cells

doi: 10.1371/journal.pone.0145217

Figure Lengend Snippet: THP-1 cells were cultured for 48h in presence or absence of 10 ng/ml TNFα. S100A8 and S100A9 mRNA expression was determined using qRT-PCR and was normalized to β-actin mRNA level. Data indicate mean ± SD of triplicate samples. Differences between treated and untreated groups are significant at ***P<0.001 by unpaired Student’s t test (A). Intracellular S100A8 and S100A9 protein expression was determined using Western blot (B). THP1 cells were treated with TNFα or left untreated. Cells were harvested either after 48 h (C) or after 24 h and 48 h (D); gently homogenized in homogenization buffer and post-nuclear supernatants (PNSs) were prepared. Using the PNSs as starting material, endosome enriched fractions were prepared using a discontinuous sucrose gradient centrifugation. Fractions were analysed by western blotting using the indicated antibodies. F1 is the interphase between 25% sucrose/homogenization buffer and F2 is the interphase between 35%/25% sucrose. (E) Immunofluorescence analysis of S100A9 localization in TNFα activated THP-1 cells; THP-1 cells cultured for 48 h with TNFα were fixed, permeabilized, stained with anti-S100A9 antibody (green) along with different vesicular marker antibodies anti-Rab5, anti-Rab7 and anti-cathepsin D (all of them marked in the red channel) and analyzed by confocal microscopy.

Article Snippet: Then antibodies raised against human S100A8 (mAb 8-5C2 from BMA Biomedicals, Augst, Switzerland), S100A9 (mAb 43/8; produced at Active Biotech AB), S100A8/S100A9 (mAb 27E10; BMA Biomedicals, Augst, Switzerland), RAGE/Fc (MAB11451; R&D Systems Minneapolis, MN, USA) or TLR4 (MAB14781; R&D Systems, Minneapolis, MN, USA) were injected for 2–3 min in 10 mM Hepes, 0.15 M NaCl and 0.005% Surfactant P20 (HBS-P), pH 7.4, containing 1 mM Ca ++ and 10 μM Zn ++ .

Techniques: Cell Culture, Expressing, Quantitative RT-PCR, Western Blot, Homogenization, Gradient Centrifugation, Immunofluorescence, Staining, Marker, Confocal Microscopy

Journal: PLoS ONE

Article Title: Vesicular Location and Transport of S100A8 and S100A9 Proteins in Monocytoid Cells

doi: 10.1371/journal.pone.0145217

Figure Lengend Snippet: THP-1 cells were cultured in triplicate either in the presence or absence of 10 ng/ml TNFα and TNFα + IL-10 (10 ng/ml). Culture supernatant was collected after 48h of incubation and S100A8/S100A9 dimer concentration was determined with ELISA following manufacturer’s protocol (A). Percent cell death was determined using LDH assay (B). In some experiments, THP-1 cells were stimulated with TNFα for 48 h in presence of methylamine (30 μM). In other experiments, cells were stimulated 12 h with TNFα without methylamine, followed by 36 h of culture with the drug. Alternatively, THP-1 cells were stimulated with TNFα; after 36 h, brefeldin A (5 μg/ml) was added and cells were cultured for another 12 h. Culture supernatant was collected after completion of incubation and S100A8/A9 heterodimer (C) and S100A9 (D) concentration was determined with ELISA following manufacturer’s protocol. Values are means of triplicates ± SD. Differences between various treatment groups are significant at **P<0.01, ***P<0.001 and ns = not significant by unpaired Student’s t test.

Article Snippet: Then antibodies raised against human S100A8 (mAb 8-5C2 from BMA Biomedicals, Augst, Switzerland), S100A9 (mAb 43/8; produced at Active Biotech AB), S100A8/S100A9 (mAb 27E10; BMA Biomedicals, Augst, Switzerland), RAGE/Fc (MAB11451; R&D Systems Minneapolis, MN, USA) or TLR4 (MAB14781; R&D Systems, Minneapolis, MN, USA) were injected for 2–3 min in 10 mM Hepes, 0.15 M NaCl and 0.005% Surfactant P20 (HBS-P), pH 7.4, containing 1 mM Ca ++ and 10 μM Zn ++ .

Techniques: Cell Culture, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Lactate Dehydrogenase Assay