goat anti syntaxin 1a  (R&D Systems)

Journal: Alzheimer's & dementia : the journal of the Alzheimer's Association

Article Title: Reduced synaptic proteins and SNARE complexes in Down syndrome with Alzheimer's disease and the Dp16 mouse Down syndrome model: Impact of APP gene dose.

doi: 10.1002/alz.12835

Figure Lengend Snippet: FIGURE 2 Reduction of SNARE complexes in AD-DS, AD, and Dp16 samples. Syntaxin 1A was present in a 35 kD monomeric form and in a 73 kD trimeric complex with SNAP25 and synaptobrevin 2 as well as other high molecular weight forms. A, D, G, Unboiled AD-DS (A), DS (D), and AD (G) SDS samples were loaded on SDS-PAGE and probed with the syntaxin 1A antibody. A boiled sample was also loaded as a control to demonstrate disruption of the complexes with heating. The arrow points to the 73 kD trimeric band. B, E, H, Profiles of relative intensities of the ladder of complexes from the representative samples marked with asterisks; the heaviest complexes are represented at the left end of the x-axis. C, F, I, Quantitation and statistical analysis of the levels of 73 kD trimeric SNARE complex in AD-DS and DS samples. Mann–Whitney test; *P < 0.05, **P < 0.01. J, Unboiled 2N, Dp16, and Dp16: App++−samples from mice aged 9 to 10 months were loaded on SDS-PAGE and probed with the syntaxin 1A antibody. Arrows point to the major 73 kD, 100–130 kD, and > 180 kD bands. K–M, Quantitation and statistical analysis of the levels of 73 kD, 100–130 kD and > 180 kD bands. One-way analysis of variance test followed by Newman–Keuls multiple comparison test. n = 3 mice for each category; *P < 0.05. C/, control for; AD-DS, Alzheimer’s disease with Down syndrome; DS, Down syndrome without Alzheimer’s disease; AD, Alzheimer’s disease; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis

Article Snippet: 3.1 Methods 3.1.1 Antibodies Mouse antibody anti-syntaxin 1A (1:5000; S0664; HPC-1) and rabbit antibody anti-APP (1:3000; A8717) were obtained from MilliporeSigma; rabbit antibody anti-SNAP25 (1:2000; 14903-1-AP), antiNPTX2 (10889-1-AP), mouse antibodies anti-synaptophysin (1:5000; 67864-1-Ig), anti-β-actin (1:10,000; 60008-1-Ig) were from Proteintech.

Techniques: High Molecular Weight, SDS Page, Control, Disruption, Quantitation Assay, MANN-WHITNEY, Comparison, Polyacrylamide Gel Electrophoresis

Journal: Alzheimer's & dementia : the journal of the Alzheimer's Association

Article Title: Reduced synaptic proteins and SNARE complexes in Down syndrome with Alzheimer's disease and the Dp16 mouse Down syndrome model: Impact of APP gene dose.

doi: 10.1002/alz.12835

Figure Lengend Snippet: FIGURE 4 Levels of selective biomarkers in the frontal cortex of AD-DS, DS, and the cortex of Dp16 mice. A, Western blotting analysis of the levels of NfL, NPTX2, and syntaxin1B in the frontal cortex of patients with DS, AD-DS, C/DS, or C/AD-DS. β-actin was used as a loading control. B, Quantitation and statistical analysis of the levels of NfL, NPTX2, and syntaxin 1B in female and male samples combined. Mann–Whitney test; *P < 0.05, **P < 0.01, ***P < 0.001. C, Western blotting analysis of the levels of NfL and NPTX2 in the cortex from 9- to 10-month-old and 20-month-old Dp16 mice. β-actin was used as a loading control. D, Quantitation and statistical analysis of the levels of NfL and NPTX2 in Dp16 mice. Unpaired Student t test; n = 4 mice for each category for 9- to 10-month-old mice, n = 3–4 mice for each category for 20-month-old mice; *P < 0.05. For all the proteins, the bands marked with (*) and ([) at the right molecular weight were quantitated as stated in the Methods section with the same policy applied to the bands for the same proteins from other samples. C/, control for; AD-DS, Alzheimer’s disease with Down syndrome; DS, Down syndrome without Alzheimer’s disease; AD, Alzheimer’s disease; NfL, neurofilament light; NPTX2, neuronal pentraxin 2

Article Snippet: 3.1 Methods 3.1.1 Antibodies Mouse antibody anti-syntaxin 1A (1:5000; S0664; HPC-1) and rabbit antibody anti-APP (1:3000; A8717) were obtained from MilliporeSigma; rabbit antibody anti-SNAP25 (1:2000; 14903-1-AP), antiNPTX2 (10889-1-AP), mouse antibodies anti-synaptophysin (1:5000; 67864-1-Ig), anti-β-actin (1:10,000; 60008-1-Ig) were from Proteintech.

Techniques: Western Blot, Control, Quantitation Assay, MANN-WHITNEY, Molecular Weight

Journal:

Article Title: The Platelet Actin Cytoskeleton Associates with SNAREs and Participates in ?-Granule Secretion †

doi: 10.1021/bi100541t

Figure Lengend Snippet: A, increased expression of P-selectin is observed following exposure of the α–granule enriched membrane fraction to 5 mM ATP plus 4 mg/ml platelet cytosol. B, P-selectin expression was monitored following incubation of the α–granule enriched membrane fraction with buffer alone (NA), with 5 mM ATP (ATP), with 4 mg/ml platelet cytosol (cytosol), or with ATP plus cytosol (ATP+cytosol). Data represent the mean ± S.D. of 3-4 experiments. Insert demonstrates release of β-thromboglobulin from α–granule-enriched fraction following exposure to 4 mg/ml platelet cytosol and 5 mM ATP. C, P-selectin expression was monitored following incubation of α–granule-enriched membrane fraction with buffer alone (NA), or in the presence of 5 mM ATP plus 4 mg/ml cytosol following addition of 25 μg/ml of either non-immune IgG (NI), anti-syntaxin 4 antibody (anti-synt-4) or anti-P-selectin cytoplasmic tail antibody (anti-PSCT). Following a 20 minute incubation, the sample was exposed to 5 mM ATP and 4 mg/ml platelet cytosol and analyzed for P-selectin expression by flow cytometry. Data represent the mean ± S.D. of 3 experiments.

Article Snippet: Goat anti–syntaxin-2 antibody directed against a 19 amino acid portion toward the C-terminus of the cytoplasmic domain of syntaxin-1A was obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, Incubation, Flow Cytometry

Journal:

Article Title: The Platelet Actin Cytoskeleton Associates with SNAREs and Participates in ?-Granule Secretion †

doi: 10.1021/bi100541t

Figure Lengend Snippet: A, platelets were lysed in 1.5 ml of Triton X-100 lysis buffer at 4°C. Samples were subject to centrifugation at 15,000g to isolate the platelet cytoskeletal actin network. The supernatant was removed and the pellet washed. Samples of the pellet (Pellet, 30% of total) and the supernatant (Sup, 1% of total) were solubilized in sample buffer and proteins within the samples separated by SDS-PAGE. SNAP-23, syntaxin-2, syntaxin-4, and VAMP-8 within the samples were detected by immunoblot analysis. B, platelets were incubated in the presence (+) or absence (−) of SFLLRN and lysed in Triton X-100 lysis buffer containing either EGTA. Lysates were then spun at 15,000g. Proteins in the pellet were separated by SDS-PAGE and analyzed for SNAP-23, syntaxin-2, syntaxin-4, and VAMP-8 by immunoblot analysis.

Article Snippet: Goat anti–syntaxin-2 antibody directed against a 19 amino acid portion toward the C-terminus of the cytoplasmic domain of syntaxin-1A was obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Lysis, Centrifugation, SDS Page, Western Blot, Incubation

Journal:

Article Title: The Platelet Actin Cytoskeleton Associates with SNAREs and Participates in ?-Granule Secretion †

doi: 10.1021/bi100541t

Figure Lengend Snippet: A, proteins were incubated alone (GST alone; synt-4-GST alone) or in the presence of polymerized platelet actin (GST, actin; synt-4-GST, actin). Samples were pelleted by centrifugation. Proteins within supernatants and pellets were separated by SDS-PAGE and visualized by Coomassie blue staining. B, recombinant GST alone, syntaxin-4-GST, α-actinin, or SNAP-23-GST were incubated in the presence (black bars) or absence (white bars) of polymerized platelet actin. Samples were pelleted and proteins within the supernatants and pellets separated by SDS-PAGE. Bands corresponding to each of the proteins were quantified by densitometry. Data represent the ratio of band density in the pellet compared to that in the supernatant and are representative of 3-5 experiments.

Article Snippet: Goat anti–syntaxin-2 antibody directed against a 19 amino acid portion toward the C-terminus of the cytoplasmic domain of syntaxin-1A was obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Incubation, Centrifugation, SDS Page, Staining, Recombinant