polyclonal rabbit anti-alpha Search Results


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  • 86
    Thermo Fisher polyclonal rabbit anti α adaptin
    siRNA silencing of <t>α-adaptin</t> inhibits MDA-MB-231 migration into EGF spots. A) Representative images treated with control siRNA, α-adaptin siRNA and caveolin1 siRNA after 14 h migration into an agarose spot containing EGF. Scale bars are 100 µm. B) Quantification of total number of cells migrated under siRNA conditions. Line denotes edge of the agarose spot. n = 22 fields of view per treatment. NSC = non-silencing control siRNA.
    Polyclonal Rabbit Anti α Adaptin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore rabbit polyclonal anti α catenin
    Immunofluorescence of sections of control ( a , c , e , g ) and MLP-null ( b , d , f , h ) papillary muscle labeled with various antibodies. a , b <t>α-catenin</t> ( green ) and α-actinin ( red ). c , d β-catenin ( green ) and connexin 43 ( red ). e , f αII spectrin ( green ) and β-catenin ( red ). g , h αII spectrin ( green ) and connexin 43 ( red ). White arrows in f and h indicate loops of spectrin label
    Rabbit Polyclonal Anti α Catenin, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology rabbit polyclonal anti re alpha
    Immunofluorescence tests for contaminating cells (100×). The results indicated that contaminating cells detected both in P0 and P3 cell cultures were few and included SECs (positive for CD31) and HSC (positive for <t>alpha-SMA),</t> but not hepatocytes (positive for CK18). (Blue: DAPI, Green: FITC beads).
    Rabbit Polyclonal Anti Re Alpha, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Cell Signaling Technology Inc anti pkc alpha rabbit polyclonal
    Immunofluorescence tests for contaminating cells (100×). The results indicated that contaminating cells detected both in P0 and P3 cell cultures were few and included SECs (positive for CD31) and HSC (positive for <t>alpha-SMA),</t> but not hepatocytes (positive for CK18). (Blue: DAPI, Green: FITC beads).
    Anti Pkc Alpha Rabbit Polyclonal, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology rabbit polyclonal anti hif3 alpha
    Immunofluorescence tests for contaminating cells (100×). The results indicated that contaminating cells detected both in P0 and P3 cell cultures were few and included SECs (positive for CD31) and HSC (positive for <t>alpha-SMA),</t> but not hepatocytes (positive for CK18). (Blue: DAPI, Green: FITC beads).
    Rabbit Polyclonal Anti Hif3 Alpha, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher rabbit polyclonal anti alpha 6
    Immunofluorescence tests for contaminating cells (100×). The results indicated that contaminating cells detected both in P0 and P3 cell cultures were few and included SECs (positive for CD31) and HSC (positive for <t>alpha-SMA),</t> but not hepatocytes (positive for CK18). (Blue: DAPI, Green: FITC beads).
    Rabbit Polyclonal Anti Alpha 6, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rabbit polyclonal anti tnf α antibodies
    Immunohistochemical staining of <t>TNF-α-positive</t> cells in the liver. Livers were removed 6 hours after alcohol administration (6 g/kg) and 7-μm cryostat section were made. Sections were incubated with a rabbit <t>polyclonal</t> anti-mouse TNF-α antibody, followed by incubation with HRP-conjugated goat anti-rabbit IgG antibody. A: Control liver. B: Alcohol-treated liver. Arrowheads : TNF-α-positive cells. Arrows : Liver sinusoid. Magnification, ×260.
    Rabbit Polyclonal Anti Tnf α Antibodies, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Santa Cruz Biotechnology rabbit polyclonal anti tubulin
    Cellular distribution of T1L μ2 full-length, aa regions (1–282) and (283–736) fused to EGFP. (A) Confocal immunofluorescence of CV-1 cells expressing T1L μ2 full-length or both aa regions (1–282) or (283–736) fused to EGFP. At 20 hpt, the cells were methanol fixed and immunostained for the detection of μ2 (specific <t>polyclonal</t> anti-μ2 serum, green) (left column) and MTs (mouse mAb anti-alpha <t>tubulin,</t> red) (middle column). The merged images are shown in the right column. Nuclei are stained with DAPI (blue). Scale bar is 20 μm. (B) Confocal immunofluorescence of CV-1 cells co-expressing T1L μNS with T1L μ2 full-length or both aa region (1–282) or (283–736) fused to EGFP. At 20 hpt, the cells were methanol fixed and immunostained for the detection of μ2 (mouse mAb anti-EGFP, green) (left column) and μNS (specific polyclonal anti-μNS serum, red) (middle column). The merged images are shown in the right column. Nuclei are stained with DAPI (blue). Scale bar is 20μm.
    Rabbit Polyclonal Anti Tubulin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 89/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology rabbit polyclonal anti alpha dystroglycan
    Cellular distribution of T1L μ2 full-length, aa regions (1–282) and (283–736) fused to EGFP. (A) Confocal immunofluorescence of CV-1 cells expressing T1L μ2 full-length or both aa regions (1–282) or (283–736) fused to EGFP. At 20 hpt, the cells were methanol fixed and immunostained for the detection of μ2 (specific <t>polyclonal</t> anti-μ2 serum, green) (left column) and MTs (mouse mAb anti-alpha <t>tubulin,</t> red) (middle column). The merged images are shown in the right column. Nuclei are stained with DAPI (blue). Scale bar is 20 μm. (B) Confocal immunofluorescence of CV-1 cells co-expressing T1L μNS with T1L μ2 full-length or both aa region (1–282) or (283–736) fused to EGFP. At 20 hpt, the cells were methanol fixed and immunostained for the detection of μ2 (mouse mAb anti-EGFP, green) (left column) and μNS (specific polyclonal anti-μNS serum, red) (middle column). The merged images are shown in the right column. Nuclei are stained with DAPI (blue). Scale bar is 20μm.
    Rabbit Polyclonal Anti Alpha Dystroglycan, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc rabbit polyclonal anti pka c alpha
    Cellular distribution of T1L μ2 full-length, aa regions (1–282) and (283–736) fused to EGFP. (A) Confocal immunofluorescence of CV-1 cells expressing T1L μ2 full-length or both aa regions (1–282) or (283–736) fused to EGFP. At 20 hpt, the cells were methanol fixed and immunostained for the detection of μ2 (specific <t>polyclonal</t> anti-μ2 serum, green) (left column) and MTs (mouse mAb anti-alpha <t>tubulin,</t> red) (middle column). The merged images are shown in the right column. Nuclei are stained with DAPI (blue). Scale bar is 20 μm. (B) Confocal immunofluorescence of CV-1 cells co-expressing T1L μNS with T1L μ2 full-length or both aa region (1–282) or (283–736) fused to EGFP. At 20 hpt, the cells were methanol fixed and immunostained for the detection of μ2 (mouse mAb anti-EGFP, green) (left column) and μNS (specific polyclonal anti-μNS serum, red) (middle column). The merged images are shown in the right column. Nuclei are stained with DAPI (blue). Scale bar is 20μm.
    Rabbit Polyclonal Anti Pka C Alpha, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore polyclonal rabbit anti α syn
    Induction of <t>α-SYN</t> aggregation and apoptosis in SHSY5Y neuroblastoma cells by oxidative stress. A , B , ThioS staining ( A ) and immunocytochemical staining ( B ) of intracytoplasmic α-SYN aggregates in SHSY5Y cells after induction of oxidative stress. Scale bars, 10 μm. These images are used for manual quantification of aggregation. C , D , Pictures taken with the IN cell analyzer for high-content analysis. C , Example of nucleus detection via DAPI staining (left) and α-SYN detection via immunocytochemical staining (middle). D , Example of nucleus detection via DAPI staining (left) and α-SYN fibril detection via ThioS staining (middle). The cell nucleus is identified based on the DAPI fluorescence (blue) and the cytoplasm via the ThioS background fluorescence (green). Intracytoplasmic α-SYN inclusions (yellow) and nuclear apoptotic fragments (pink) are detected based on a high-intensity compared with the background staining in the cytoplasm and the nucleus, respectively. E , Percentage of cells containing α-SYN aggregates determined manually after induction of oxidative stress. Both wild-type SHSY5Y cells and α-SYN-overexpressing cells were analyzed. A luciferase-overexpressing cell line was used as a negative control. SEM is shown for each condition. F , High-content analysis determining percentage of cells with (1) α-SYN aggregates and (2) nuclear condensation in wild-type SHSY5Y cells with and without stress and in α-SYN-overexpressing SHSY5Y cells with stress. SEM is shown for each condition. G , Histogram of the area distribution of α-SYN aggregates per cell in the cells containing aggregates (high-content analysis). The percentage of cells with α-SYN aggregates for these cell lines is shown in F .
    Polyclonal Rabbit Anti α Syn, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Bioss rabbit polyclonal anti α
    Induction of <t>α-SYN</t> aggregation and apoptosis in SHSY5Y neuroblastoma cells by oxidative stress. A , B , ThioS staining ( A ) and immunocytochemical staining ( B ) of intracytoplasmic α-SYN aggregates in SHSY5Y cells after induction of oxidative stress. Scale bars, 10 μm. These images are used for manual quantification of aggregation. C , D , Pictures taken with the IN cell analyzer for high-content analysis. C , Example of nucleus detection via DAPI staining (left) and α-SYN detection via immunocytochemical staining (middle). D , Example of nucleus detection via DAPI staining (left) and α-SYN fibril detection via ThioS staining (middle). The cell nucleus is identified based on the DAPI fluorescence (blue) and the cytoplasm via the ThioS background fluorescence (green). Intracytoplasmic α-SYN inclusions (yellow) and nuclear apoptotic fragments (pink) are detected based on a high-intensity compared with the background staining in the cytoplasm and the nucleus, respectively. E , Percentage of cells containing α-SYN aggregates determined manually after induction of oxidative stress. Both wild-type SHSY5Y cells and α-SYN-overexpressing cells were analyzed. A luciferase-overexpressing cell line was used as a negative control. SEM is shown for each condition. F , High-content analysis determining percentage of cells with (1) α-SYN aggregates and (2) nuclear condensation in wild-type SHSY5Y cells with and without stress and in α-SYN-overexpressing SHSY5Y cells with stress. SEM is shown for each condition. G , Histogram of the area distribution of α-SYN aggregates per cell in the cells containing aggregates (high-content analysis). The percentage of cells with α-SYN aggregates for these cell lines is shown in F .
    Rabbit Polyclonal Anti α, supplied by Bioss, used in various techniques. Bioz Stars score: 88/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore polyclonal rabbit anti α synuclein antibody
    Annexin A5 expression decreases the number of <t>α-synuclein</t> inclusions in vivo. (A) Representative fluorescent images of head muscles of transgenic young adult animals expressing either α-synuclein::YFP alone (the pkIs2386 transgene) or
    Polyclonal Rabbit Anti α Synuclein Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Proteintech rabbit alpha synuclein polyclonal
    The MetaBlue blue autofluorescence signals colocalized with Aβ and <t>alpha</t> hemoglobin HBA staining in the red blood cells, CAA, dense-core plaques and diffusive plaques. A : MetaBlue signals colocalized with Aβ in the red blood cells ( i ), CAA ( ii ), dense-core plaques ( iii ) and diffusive plaques ( iv ), indicated by arrows. B : MetaBlue signals colocalized with alpha hemoglobin HBA staining in the red blood cells ( i ), CAA ( ii ), dense-core plaques ( iii ) and diffusive plaques ( iv ), indicated by arrows. Scale bars, 50 μm.
    Rabbit Alpha Synuclein Polyclonal, supplied by Proteintech, used in various techniques. Bioz Stars score: 91/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore rabbit polyclonal α tubulin
    Dystonin-a2–MAP1B interaction maintains <t>Ac–α-tubulin</t> and promotes flux through the secretory pathway. (A) Western blot analysis of 293T cell lysate shows that MAP1B overexpression maintains Ac–α-tubulin after dystonin-a2 depletion relative to empty vector (EV) control. (B) MAP1B overexpression prevents Golgi fragmentation after dystonin-a2 depletion in 293Ts. Arrows point to intact Golgi in an siA2-treated cell that is positive for MAP1B-GFP expression. Arrowheads point to fragmented Golgi in a neighboring cell that was not transfected with the MAP1B-GFP construct. (C) MAP1B overexpression promotes VSVG trafficking to the Golgi and PM after dystonin-a2 depletion relative to empty vector control. Scramble siRNA-treated cells (top and A) are labeled C. The arrowhead identifies aberrantly localized VSVG molecules, whereas arrows identify normal VSVG localization. (D and E) Quantification of MAP1B impact on VSVG accumulation in the Golgi (D) and at the PM (E; ANOVA posthoc Tukey; n = 3). (F) MAP1B antigenic labeling of WT primary sensory neurons shows a perinuclear accumulation that is lost in dt 27J neurons (arrows). (G) MAP1B expression restores GLuc flux through the secretory pathway (time = 1 h) in P15 dt 27J sensory neurons (ANOVA posthoc Tukey; n = 6). Error bars show means ± SEM. Cont, control; Tub, tubulin. **, P
    Rabbit Polyclonal α Tubulin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies rabbit polyclonal anti alpha fetoprotein
    Spontaneous differentiation of mESCs. EB s from all cell lines (A) and inmunofluorescence assays of differentiation markers in J1 and CMT cell lines at early and late passages (B). (a), (b) EBs in bacteriological dishes from J1 and E14 cell lines, respectively. (c) Cell migration and differentiation from attached EBs obtained from the CMT cell line (two images overlapped at 4× magnification). (B) CMT and J1 cell lines (chromosomally stable and unstable, respectively) maintained a functional pluripotency throughout the study. Both mESCs expressed markers of the three germ layers after at least 10 days of spontaneous differentiation. AFP : <t>Alpha</t> <t>Fetoprotein,</t> endodermal marker. SMA : alpha Smooth Muscle Actin, mesodermal marker. Nest : Nestin, ectodermal marker. A and B magnification:10×. Other magnifications are shown on the pictures. Images obtained from independent experiments.
    Rabbit Polyclonal Anti Alpha Fetoprotein, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 86/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam rabbit polyclonal anti alpha tubulin
    Detection of Golgi-derived MTs using the ice recovery assay. (A–F) Immunofluorescence images of RPE cells expressing Centrin-GFP (green) stained for <t>alpha-tubulin</t> (red) and the Golgi marker GM130 (blue). Maximum intensity projection of a confocal
    Rabbit Polyclonal Anti Alpha Tubulin, supplied by Abcam, used in various techniques. Bioz Stars score: 91/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam rabbit polyclonal anti insulin receptor alpha
    Detection of Golgi-derived MTs using the ice recovery assay. (A–F) Immunofluorescence images of RPE cells expressing Centrin-GFP (green) stained for <t>alpha-tubulin</t> (red) and the Golgi marker GM130 (blue). Maximum intensity projection of a confocal
    Rabbit Polyclonal Anti Insulin Receptor Alpha, supplied by Abcam, used in various techniques. Bioz Stars score: 88/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc rabbit polyclonal anti phospho cpla2 alpha
    Detection of Golgi-derived MTs using the ice recovery assay. (A–F) Immunofluorescence images of RPE cells expressing Centrin-GFP (green) stained for <t>alpha-tubulin</t> (red) and the Golgi marker GM130 (blue). Maximum intensity projection of a confocal
    Rabbit Polyclonal Anti Phospho Cpla2 Alpha, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    siRNA silencing of α-adaptin inhibits MDA-MB-231 migration into EGF spots. A) Representative images treated with control siRNA, α-adaptin siRNA and caveolin1 siRNA after 14 h migration into an agarose spot containing EGF. Scale bars are 100 µm. B) Quantification of total number of cells migrated under siRNA conditions. Line denotes edge of the agarose spot. n = 22 fields of view per treatment. NSC = non-silencing control siRNA.

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: Polarised Clathrin-Mediated Endocytosis of EGFR During Chemotactic Invasion

    doi: 10.1111/tra.12165

    Figure Lengend Snippet: siRNA silencing of α-adaptin inhibits MDA-MB-231 migration into EGF spots. A) Representative images treated with control siRNA, α-adaptin siRNA and caveolin1 siRNA after 14 h migration into an agarose spot containing EGF. Scale bars are 100 µm. B) Quantification of total number of cells migrated under siRNA conditions. Line denotes edge of the agarose spot. n = 22 fields of view per treatment. NSC = non-silencing control siRNA.

    Article Snippet: Primary antibodies used were polyclonal rabbit anti-α-adaptin (Life Technologies), polyclonal rabbit anti-caveolin1 (BD Biosciences) and monoclonal mouse anti-tubulin (Sigma).

    Techniques: Multiple Displacement Amplification, Migration

    siRNA silencing of α-adaptin does not inhibit focal adhesion disassembly during wound healing. A) Disappearance of paxillin-mRFP over time using NSC and α-adaptin siRNA. B) Graph showing mean focal adhesion disassembly time for cells treated with NSC and α-adaptin siRNA. n ≥ 100 focal adhesions from 20 cells per treatment. C) Wound healing assay with α-adaptin knock-down. Representative images of wounds at time 0 and after 14 h. Lines denote edge of wound at both time points. Distances between cells at time 0 and 14 h were measured at various points in each image to give an average distance migrated in NSC and α-adaptin siRNA treated cells. D) Quantification of distance moved by siRNA treated cells. n = 22 fields of view per treatment.

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: Polarised Clathrin-Mediated Endocytosis of EGFR During Chemotactic Invasion

    doi: 10.1111/tra.12165

    Figure Lengend Snippet: siRNA silencing of α-adaptin does not inhibit focal adhesion disassembly during wound healing. A) Disappearance of paxillin-mRFP over time using NSC and α-adaptin siRNA. B) Graph showing mean focal adhesion disassembly time for cells treated with NSC and α-adaptin siRNA. n ≥ 100 focal adhesions from 20 cells per treatment. C) Wound healing assay with α-adaptin knock-down. Representative images of wounds at time 0 and after 14 h. Lines denote edge of wound at both time points. Distances between cells at time 0 and 14 h were measured at various points in each image to give an average distance migrated in NSC and α-adaptin siRNA treated cells. D) Quantification of distance moved by siRNA treated cells. n = 22 fields of view per treatment.

    Article Snippet: Primary antibodies used were polyclonal rabbit anti-α-adaptin (Life Technologies), polyclonal rabbit anti-caveolin1 (BD Biosciences) and monoclonal mouse anti-tubulin (Sigma).

    Techniques: Wound Healing Assay

    Immunofluorescence of sections of control ( a , c , e , g ) and MLP-null ( b , d , f , h ) papillary muscle labeled with various antibodies. a , b α-catenin ( green ) and α-actinin ( red ). c , d β-catenin ( green ) and connexin 43 ( red ). e , f αII spectrin ( green ) and β-catenin ( red ). g , h αII spectrin ( green ) and connexin 43 ( red ). White arrows in f and h indicate loops of spectrin label

    Journal: Cellular and Molecular Life Sciences

    Article Title: Cardiomyocyte growth and sarcomerogenesis at the intercalated disc

    doi: 10.1007/s00018-013-1374-5

    Figure Lengend Snippet: Immunofluorescence of sections of control ( a , c , e , g ) and MLP-null ( b , d , f , h ) papillary muscle labeled with various antibodies. a , b α-catenin ( green ) and α-actinin ( red ). c , d β-catenin ( green ) and connexin 43 ( red ). e , f αII spectrin ( green ) and β-catenin ( red ). g , h αII spectrin ( green ) and connexin 43 ( red ). White arrows in f and h indicate loops of spectrin label

    Article Snippet: Rabbit polyclonal anti-α-catenin (C-2081) was from Sigma-Aldrich, UK.

    Techniques: Immunofluorescence, Labeling

    Immunofluorescence tests for contaminating cells (100×). The results indicated that contaminating cells detected both in P0 and P3 cell cultures were few and included SECs (positive for CD31) and HSC (positive for alpha-SMA), but not hepatocytes (positive for CK18). (Blue: DAPI, Green: FITC beads).

    Journal: PLoS ONE

    Article Title: A New Method to Isolate and Culture Rat Kupffer Cells

    doi: 10.1371/journal.pone.0070832

    Figure Lengend Snippet: Immunofluorescence tests for contaminating cells (100×). The results indicated that contaminating cells detected both in P0 and P3 cell cultures were few and included SECs (positive for CD31) and HSC (positive for alpha-SMA), but not hepatocytes (positive for CK18). (Blue: DAPI, Green: FITC beads).

    Article Snippet: After 1 day of culture, the slides were rinsedwith PBS and fixed with 4% paraformaldehyde at room temperature for 15 min. After permeabilization with 0.1% Triton X-100 for 10 min followed by blocking with 10% normal goat serum for 30 min, the cells were incubated with the following antibodies overnight at 4°C: rabbit polyclonal anti-CD68 (ED-1, Bioss, China), rabbit polyclonal anti-CD163 (ED-2, Bioss, China), rabbit polyclonal anti-CK18 (Bioss, China), rabbit polyclonal anti-CD31 (Bioss, China) and rabbit polyclonal anti- alpha-SMA (Santa Cruz, USA).

    Techniques: Immunofluorescence

    Immunohistochemical staining of TNF-α-positive cells in the liver. Livers were removed 6 hours after alcohol administration (6 g/kg) and 7-μm cryostat section were made. Sections were incubated with a rabbit polyclonal anti-mouse TNF-α antibody, followed by incubation with HRP-conjugated goat anti-rabbit IgG antibody. A: Control liver. B: Alcohol-treated liver. Arrowheads : TNF-α-positive cells. Arrows : Liver sinusoid. Magnification, ×260.

    Journal: The American Journal of Pathology

    Article Title: A Critical Involvement of Oxidative Stress in Acute Alcohol-Induced Hepatic TNF-? Production

    doi:

    Figure Lengend Snippet: Immunohistochemical staining of TNF-α-positive cells in the liver. Livers were removed 6 hours after alcohol administration (6 g/kg) and 7-μm cryostat section were made. Sections were incubated with a rabbit polyclonal anti-mouse TNF-α antibody, followed by incubation with HRP-conjugated goat anti-rabbit IgG antibody. A: Control liver. B: Alcohol-treated liver. Arrowheads : TNF-α-positive cells. Arrows : Liver sinusoid. Magnification, ×260.

    Article Snippet: Sections were incubated with polyclonal rabbit anti-TNF-α antibody (BioSource International, Inc.) overnight at 4°C, followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (Zymed Laboratories, Inc., San Francisco, CA) for 1 hour.

    Techniques: Immunohistochemistry, Staining, Incubation

    Cellular distribution of T1L μ2 full-length, aa regions (1–282) and (283–736) fused to EGFP. (A) Confocal immunofluorescence of CV-1 cells expressing T1L μ2 full-length or both aa regions (1–282) or (283–736) fused to EGFP. At 20 hpt, the cells were methanol fixed and immunostained for the detection of μ2 (specific polyclonal anti-μ2 serum, green) (left column) and MTs (mouse mAb anti-alpha tubulin, red) (middle column). The merged images are shown in the right column. Nuclei are stained with DAPI (blue). Scale bar is 20 μm. (B) Confocal immunofluorescence of CV-1 cells co-expressing T1L μNS with T1L μ2 full-length or both aa region (1–282) or (283–736) fused to EGFP. At 20 hpt, the cells were methanol fixed and immunostained for the detection of μ2 (mouse mAb anti-EGFP, green) (left column) and μNS (specific polyclonal anti-μNS serum, red) (middle column). The merged images are shown in the right column. Nuclei are stained with DAPI (blue). Scale bar is 20μm.

    Journal: PLoS ONE

    Article Title: Dissection of mammalian orthoreovirus µ2 reveals a self-associative domain required for binding to microtubules but not to factory matrix protein µNS

    doi: 10.1371/journal.pone.0184356

    Figure Lengend Snippet: Cellular distribution of T1L μ2 full-length, aa regions (1–282) and (283–736) fused to EGFP. (A) Confocal immunofluorescence of CV-1 cells expressing T1L μ2 full-length or both aa regions (1–282) or (283–736) fused to EGFP. At 20 hpt, the cells were methanol fixed and immunostained for the detection of μ2 (specific polyclonal anti-μ2 serum, green) (left column) and MTs (mouse mAb anti-alpha tubulin, red) (middle column). The merged images are shown in the right column. Nuclei are stained with DAPI (blue). Scale bar is 20 μm. (B) Confocal immunofluorescence of CV-1 cells co-expressing T1L μNS with T1L μ2 full-length or both aa region (1–282) or (283–736) fused to EGFP. At 20 hpt, the cells were methanol fixed and immunostained for the detection of μ2 (mouse mAb anti-EGFP, green) (left column) and μNS (specific polyclonal anti-μNS serum, red) (middle column). The merged images are shown in the right column. Nuclei are stained with DAPI (blue). Scale bar is 20μm.

    Article Snippet: Rabbit polyclonal anti-tubulin (H-300) and mouse MAb anti-GFP (B-2) were obtained from Santa Cruz Biotechnology, Inc. Rabbit polyclonal anti-GFP (ab290) was obtained from Abcam.

    Techniques: Immunofluorescence, Expressing, Staining

    μ2 region 1 to 373 fused to HA tag is necessary for MT-association. (A) Schematic representation of T1L μ2 N-terminal deletion mutants fused to an HA tag (not to scale). Positive (+) or negative (-) MT-association phenotype is indicated at right. (B) Confocal immunofluorescence of CV-1 cells expressing μ2 or its deletion mutants with HA tag. At 20 hpt, cells were methanol fixed and immunostained for the detection of μ2 or its deletion mutants (mouse mAb anti-HA, green) (right upper panel) and MTs (specific polyclonal anti-alpha tubulin serum, red) (right lower panel). A Merged image is shown at the left of each panel. Nuclei are stained with DAPI (blue). Scale bar is 20 μm. The percentage of positive cells HA-tagged proteins associating to MTs is indicated at the left bottom of each merged image.

    Journal: PLoS ONE

    Article Title: Dissection of mammalian orthoreovirus µ2 reveals a self-associative domain required for binding to microtubules but not to factory matrix protein µNS

    doi: 10.1371/journal.pone.0184356

    Figure Lengend Snippet: μ2 region 1 to 373 fused to HA tag is necessary for MT-association. (A) Schematic representation of T1L μ2 N-terminal deletion mutants fused to an HA tag (not to scale). Positive (+) or negative (-) MT-association phenotype is indicated at right. (B) Confocal immunofluorescence of CV-1 cells expressing μ2 or its deletion mutants with HA tag. At 20 hpt, cells were methanol fixed and immunostained for the detection of μ2 or its deletion mutants (mouse mAb anti-HA, green) (right upper panel) and MTs (specific polyclonal anti-alpha tubulin serum, red) (right lower panel). A Merged image is shown at the left of each panel. Nuclei are stained with DAPI (blue). Scale bar is 20 μm. The percentage of positive cells HA-tagged proteins associating to MTs is indicated at the left bottom of each merged image.

    Article Snippet: Rabbit polyclonal anti-tubulin (H-300) and mouse MAb anti-GFP (B-2) were obtained from Santa Cruz Biotechnology, Inc. Rabbit polyclonal anti-GFP (ab290) was obtained from Abcam.

    Techniques: Immunofluorescence, Expressing, Staining

    Point mutations in a repetitive motif in μ2 region, 283 to 325, abrogate MT-association. (A) Schematic representation of the amino acidic sequence in T1L μ2 region 283 to 325. The Asp in each Val-Asp-Val motif is underlined. In the scheme, the μ2 is fused at the C-terminus to an HA tag. (B) Confocal immunofluorescence of CV-1 cells expressing μ2-HA wild type or containing the specified point mutation. At 20 hpt, cells were methanol fixed and immunostained for detection of μ2 (mouse mAb anti-HA, green) (right upper panel) and MTs (specific polyclonal anti-alpha tubulin serum, red)(right lower panel). A Merged image is shown at the left of each panel. Nuclei are stained with DAPI (blue). Scale bar is 20 μm.

    Journal: PLoS ONE

    Article Title: Dissection of mammalian orthoreovirus µ2 reveals a self-associative domain required for binding to microtubules but not to factory matrix protein µNS

    doi: 10.1371/journal.pone.0184356

    Figure Lengend Snippet: Point mutations in a repetitive motif in μ2 region, 283 to 325, abrogate MT-association. (A) Schematic representation of the amino acidic sequence in T1L μ2 region 283 to 325. The Asp in each Val-Asp-Val motif is underlined. In the scheme, the μ2 is fused at the C-terminus to an HA tag. (B) Confocal immunofluorescence of CV-1 cells expressing μ2-HA wild type or containing the specified point mutation. At 20 hpt, cells were methanol fixed and immunostained for detection of μ2 (mouse mAb anti-HA, green) (right upper panel) and MTs (specific polyclonal anti-alpha tubulin serum, red)(right lower panel). A Merged image is shown at the left of each panel. Nuclei are stained with DAPI (blue). Scale bar is 20 μm.

    Article Snippet: Rabbit polyclonal anti-tubulin (H-300) and mouse MAb anti-GFP (B-2) were obtained from Santa Cruz Biotechnology, Inc. Rabbit polyclonal anti-GFP (ab290) was obtained from Abcam.

    Techniques: Sequencing, Immunofluorescence, Expressing, Mutagenesis, Staining

    Induction of α-SYN aggregation and apoptosis in SHSY5Y neuroblastoma cells by oxidative stress. A , B , ThioS staining ( A ) and immunocytochemical staining ( B ) of intracytoplasmic α-SYN aggregates in SHSY5Y cells after induction of oxidative stress. Scale bars, 10 μm. These images are used for manual quantification of aggregation. C , D , Pictures taken with the IN cell analyzer for high-content analysis. C , Example of nucleus detection via DAPI staining (left) and α-SYN detection via immunocytochemical staining (middle). D , Example of nucleus detection via DAPI staining (left) and α-SYN fibril detection via ThioS staining (middle). The cell nucleus is identified based on the DAPI fluorescence (blue) and the cytoplasm via the ThioS background fluorescence (green). Intracytoplasmic α-SYN inclusions (yellow) and nuclear apoptotic fragments (pink) are detected based on a high-intensity compared with the background staining in the cytoplasm and the nucleus, respectively. E , Percentage of cells containing α-SYN aggregates determined manually after induction of oxidative stress. Both wild-type SHSY5Y cells and α-SYN-overexpressing cells were analyzed. A luciferase-overexpressing cell line was used as a negative control. SEM is shown for each condition. F , High-content analysis determining percentage of cells with (1) α-SYN aggregates and (2) nuclear condensation in wild-type SHSY5Y cells with and without stress and in α-SYN-overexpressing SHSY5Y cells with stress. SEM is shown for each condition. G , Histogram of the area distribution of α-SYN aggregates per cell in the cells containing aggregates (high-content analysis). The percentage of cells with α-SYN aggregates for these cell lines is shown in F .

    Journal: The Journal of Neuroscience

    Article Title: Inhibition of FK506 Binding Proteins Reduces α-Synuclein Aggregation and Parkinson's Disease-Like Pathology

    doi: 10.1523/JNEUROSCI.5983-09.2010

    Figure Lengend Snippet: Induction of α-SYN aggregation and apoptosis in SHSY5Y neuroblastoma cells by oxidative stress. A , B , ThioS staining ( A ) and immunocytochemical staining ( B ) of intracytoplasmic α-SYN aggregates in SHSY5Y cells after induction of oxidative stress. Scale bars, 10 μm. These images are used for manual quantification of aggregation. C , D , Pictures taken with the IN cell analyzer for high-content analysis. C , Example of nucleus detection via DAPI staining (left) and α-SYN detection via immunocytochemical staining (middle). D , Example of nucleus detection via DAPI staining (left) and α-SYN fibril detection via ThioS staining (middle). The cell nucleus is identified based on the DAPI fluorescence (blue) and the cytoplasm via the ThioS background fluorescence (green). Intracytoplasmic α-SYN inclusions (yellow) and nuclear apoptotic fragments (pink) are detected based on a high-intensity compared with the background staining in the cytoplasm and the nucleus, respectively. E , Percentage of cells containing α-SYN aggregates determined manually after induction of oxidative stress. Both wild-type SHSY5Y cells and α-SYN-overexpressing cells were analyzed. A luciferase-overexpressing cell line was used as a negative control. SEM is shown for each condition. F , High-content analysis determining percentage of cells with (1) α-SYN aggregates and (2) nuclear condensation in wild-type SHSY5Y cells with and without stress and in α-SYN-overexpressing SHSY5Y cells with stress. SEM is shown for each condition. G , Histogram of the area distribution of α-SYN aggregates per cell in the cells containing aggregates (high-content analysis). The percentage of cells with α-SYN aggregates for these cell lines is shown in F .

    Article Snippet: Briefly, sections were exposed to 3% hydrogen peroxide and incubated overnight with a polyclonal rabbit anti-α-SYN (1:5000; Millipore Bioscience Research Reagents) in 10% normal swine serum.

    Techniques: Staining, High Content Screening, Fluorescence, Luciferase, Negative Control

    Stable FKBP12 knockdown inhibits α-SYN aggregation and apoptosis in SHSY5Y cells. A , Left, Western blot showing stable FKBP12 knockdown in wild-type SHSY5Y cells after transduction with LV encoding shFKBP12 or shFKBP12_MM, followed by selection with hygromycin. In each lane, cell extract containing 10 μg of total protein was loaded and FKBP12 expression was visualized. Lane 1, Untreated SHSY5Y cells. Lane 2, FKBP12-overexpressing SHSY5Y. Lane 3, Knockdown of endogenous FKBP12 by shFKBP12. Lane 4, SHSY5Y expressing shFKBP12_MM. GAPDH expression is shown as loading control. FKBP52 expression is unaffected by FKBP12 overexpression or knockdown. Right, Stable FKBP12 knockdown in SHSY5Y cells overexpressing α-SYN. Lane 1, FKBP12-overexpressing α-SYN–SHSY5Y. Lane 2, Knockdown of endogenous FKBP12. Lane 3, α-SYN–SHSY5Y expressing shFKBP12_MM. Lane 4, α-SYN–SHSY5Y. B , High-content analysis of α-SYN aggregation and apoptosis in SHSY5Y cells and α-SYN–SHSY5Y cells after knockdown of FKBP12. Error bars indicate the SEM. p values are indicated compared with control condition (cell lines expressing shFKBP12_MM).

    Journal: The Journal of Neuroscience

    Article Title: Inhibition of FK506 Binding Proteins Reduces α-Synuclein Aggregation and Parkinson's Disease-Like Pathology

    doi: 10.1523/JNEUROSCI.5983-09.2010

    Figure Lengend Snippet: Stable FKBP12 knockdown inhibits α-SYN aggregation and apoptosis in SHSY5Y cells. A , Left, Western blot showing stable FKBP12 knockdown in wild-type SHSY5Y cells after transduction with LV encoding shFKBP12 or shFKBP12_MM, followed by selection with hygromycin. In each lane, cell extract containing 10 μg of total protein was loaded and FKBP12 expression was visualized. Lane 1, Untreated SHSY5Y cells. Lane 2, FKBP12-overexpressing SHSY5Y. Lane 3, Knockdown of endogenous FKBP12 by shFKBP12. Lane 4, SHSY5Y expressing shFKBP12_MM. GAPDH expression is shown as loading control. FKBP52 expression is unaffected by FKBP12 overexpression or knockdown. Right, Stable FKBP12 knockdown in SHSY5Y cells overexpressing α-SYN. Lane 1, FKBP12-overexpressing α-SYN–SHSY5Y. Lane 2, Knockdown of endogenous FKBP12. Lane 3, α-SYN–SHSY5Y expressing shFKBP12_MM. Lane 4, α-SYN–SHSY5Y. B , High-content analysis of α-SYN aggregation and apoptosis in SHSY5Y cells and α-SYN–SHSY5Y cells after knockdown of FKBP12. Error bars indicate the SEM. p values are indicated compared with control condition (cell lines expressing shFKBP12_MM).

    Article Snippet: Briefly, sections were exposed to 3% hydrogen peroxide and incubated overnight with a polyclonal rabbit anti-α-SYN (1:5000; Millipore Bioscience Research Reagents) in 10% normal swine serum.

    Techniques: Western Blot, Transduction, Selection, Expressing, Over Expression, High Content Screening

    FKBP overexpression enhances α-SYN aggregation and apoptosis in SHSY5Y cells. A , Expression of α-SYN, FKBP12, FKBP52, and luciferase in stable cell lines. Western blot of cell extracts of the different stable cell lines generated. Lanes 1, 3, 5, 7, Untransduced SHSY5Y cells. Lane 2, SHSY5Y cells overexpressing α-SYN (14.4 kDa). Lane 4, SHSY5Y cells overexpressing FKBP12 (12 kDa). Lane 6, SHSY5Y cells overexpressing FKBP52 (52 kDa). Lane 8, SHSY5Y cells overexpressing luciferase (61 kDa). α-Tubulin expression is shown below to confirm equal loading. B , Subcellular localization of FKBP12 and α-SYN aggregates in overexpression cell lines. Fixed and stained SHSY5Y cells overexpressing FKBP12 exposed to oxidative stress for 48 h. Left, ThioS (green) stains α-SYN fibrils (arrows). Middle, Immunocytochemical FKBP12 staining is shown in red. Right, Overlay image. C , Subcellular localization of FKBP52 and α-SYN aggregates in overexpression cell lines. Fixed and stained SHSY5Y cells overexpressing FKBP52 exposed to oxidative stress for 48 h. Left, ThioS (green) stains α-SYN fibrils (arrows). Middle, Immunocytochemical FKBP52 staining is shown in red. Right, Overlap image. D , Analysis of the effect of FKBP12 and FKBP52 overexpression on the aggregation of α-synuclein. Cells were subjected to oxidative stress for 48 h in the presence or absence of FK506 before ThioS staining. WT SHSY5Y cells and luciferase-overexpressing cells are shown as negative controls. The percentage of ThioS-positive cells was determined in the different conditions. p values are indicated compared with control condition (cell lines without FK506 treatment) or as indicated in the graph. E , High-content analysis of the effect of FKBP12 overexpression on the aggregation of α-SYN in both WT SHSY5Y and α-SYN–SHSY5Y. The error bars show the SEM for each condition. p values are indicated compared with control condition (cell lines with endogenous FKBP12 expression).

    Journal: The Journal of Neuroscience

    Article Title: Inhibition of FK506 Binding Proteins Reduces α-Synuclein Aggregation and Parkinson's Disease-Like Pathology

    doi: 10.1523/JNEUROSCI.5983-09.2010

    Figure Lengend Snippet: FKBP overexpression enhances α-SYN aggregation and apoptosis in SHSY5Y cells. A , Expression of α-SYN, FKBP12, FKBP52, and luciferase in stable cell lines. Western blot of cell extracts of the different stable cell lines generated. Lanes 1, 3, 5, 7, Untransduced SHSY5Y cells. Lane 2, SHSY5Y cells overexpressing α-SYN (14.4 kDa). Lane 4, SHSY5Y cells overexpressing FKBP12 (12 kDa). Lane 6, SHSY5Y cells overexpressing FKBP52 (52 kDa). Lane 8, SHSY5Y cells overexpressing luciferase (61 kDa). α-Tubulin expression is shown below to confirm equal loading. B , Subcellular localization of FKBP12 and α-SYN aggregates in overexpression cell lines. Fixed and stained SHSY5Y cells overexpressing FKBP12 exposed to oxidative stress for 48 h. Left, ThioS (green) stains α-SYN fibrils (arrows). Middle, Immunocytochemical FKBP12 staining is shown in red. Right, Overlay image. C , Subcellular localization of FKBP52 and α-SYN aggregates in overexpression cell lines. Fixed and stained SHSY5Y cells overexpressing FKBP52 exposed to oxidative stress for 48 h. Left, ThioS (green) stains α-SYN fibrils (arrows). Middle, Immunocytochemical FKBP52 staining is shown in red. Right, Overlap image. D , Analysis of the effect of FKBP12 and FKBP52 overexpression on the aggregation of α-synuclein. Cells were subjected to oxidative stress for 48 h in the presence or absence of FK506 before ThioS staining. WT SHSY5Y cells and luciferase-overexpressing cells are shown as negative controls. The percentage of ThioS-positive cells was determined in the different conditions. p values are indicated compared with control condition (cell lines without FK506 treatment) or as indicated in the graph. E , High-content analysis of the effect of FKBP12 overexpression on the aggregation of α-SYN in both WT SHSY5Y and α-SYN–SHSY5Y. The error bars show the SEM for each condition. p values are indicated compared with control condition (cell lines with endogenous FKBP12 expression).

    Article Snippet: Briefly, sections were exposed to 3% hydrogen peroxide and incubated overnight with a polyclonal rabbit anti-α-SYN (1:5000; Millipore Bioscience Research Reagents) in 10% normal swine serum.

    Techniques: Over Expression, Expressing, Luciferase, Stable Transfection, Western Blot, Generated, Staining, High Content Screening

    Transient FKBP knockdown inhibits α-SYN aggregation and apoptosis in SHSY5Y cells. A , Western blot showing knockdown of FKBP12 in SHSY5Y cells. In each lane, 10 μg of total protein was loaded, and FKBP12 expression was visualized by immunoblot. Lane 1, Extract of untreated SHSY5Y cells. Lanes 2–6, Cell extracts made 1, 2, 3, 5, and 7 d after transfection with siFKBP12_86. Lane 7, Extract of untreated SHSY5Y cells. Lanes 8–12, Cell extracts made 1, 2, 3, 5, and 7 d after transfection with siFKBP12_121. B , Western blot of FKBP52 in the same cell extracts (3 d after transfection) with unaltered levels of FKBP52 after knockdown of FKBP12. GAPDH blotting controls for equal loading. C , Western blot showing FKBP52 knockdown in SHSY5Y cells. In each lane, cell extract containing 10 μg of total protein was loaded and FKBP52 expression was visualized. Lane 1, Extract of untreated SHSY5Y cells. Lanes 2–6, Cell extracts made 1, 2, 3, 5, and 7 d after transfection with siFKBP52_446. Lane 7, Extract of untreated SHSY5Y cells. Lanes 8–12, Cell extracts made 1, 2, 3, 5, and 7 d after transfection with siFKBP52_1023. D , Western blot of FKBP12 in the same cell extracts (3 d after transfection) with unaltered levels of FKBP12 after knockdown of FKBP52. α-Tubulin or GAPDH blotting confirm equal loading. E , After 6 h of incubation with respective siRNA molecules, oxidative stress was induced for 72 h. SHSY5Y cells without siRNA treatment were used as a control. The percentage of ThioS-positive α-SYN aggregates was determined. SEM is shown for each condition. p values are indicated compared with control condition (mock transfection).

    Journal: The Journal of Neuroscience

    Article Title: Inhibition of FK506 Binding Proteins Reduces α-Synuclein Aggregation and Parkinson's Disease-Like Pathology

    doi: 10.1523/JNEUROSCI.5983-09.2010

    Figure Lengend Snippet: Transient FKBP knockdown inhibits α-SYN aggregation and apoptosis in SHSY5Y cells. A , Western blot showing knockdown of FKBP12 in SHSY5Y cells. In each lane, 10 μg of total protein was loaded, and FKBP12 expression was visualized by immunoblot. Lane 1, Extract of untreated SHSY5Y cells. Lanes 2–6, Cell extracts made 1, 2, 3, 5, and 7 d after transfection with siFKBP12_86. Lane 7, Extract of untreated SHSY5Y cells. Lanes 8–12, Cell extracts made 1, 2, 3, 5, and 7 d after transfection with siFKBP12_121. B , Western blot of FKBP52 in the same cell extracts (3 d after transfection) with unaltered levels of FKBP52 after knockdown of FKBP12. GAPDH blotting controls for equal loading. C , Western blot showing FKBP52 knockdown in SHSY5Y cells. In each lane, cell extract containing 10 μg of total protein was loaded and FKBP52 expression was visualized. Lane 1, Extract of untreated SHSY5Y cells. Lanes 2–6, Cell extracts made 1, 2, 3, 5, and 7 d after transfection with siFKBP52_446. Lane 7, Extract of untreated SHSY5Y cells. Lanes 8–12, Cell extracts made 1, 2, 3, 5, and 7 d after transfection with siFKBP52_1023. D , Western blot of FKBP12 in the same cell extracts (3 d after transfection) with unaltered levels of FKBP12 after knockdown of FKBP52. α-Tubulin or GAPDH blotting confirm equal loading. E , After 6 h of incubation with respective siRNA molecules, oxidative stress was induced for 72 h. SHSY5Y cells without siRNA treatment were used as a control. The percentage of ThioS-positive α-SYN aggregates was determined. SEM is shown for each condition. p values are indicated compared with control condition (mock transfection).

    Article Snippet: Briefly, sections were exposed to 3% hydrogen peroxide and incubated overnight with a polyclonal rabbit anti-α-SYN (1:5000; Millipore Bioscience Research Reagents) in 10% normal swine serum.

    Techniques: Western Blot, Expressing, Transfection, Incubation

    FK506 inhibits α-SYN aggregation and apoptosis in SHSY5Y cells. A , Effect of FK506 on α-SYN aggregation in α-SYN-overexpressing SHSY5Y cells. Cells were stained with ThioS after oxidative stress induction and counted for ThioS-positive inclusions. B , Dose-dependent inhibition of α-SYN aggregation and apoptosis by FK506 determined by high-content analysis. Cells were stained with DAPI and ThioS after induction of oxidative stress. SEM is shown for each condition. p values are indicated compared with control condition (DMSO vehicle without FK506).

    Journal: The Journal of Neuroscience

    Article Title: Inhibition of FK506 Binding Proteins Reduces α-Synuclein Aggregation and Parkinson's Disease-Like Pathology

    doi: 10.1523/JNEUROSCI.5983-09.2010

    Figure Lengend Snippet: FK506 inhibits α-SYN aggregation and apoptosis in SHSY5Y cells. A , Effect of FK506 on α-SYN aggregation in α-SYN-overexpressing SHSY5Y cells. Cells were stained with ThioS after oxidative stress induction and counted for ThioS-positive inclusions. B , Dose-dependent inhibition of α-SYN aggregation and apoptosis by FK506 determined by high-content analysis. Cells were stained with DAPI and ThioS after induction of oxidative stress. SEM is shown for each condition. p values are indicated compared with control condition (DMSO vehicle without FK506).

    Article Snippet: Briefly, sections were exposed to 3% hydrogen peroxide and incubated overnight with a polyclonal rabbit anti-α-SYN (1:5000; Millipore Bioscience Research Reagents) in 10% normal swine serum.

    Techniques: Staining, Inhibition, High Content Screening

    FK506 treatment reduces α-SYN aggregation and neurodegeneration in mouse brain. A ). Cytoplasmic α-SYN inclusions are indicated with arrows. B , Immunohistochemical staining of α-SYN in the striatum of mice injected with α-SYN encoding LV. Inlay images show high-magnification views of two α-SYN-positive neurons without (left) or with (right) α-SYN inclusions. C , Immunofluorescent staining for α-SYN and ubiquitin in the striatum 5 months after injection with α-SYN LV. Detailed pictures on the right show colocalization of α-SYN (green) and ubiquitin (red) in the inclusions. D , Number of α-SYN aggregates in α-SYN-overexpressing neurons in the striatum. The fraction of α-SYN-positive neurons that contain inclusions was determined. FK506 treatment decreases the number of α-SYN-positive neurons. Error bars show SEM ( n = 6–7). E , Survival of α-SYN-overexpressing cells in the striatum after FK506 treatment. The α-SYN-positive area was quantified using the Cavalieri method as a measure for the number of α-SYN-overexpressing cells. In the groups treated with FK506, more α-SYN-overexpressing cells survive. Error bars show the SEM ( n = 6–7).

    Journal: The Journal of Neuroscience

    Article Title: Inhibition of FK506 Binding Proteins Reduces α-Synuclein Aggregation and Parkinson's Disease-Like Pathology

    doi: 10.1523/JNEUROSCI.5983-09.2010

    Figure Lengend Snippet: FK506 treatment reduces α-SYN aggregation and neurodegeneration in mouse brain. A ). Cytoplasmic α-SYN inclusions are indicated with arrows. B , Immunohistochemical staining of α-SYN in the striatum of mice injected with α-SYN encoding LV. Inlay images show high-magnification views of two α-SYN-positive neurons without (left) or with (right) α-SYN inclusions. C , Immunofluorescent staining for α-SYN and ubiquitin in the striatum 5 months after injection with α-SYN LV. Detailed pictures on the right show colocalization of α-SYN (green) and ubiquitin (red) in the inclusions. D , Number of α-SYN aggregates in α-SYN-overexpressing neurons in the striatum. The fraction of α-SYN-positive neurons that contain inclusions was determined. FK506 treatment decreases the number of α-SYN-positive neurons. Error bars show SEM ( n = 6–7). E , Survival of α-SYN-overexpressing cells in the striatum after FK506 treatment. The α-SYN-positive area was quantified using the Cavalieri method as a measure for the number of α-SYN-overexpressing cells. In the groups treated with FK506, more α-SYN-overexpressing cells survive. Error bars show the SEM ( n = 6–7).

    Article Snippet: Briefly, sections were exposed to 3% hydrogen peroxide and incubated overnight with a polyclonal rabbit anti-α-SYN (1:5000; Millipore Bioscience Research Reagents) in 10% normal swine serum.

    Techniques: Immunohistochemistry, Staining, Mouse Assay, Injection

    Annexin A5 expression decreases the number of α-synuclein inclusions in vivo. (A) Representative fluorescent images of head muscles of transgenic young adult animals expressing either α-synuclein::YFP alone (the pkIs2386 transgene) or

    Journal: Biochemistry

    Article Title: Annexin A5 Directly Interacts with Amyloidogenic Proteins and Reduces Their Toxicity †

    doi: 10.1021/bi900608m

    Figure Lengend Snippet: Annexin A5 expression decreases the number of α-synuclein inclusions in vivo. (A) Representative fluorescent images of head muscles of transgenic young adult animals expressing either α-synuclein::YFP alone (the pkIs2386 transgene) or

    Article Snippet: Membranes were blocked with 10% nonfat dry milk in 0.1% Tween/Tris-buffered saline (TBS-T) and incubated with TBS-T containing polyclonal rabbit anti-α-synuclein antibody ( :1000) (Chemicon International, Temecula, CA) and monoclonal mouse anti-actin antibody (Calbiochem, Darmstadt, Germany).

    Techniques: Expressing, In Vivo, Transgenic Assay

    EPR spectra of R1-labeled h-IAPP and 115ter α-synuclein in the presence of annexin A5. To assess the effect of annexin A5 on amyloid misfolding, 20 μ M annexin A5 was added to 100 μ M spin-labeled (A) h-IAPP (21R1) or (B) 115ter

    Journal: Biochemistry

    Article Title: Annexin A5 Directly Interacts with Amyloidogenic Proteins and Reduces Their Toxicity †

    doi: 10.1021/bi900608m

    Figure Lengend Snippet: EPR spectra of R1-labeled h-IAPP and 115ter α-synuclein in the presence of annexin A5. To assess the effect of annexin A5 on amyloid misfolding, 20 μ M annexin A5 was added to 100 μ M spin-labeled (A) h-IAPP (21R1) or (B) 115ter

    Article Snippet: Membranes were blocked with 10% nonfat dry milk in 0.1% Tween/Tris-buffered saline (TBS-T) and incubated with TBS-T containing polyclonal rabbit anti-α-synuclein antibody ( :1000) (Chemicon International, Temecula, CA) and monoclonal mouse anti-actin antibody (Calbiochem, Darmstadt, Germany).

    Techniques: Electron Paramagnetic Resonance, Labeling

    Expression of α-synuclein in annexin A5 transgenic C. elegans . (A) Immunoblots of total protein extracts from control animals containing no α-synuclein or annexin A5 (lane 1), α-synuclein transgene (lane 2), and α-synuclein

    Journal: Biochemistry

    Article Title: Annexin A5 Directly Interacts with Amyloidogenic Proteins and Reduces Their Toxicity †

    doi: 10.1021/bi900608m

    Figure Lengend Snippet: Expression of α-synuclein in annexin A5 transgenic C. elegans . (A) Immunoblots of total protein extracts from control animals containing no α-synuclein or annexin A5 (lane 1), α-synuclein transgene (lane 2), and α-synuclein

    Article Snippet: Membranes were blocked with 10% nonfat dry milk in 0.1% Tween/Tris-buffered saline (TBS-T) and incubated with TBS-T containing polyclonal rabbit anti-α-synuclein antibody ( :1000) (Chemicon International, Temecula, CA) and monoclonal mouse anti-actin antibody (Calbiochem, Darmstadt, Germany).

    Techniques: Expressing, Transgenic Assay, Western Blot

    Generation of mice overproducing human α-synuclein in a Parkin-deficient background. (A) Breeding strategy. Parental homozygous parkin −/− and hA30Pα-syn +/+ mice were intercrossed. The breeding of the double heterozygous mice (F1) led to the generation of littermates of the nine expected genotypes, four of which, highlighted by the surrounding frames, were used for subsequent analyses. (B) Representative western blot analysis of brain extracts of 17 months-old mice showing the expression of the endogenous parkin gene and of the hA30Pα-syn transgene, normalized to actin levels.

    Journal: PLoS ONE

    Article Title: Parkin Deficiency Delays Motor Decline and Disease Manifestation in a Mouse Model of Synucleinopathy

    doi: 10.1371/journal.pone.0006629

    Figure Lengend Snippet: Generation of mice overproducing human α-synuclein in a Parkin-deficient background. (A) Breeding strategy. Parental homozygous parkin −/− and hA30Pα-syn +/+ mice were intercrossed. The breeding of the double heterozygous mice (F1) led to the generation of littermates of the nine expected genotypes, four of which, highlighted by the surrounding frames, were used for subsequent analyses. (B) Representative western blot analysis of brain extracts of 17 months-old mice showing the expression of the endogenous parkin gene and of the hA30Pα-syn transgene, normalized to actin levels.

    Article Snippet: The proteins were electrotransferred onto nitrocellulose membranes and probed with: monoclonal anti-Parkin (MAB5512, clone PRK8, Chemicon), anti PS129 -α-synuclein (clone PSyn#64, WAKO; clone EP1536Y, Abcam), anti-α-tubulin (clone DM 1A, Sigma), anti-FLAG (M2, Sigma); polyclonal anti-α-synuclein (SA3400, Affiniti) and anti-actin (A2066, Sigma), anti-GST (GE Healthcare/Amersham).

    Techniques: Mouse Assay, Western Blot, Expressing

    Phosphorylated α-synuclein is not a Parkin substrate. Analysis of a representative GST-pull-down assay performed by incubating GST-Parkin [26] or GST with the TBS-soluble protein fraction from a symptomatic hA30Pα-syn mouse. Antibodies against human α-synuclein, P S129 -α-synuclein or α-tubulin were used for western blotting. GST-Parkin interacts specifically with α-tubulin [54] , but not with α-synuclein or P S129 -α-synuclein. The asterisk indicates the GST-moiety resulting from the degradation of the proteolytically unstable recombinant GST-Parkin protein. (B) Results of an in vitro ubiquitylation assay performed by incubating GST-Parkin, or the enzymatically inactive GST-Parkin C418R variant, with ubiquitin-activating enzyme, ubiquitin-conjugating enzyme, UbcH7, FLAG-tagged ubiquitin and ATP, in the presence or absence of in vitro -phosphorylated α-synuclein (P S87,129 -α-syn). Once the reaction was completed, GST-Parkin was immobilized on glutathione-sepharose beads; the beads and the supernatant fraction were analyzed by western blotting with anti-FLAG, P S129 -α-synuclein, or anti-Parkin antibodies. GST-Parkin (but not GST-Parkin C418R ) promoted its own ubiquitylation in a dose-dependent manner, as demonstrated by the amounts of ubiquitylated species in the bead fraction being proportional to the amounts of GST-Parkin used for the ubiquitylation reactions. In addition, GST-Parkin (but not GST-Parkin C418R ) promoted the formation of ubiquitin chains, as indicated by the presence of a ladder of regularly spaced ubiquitin-immunoreactive protein bands in the supernatant fraction. However, GST-Parkin did not promote the ubiquitylation of P S129 -α-synuclein: P S129 -α-synuclein-immunoreactive proteins with higher apparent molecular mass than the monomer were not detected in the supernatant fraction at any of the GST-Parkin concentrations used in the assay (P S129 -α-syn); moreover, we did not observe any ubiquitin-immunoreactive bands specific for P S129 -α-synuclein in the supernatant fraction (FLAG). The asterisk indicates a FLAG immunoreactive band most likely corresponding to a thioester adduct formed between the E2 enzyme UbcH7 and FLAG-tagged ubiquitin in the presence of the E1 enzyme.

    Journal: PLoS ONE

    Article Title: Parkin Deficiency Delays Motor Decline and Disease Manifestation in a Mouse Model of Synucleinopathy

    doi: 10.1371/journal.pone.0006629

    Figure Lengend Snippet: Phosphorylated α-synuclein is not a Parkin substrate. Analysis of a representative GST-pull-down assay performed by incubating GST-Parkin [26] or GST with the TBS-soluble protein fraction from a symptomatic hA30Pα-syn mouse. Antibodies against human α-synuclein, P S129 -α-synuclein or α-tubulin were used for western blotting. GST-Parkin interacts specifically with α-tubulin [54] , but not with α-synuclein or P S129 -α-synuclein. The asterisk indicates the GST-moiety resulting from the degradation of the proteolytically unstable recombinant GST-Parkin protein. (B) Results of an in vitro ubiquitylation assay performed by incubating GST-Parkin, or the enzymatically inactive GST-Parkin C418R variant, with ubiquitin-activating enzyme, ubiquitin-conjugating enzyme, UbcH7, FLAG-tagged ubiquitin and ATP, in the presence or absence of in vitro -phosphorylated α-synuclein (P S87,129 -α-syn). Once the reaction was completed, GST-Parkin was immobilized on glutathione-sepharose beads; the beads and the supernatant fraction were analyzed by western blotting with anti-FLAG, P S129 -α-synuclein, or anti-Parkin antibodies. GST-Parkin (but not GST-Parkin C418R ) promoted its own ubiquitylation in a dose-dependent manner, as demonstrated by the amounts of ubiquitylated species in the bead fraction being proportional to the amounts of GST-Parkin used for the ubiquitylation reactions. In addition, GST-Parkin (but not GST-Parkin C418R ) promoted the formation of ubiquitin chains, as indicated by the presence of a ladder of regularly spaced ubiquitin-immunoreactive protein bands in the supernatant fraction. However, GST-Parkin did not promote the ubiquitylation of P S129 -α-synuclein: P S129 -α-synuclein-immunoreactive proteins with higher apparent molecular mass than the monomer were not detected in the supernatant fraction at any of the GST-Parkin concentrations used in the assay (P S129 -α-syn); moreover, we did not observe any ubiquitin-immunoreactive bands specific for P S129 -α-synuclein in the supernatant fraction (FLAG). The asterisk indicates a FLAG immunoreactive band most likely corresponding to a thioester adduct formed between the E2 enzyme UbcH7 and FLAG-tagged ubiquitin in the presence of the E1 enzyme.

    Article Snippet: The proteins were electrotransferred onto nitrocellulose membranes and probed with: monoclonal anti-Parkin (MAB5512, clone PRK8, Chemicon), anti PS129 -α-synuclein (clone PSyn#64, WAKO; clone EP1536Y, Abcam), anti-α-tubulin (clone DM 1A, Sigma), anti-FLAG (M2, Sigma); polyclonal anti-α-synuclein (SA3400, Affiniti) and anti-actin (A2066, Sigma), anti-GST (GE Healthcare/Amersham).

    Techniques: Pull Down Assay, Western Blot, Recombinant, In Vitro, Ubiquitin Assay, Variant Assay

    P S129 -α-synuclein-immunoreactivity is associated with activation of caspase 9. (A) Examples of P S129 -α-synuclein-positive neurons with abnormally shaped nuclei and dysmorphic or abnormally swollen somata and proximal dendrites in the brainstem of symptomatic hA30Pα-syn mice. The upper and lower right micrographs correspond to a higher magnification of the regions outlined by the frames in the left micrograph. (B) Double immunofluorescent labelling showing caspase 9 activation in representative P S129 -α-synuclein- or ubiquitin-positive neurons of the spinal cord. Scale bars indicate 30 µm in (A) and 15 µm in (B).

    Journal: PLoS ONE

    Article Title: Parkin Deficiency Delays Motor Decline and Disease Manifestation in a Mouse Model of Synucleinopathy

    doi: 10.1371/journal.pone.0006629

    Figure Lengend Snippet: P S129 -α-synuclein-immunoreactivity is associated with activation of caspase 9. (A) Examples of P S129 -α-synuclein-positive neurons with abnormally shaped nuclei and dysmorphic or abnormally swollen somata and proximal dendrites in the brainstem of symptomatic hA30Pα-syn mice. The upper and lower right micrographs correspond to a higher magnification of the regions outlined by the frames in the left micrograph. (B) Double immunofluorescent labelling showing caspase 9 activation in representative P S129 -α-synuclein- or ubiquitin-positive neurons of the spinal cord. Scale bars indicate 30 µm in (A) and 15 µm in (B).

    Article Snippet: The proteins were electrotransferred onto nitrocellulose membranes and probed with: monoclonal anti-Parkin (MAB5512, clone PRK8, Chemicon), anti PS129 -α-synuclein (clone PSyn#64, WAKO; clone EP1536Y, Abcam), anti-α-tubulin (clone DM 1A, Sigma), anti-FLAG (M2, Sigma); polyclonal anti-α-synuclein (SA3400, Affiniti) and anti-actin (A2066, Sigma), anti-GST (GE Healthcare/Amersham).

    Techniques: Activation Assay, Mouse Assay

    The frequency of P S129 -α-synuclein-positive structures immunolabelled with anti-ubiquitin antibodies is lower in the absence than presence of Parkin. (A) Confocal scanning laser micrographs of a brainstem section from a representative symptomatic hA30Pα-syn mouse illustrating colocalization of ubiquitin staining with P S129 -α-synuclein-positive deposits (top panel). Note that ubiquitin was present in most of the swollen P S129 -α-synuclein-positive neurites (arrows). Synphilin-1-immunoreactivity was not found within the P S129 -α-synuclein-positive deposits (middle panel), whereas it was detected in nerve processes in a representative region of the cerebellum (lower panel). gr: granular cell layer; P: Purkinje cell layer; mol: molecular cell layer. Scale bars indicate 25 µm. (B) Percentage of P S129 -α-synuclein-immunopositive neurites and cell bodies co-labelled for ubiquitin in a representative region of the brainstem of symptomatic hA30Pα-syn mice with or without Parkin (n = 4–5). *, p

    Journal: PLoS ONE

    Article Title: Parkin Deficiency Delays Motor Decline and Disease Manifestation in a Mouse Model of Synucleinopathy

    doi: 10.1371/journal.pone.0006629

    Figure Lengend Snippet: The frequency of P S129 -α-synuclein-positive structures immunolabelled with anti-ubiquitin antibodies is lower in the absence than presence of Parkin. (A) Confocal scanning laser micrographs of a brainstem section from a representative symptomatic hA30Pα-syn mouse illustrating colocalization of ubiquitin staining with P S129 -α-synuclein-positive deposits (top panel). Note that ubiquitin was present in most of the swollen P S129 -α-synuclein-positive neurites (arrows). Synphilin-1-immunoreactivity was not found within the P S129 -α-synuclein-positive deposits (middle panel), whereas it was detected in nerve processes in a representative region of the cerebellum (lower panel). gr: granular cell layer; P: Purkinje cell layer; mol: molecular cell layer. Scale bars indicate 25 µm. (B) Percentage of P S129 -α-synuclein-immunopositive neurites and cell bodies co-labelled for ubiquitin in a representative region of the brainstem of symptomatic hA30Pα-syn mice with or without Parkin (n = 4–5). *, p

    Article Snippet: The proteins were electrotransferred onto nitrocellulose membranes and probed with: monoclonal anti-Parkin (MAB5512, clone PRK8, Chemicon), anti PS129 -α-synuclein (clone PSyn#64, WAKO; clone EP1536Y, Abcam), anti-α-tubulin (clone DM 1A, Sigma), anti-FLAG (M2, Sigma); polyclonal anti-α-synuclein (SA3400, Affiniti) and anti-actin (A2066, Sigma), anti-GST (GE Healthcare/Amersham).

    Techniques: Staining, Mouse Assay

    Distribution of P S129 -α-synuclein- and ubiquitin-immunolabelling in symptomatic hA30Pα-syn mice. (A) Schematic illustration of the distribution and density of P S129 -α-synuclein deposits on representative brain coronal sections of symptomatic mice. Similar distributions were observed in hA30Pα-syn mice with or without Parkin. Regions immunolabelled in most animals are indicated in red, whereas those less frequently stained are in grey. (B) Representative micrographs illustrating P S129 -α-synuclein-immunolabelling in the brainstem (bregma −2.9) of symptomatic hA30Pα-syn mice with and without Parkin, and in a healthy hA30Pα-syn control mouse. Quantitative analysis of the density of P S129 -α-synuclein-labelling in a representative region of the brainstem of symptomatic mice (n = 5 per genotype). (C) Representative micrographs illustrating typical ubiquitin-immunoreactivity in the presence or absence of Parkin in the brainstem of symptomatic mice, and of a healthy hA30Pα-syn control mouse. 1- superior colliculus , 2- pretectal nuclei, 3- geniculate nuclei, 4- periaqueductal grey layer, 5- zona incerta , 6- ventral tegmental area, 7- SN pars reticulata , 8- cerebellar white layer, 9- lateral cerebellar nuclei, 10- vestibular nuclei, 11- cochlear nuclei 12- trigeminal nuclei, 13- pontine reticular nuclei, 14- parvicellular reticular nuclei. Scale bars indicate 200 µm.

    Journal: PLoS ONE

    Article Title: Parkin Deficiency Delays Motor Decline and Disease Manifestation in a Mouse Model of Synucleinopathy

    doi: 10.1371/journal.pone.0006629

    Figure Lengend Snippet: Distribution of P S129 -α-synuclein- and ubiquitin-immunolabelling in symptomatic hA30Pα-syn mice. (A) Schematic illustration of the distribution and density of P S129 -α-synuclein deposits on representative brain coronal sections of symptomatic mice. Similar distributions were observed in hA30Pα-syn mice with or without Parkin. Regions immunolabelled in most animals are indicated in red, whereas those less frequently stained are in grey. (B) Representative micrographs illustrating P S129 -α-synuclein-immunolabelling in the brainstem (bregma −2.9) of symptomatic hA30Pα-syn mice with and without Parkin, and in a healthy hA30Pα-syn control mouse. Quantitative analysis of the density of P S129 -α-synuclein-labelling in a representative region of the brainstem of symptomatic mice (n = 5 per genotype). (C) Representative micrographs illustrating typical ubiquitin-immunoreactivity in the presence or absence of Parkin in the brainstem of symptomatic mice, and of a healthy hA30Pα-syn control mouse. 1- superior colliculus , 2- pretectal nuclei, 3- geniculate nuclei, 4- periaqueductal grey layer, 5- zona incerta , 6- ventral tegmental area, 7- SN pars reticulata , 8- cerebellar white layer, 9- lateral cerebellar nuclei, 10- vestibular nuclei, 11- cochlear nuclei 12- trigeminal nuclei, 13- pontine reticular nuclei, 14- parvicellular reticular nuclei. Scale bars indicate 200 µm.

    Article Snippet: The proteins were electrotransferred onto nitrocellulose membranes and probed with: monoclonal anti-Parkin (MAB5512, clone PRK8, Chemicon), anti PS129 -α-synuclein (clone PSyn#64, WAKO; clone EP1536Y, Abcam), anti-α-tubulin (clone DM 1A, Sigma), anti-FLAG (M2, Sigma); polyclonal anti-α-synuclein (SA3400, Affiniti) and anti-actin (A2066, Sigma), anti-GST (GE Healthcare/Amersham).

    Techniques: Mouse Assay, Staining

    The solubility of P S129 -α-synuclein but not P S87 -α-synuclein species is decreased in the brains of end-stage symptomatic hA30Pα-syn mice. (A) Representative western blot using protein-specific antibodies illustrating recognition of human α-synuclein and its isoforms phosphorylated at serine 87 or 129, in the TBS-soluble brain fraction of a symptomatic transgenic hA30Pα-syn mouse. These proteins were not detected in the TBS- or SDS-soluble brain fractions of an aged wild-type control animal (left panel). The anti-P S87 - and anti-P S129 -α-synuclein antibodies detected specifically normal recombinant α-synuclein (P S87,129 α-syn), phosphorylated in vitro by casein kinase 1 as described previously [42] , but not the non phosphorylated protein (α-syn). As expected, the anti-P S87 -α-synuclein antibody also recognized the in vitro -phosphorylated S129A α-synuclein variant (S129A P S87 α-syn), but not phosphorylated S87A α-synuclein (S87A P S129 α-syn). In contrast, the anti-P S129 -α-synuclein antibody recognized S87A P S129 α-syn but not S129A P S87 α-syn. (B) Representative western blots illustrating α-synuclein (top panel), P S87 -α-synuclein and P S129 -α-synuclein (middle and bottom panels) protein abundance in TBS-soluble and -insoluble (SDS) brain fractions from symptomatic (S) and non symptomatic (ns) mice with and without Parkin. (C) Quantitative analysis of human α-synuclein, P S87 -α-synuclein and P S129 -α-synuclein solubility in symptomatic and non symptomatic hA30Pα-syn mice with (ns: n = 4; S: n = 13−8) and without (ns: n = 4; S: n = 11−8) Parkin. The relative protein amount was defined as the ratio between the amount of protein normalized to α-tubulin in the SDS fraction, and the total amount of protein normalized to -tubulin in the SDS and TBS fractions. The abundance of P S129 -α-synuclein in the SDS brain fraction was significantly higher in symptomatic mice in the presence or absence of Parkin than in healthy mice. *, p

    Journal: PLoS ONE

    Article Title: Parkin Deficiency Delays Motor Decline and Disease Manifestation in a Mouse Model of Synucleinopathy

    doi: 10.1371/journal.pone.0006629

    Figure Lengend Snippet: The solubility of P S129 -α-synuclein but not P S87 -α-synuclein species is decreased in the brains of end-stage symptomatic hA30Pα-syn mice. (A) Representative western blot using protein-specific antibodies illustrating recognition of human α-synuclein and its isoforms phosphorylated at serine 87 or 129, in the TBS-soluble brain fraction of a symptomatic transgenic hA30Pα-syn mouse. These proteins were not detected in the TBS- or SDS-soluble brain fractions of an aged wild-type control animal (left panel). The anti-P S87 - and anti-P S129 -α-synuclein antibodies detected specifically normal recombinant α-synuclein (P S87,129 α-syn), phosphorylated in vitro by casein kinase 1 as described previously [42] , but not the non phosphorylated protein (α-syn). As expected, the anti-P S87 -α-synuclein antibody also recognized the in vitro -phosphorylated S129A α-synuclein variant (S129A P S87 α-syn), but not phosphorylated S87A α-synuclein (S87A P S129 α-syn). In contrast, the anti-P S129 -α-synuclein antibody recognized S87A P S129 α-syn but not S129A P S87 α-syn. (B) Representative western blots illustrating α-synuclein (top panel), P S87 -α-synuclein and P S129 -α-synuclein (middle and bottom panels) protein abundance in TBS-soluble and -insoluble (SDS) brain fractions from symptomatic (S) and non symptomatic (ns) mice with and without Parkin. (C) Quantitative analysis of human α-synuclein, P S87 -α-synuclein and P S129 -α-synuclein solubility in symptomatic and non symptomatic hA30Pα-syn mice with (ns: n = 4; S: n = 13−8) and without (ns: n = 4; S: n = 11−8) Parkin. The relative protein amount was defined as the ratio between the amount of protein normalized to α-tubulin in the SDS fraction, and the total amount of protein normalized to -tubulin in the SDS and TBS fractions. The abundance of P S129 -α-synuclein in the SDS brain fraction was significantly higher in symptomatic mice in the presence or absence of Parkin than in healthy mice. *, p

    Article Snippet: The proteins were electrotransferred onto nitrocellulose membranes and probed with: monoclonal anti-Parkin (MAB5512, clone PRK8, Chemicon), anti PS129 -α-synuclein (clone PSyn#64, WAKO; clone EP1536Y, Abcam), anti-α-tubulin (clone DM 1A, Sigma), anti-FLAG (M2, Sigma); polyclonal anti-α-synuclein (SA3400, Affiniti) and anti-actin (A2066, Sigma), anti-GST (GE Healthcare/Amersham).

    Techniques: Solubility, Mouse Assay, Western Blot, Transgenic Assay, Recombinant, In Vitro, Variant Assay

    Parkin deficiency mitigates neuritic pathology in end-stage symptomatic hA30Pα-syn mice. (A) Representative micrographs illustrating P S129 -α-synuclein and ubiquitin immunoreactivities in the brainstem of hA30Pα-syn symptomatic mice. Swollen, spherical (black arrowheads) and elongated (black arrows) neuritic profiles, thin, thread-like neurites (white arrowhead), rare compact inclusions (white arrow), and immunostaining throughout the somatodendritic compartment (stars) were observed. The two upper right micrographs correspond to a higher magnification of the regions outlined by the frames in the left micrograph. Scale bars indicate 20 µm. (B) Representative micrographs illustrating P S129 -α-synuclein and ubiquitin immunoreactivities in the grey matter of the cervical spinal cord from hA30Pα-syn mice with (left column) and without Parkin (right column). Note the abundance of aberrantly swollen neuritic processes (white arrows) in the presence of Parkin. Scale bar indicates 60 µm.

    Journal: PLoS ONE

    Article Title: Parkin Deficiency Delays Motor Decline and Disease Manifestation in a Mouse Model of Synucleinopathy

    doi: 10.1371/journal.pone.0006629

    Figure Lengend Snippet: Parkin deficiency mitigates neuritic pathology in end-stage symptomatic hA30Pα-syn mice. (A) Representative micrographs illustrating P S129 -α-synuclein and ubiquitin immunoreactivities in the brainstem of hA30Pα-syn symptomatic mice. Swollen, spherical (black arrowheads) and elongated (black arrows) neuritic profiles, thin, thread-like neurites (white arrowhead), rare compact inclusions (white arrow), and immunostaining throughout the somatodendritic compartment (stars) were observed. The two upper right micrographs correspond to a higher magnification of the regions outlined by the frames in the left micrograph. Scale bars indicate 20 µm. (B) Representative micrographs illustrating P S129 -α-synuclein and ubiquitin immunoreactivities in the grey matter of the cervical spinal cord from hA30Pα-syn mice with (left column) and without Parkin (right column). Note the abundance of aberrantly swollen neuritic processes (white arrows) in the presence of Parkin. Scale bar indicates 60 µm.

    Article Snippet: The proteins were electrotransferred onto nitrocellulose membranes and probed with: monoclonal anti-Parkin (MAB5512, clone PRK8, Chemicon), anti PS129 -α-synuclein (clone PSyn#64, WAKO; clone EP1536Y, Abcam), anti-α-tubulin (clone DM 1A, Sigma), anti-FLAG (M2, Sigma); polyclonal anti-α-synuclein (SA3400, Affiniti) and anti-actin (A2066, Sigma), anti-GST (GE Healthcare/Amersham).

    Techniques: Mouse Assay, Immunostaining

    The MetaBlue blue autofluorescence signals colocalized with Aβ and alpha hemoglobin HBA staining in the red blood cells, CAA, dense-core plaques and diffusive plaques. A : MetaBlue signals colocalized with Aβ in the red blood cells ( i ), CAA ( ii ), dense-core plaques ( iii ) and diffusive plaques ( iv ), indicated by arrows. B : MetaBlue signals colocalized with alpha hemoglobin HBA staining in the red blood cells ( i ), CAA ( ii ), dense-core plaques ( iii ) and diffusive plaques ( iv ), indicated by arrows. Scale bars, 50 μm.

    Journal: bioRxiv

    Article Title: Senile plaques in Alzheimer’s disease are primarily induced by cerebral vessel leakage of red blood cells and other blood contents

    doi: 10.1101/2020.05.17.100941

    Figure Lengend Snippet: The MetaBlue blue autofluorescence signals colocalized with Aβ and alpha hemoglobin HBA staining in the red blood cells, CAA, dense-core plaques and diffusive plaques. A : MetaBlue signals colocalized with Aβ in the red blood cells ( i ), CAA ( ii ), dense-core plaques ( iii ) and diffusive plaques ( iv ), indicated by arrows. B : MetaBlue signals colocalized with alpha hemoglobin HBA staining in the red blood cells ( i ), CAA ( ii ), dense-core plaques ( iii ) and diffusive plaques ( iv ), indicated by arrows. Scale bars, 50 μm.

    Article Snippet: List of antibodies for immunocytochemistry The following primary antibodies and dilutions have been used in this study: MDA (Abcam ab6463, 1:200), UBB (Proteintech 10201-2-AP, 1:200), Lamp2 (Proteintech 66301-1-Ig, 1:100), LC3 (This antibody detects both LC3A and LC3B) (Proteintech 66139-1-Ig, 1:500), phos-TAU (Abcam ab151559, 1:200), Aβ (Abcam ab201061, 1:200), Aβ/APP (CST #2450, 1:200), ColIV(Abcam ab236640, 1:200), LRP1 (Abcam ab92544, 1:200), ApoE (Abcam ab183597, 1:200), CD42b (Abcam ab210407, 1:200), HBA (Abcam ab92492, 1:200), GFAP (Abcam ab33922, 1:200), Iba1 (Abcam ab178847, 1:200), αSyn (Proteintech 10842-1-AP, 1:200).

    Techniques: Staining

    The colocalization of Aβ and red blood cell alpha Hemoglobin (HBA). ( i ) Aβ staining of a single red blood cell marked with HBA in the center of a senile plaque. ( ii ) Aβ staining of red blood cells marked with HBA during red blood cell extravasation. ( iii ) Aβ staining of red blood cells marked with HBA during intravascular hemolysis with the disappearance of clear round shape structure of red blood cells. The arrow indicated Aβ and HBA co-staining. The dashed lines in (ii) indicated the outline of a blood vessel. Scale bar, 50 μm.

    Journal: bioRxiv

    Article Title: Senile plaques in Alzheimer’s disease are primarily induced by cerebral vessel leakage of red blood cells and other blood contents

    doi: 10.1101/2020.05.17.100941

    Figure Lengend Snippet: The colocalization of Aβ and red blood cell alpha Hemoglobin (HBA). ( i ) Aβ staining of a single red blood cell marked with HBA in the center of a senile plaque. ( ii ) Aβ staining of red blood cells marked with HBA during red blood cell extravasation. ( iii ) Aβ staining of red blood cells marked with HBA during intravascular hemolysis with the disappearance of clear round shape structure of red blood cells. The arrow indicated Aβ and HBA co-staining. The dashed lines in (ii) indicated the outline of a blood vessel. Scale bar, 50 μm.

    Article Snippet: List of antibodies for immunocytochemistry The following primary antibodies and dilutions have been used in this study: MDA (Abcam ab6463, 1:200), UBB (Proteintech 10201-2-AP, 1:200), Lamp2 (Proteintech 66301-1-Ig, 1:100), LC3 (This antibody detects both LC3A and LC3B) (Proteintech 66139-1-Ig, 1:500), phos-TAU (Abcam ab151559, 1:200), Aβ (Abcam ab201061, 1:200), Aβ/APP (CST #2450, 1:200), ColIV(Abcam ab236640, 1:200), LRP1 (Abcam ab92544, 1:200), ApoE (Abcam ab183597, 1:200), CD42b (Abcam ab210407, 1:200), HBA (Abcam ab92492, 1:200), GFAP (Abcam ab33922, 1:200), Iba1 (Abcam ab178847, 1:200), αSyn (Proteintech 10842-1-AP, 1:200).

    Techniques: Staining

    Dystonin-a2–MAP1B interaction maintains Ac–α-tubulin and promotes flux through the secretory pathway. (A) Western blot analysis of 293T cell lysate shows that MAP1B overexpression maintains Ac–α-tubulin after dystonin-a2 depletion relative to empty vector (EV) control. (B) MAP1B overexpression prevents Golgi fragmentation after dystonin-a2 depletion in 293Ts. Arrows point to intact Golgi in an siA2-treated cell that is positive for MAP1B-GFP expression. Arrowheads point to fragmented Golgi in a neighboring cell that was not transfected with the MAP1B-GFP construct. (C) MAP1B overexpression promotes VSVG trafficking to the Golgi and PM after dystonin-a2 depletion relative to empty vector control. Scramble siRNA-treated cells (top and A) are labeled C. The arrowhead identifies aberrantly localized VSVG molecules, whereas arrows identify normal VSVG localization. (D and E) Quantification of MAP1B impact on VSVG accumulation in the Golgi (D) and at the PM (E; ANOVA posthoc Tukey; n = 3). (F) MAP1B antigenic labeling of WT primary sensory neurons shows a perinuclear accumulation that is lost in dt 27J neurons (arrows). (G) MAP1B expression restores GLuc flux through the secretory pathway (time = 1 h) in P15 dt 27J sensory neurons (ANOVA posthoc Tukey; n = 6). Error bars show means ± SEM. Cont, control; Tub, tubulin. **, P

    Journal: The Journal of Cell Biology

    Article Title: Microtubule stability, Golgi organization, and transport flux require dystonin-a2-MAP1B interaction

    doi: 10.1083/jcb.201107096

    Figure Lengend Snippet: Dystonin-a2–MAP1B interaction maintains Ac–α-tubulin and promotes flux through the secretory pathway. (A) Western blot analysis of 293T cell lysate shows that MAP1B overexpression maintains Ac–α-tubulin after dystonin-a2 depletion relative to empty vector (EV) control. (B) MAP1B overexpression prevents Golgi fragmentation after dystonin-a2 depletion in 293Ts. Arrows point to intact Golgi in an siA2-treated cell that is positive for MAP1B-GFP expression. Arrowheads point to fragmented Golgi in a neighboring cell that was not transfected with the MAP1B-GFP construct. (C) MAP1B overexpression promotes VSVG trafficking to the Golgi and PM after dystonin-a2 depletion relative to empty vector control. Scramble siRNA-treated cells (top and A) are labeled C. The arrowhead identifies aberrantly localized VSVG molecules, whereas arrows identify normal VSVG localization. (D and E) Quantification of MAP1B impact on VSVG accumulation in the Golgi (D) and at the PM (E; ANOVA posthoc Tukey; n = 3). (F) MAP1B antigenic labeling of WT primary sensory neurons shows a perinuclear accumulation that is lost in dt 27J neurons (arrows). (G) MAP1B expression restores GLuc flux through the secretory pathway (time = 1 h) in P15 dt 27J sensory neurons (ANOVA posthoc Tukey; n = 6). Error bars show means ± SEM. Cont, control; Tub, tubulin. **, P

    Article Snippet: Primary antibodies used were rabbit polyclonal α-tubulin (1:2,000; Sigma-Aldrich), MAP1B (1:500; Sigma-Aldrich), mouse monoclonal anti–α-tubulin (IgG1; 1:2,000; Oncogene), anti–Ac–α-tubulin (IgG2B; 1:2,000; Sigma-Aldrich), anti-GM130 (1:500; BD), antipericentrin (1:1,000; Abcam), anti–c-myc (1:800; Santa Cruz Biotechnology, Inc.), and anti-VSVG (1:100; gift from X. Zha).

    Techniques: Western Blot, Over Expression, Plasmid Preparation, Expressing, Transfection, Construct, Labeling

    Impaired MT acetylation results in Golgi fragmentation after loss of dystonin-a2. (A) Silencing of dystonin-a2 in 293T cells results in decreased MT acetylation coupled with reorganization of the Golgi complex. cis-Golgi (GM130) tightly associates with Ac–α-tubulin (arrows vs. arrowheads). (B) 3D rendering of P15 primary sensory neurons from WT and dt 27J mice. dt 27J sensory neurons show reduced Ac–α-tubulin coupled to a decrease GM130 antigenic labeling in the cell soma. (C and D) Maintenance of MT acetylation status with TSA prevents Golgi fragmentation after depletion of dystonin-a2 (ANOVA posthoc Tukey; *, P

    Journal: The Journal of Cell Biology

    Article Title: Microtubule stability, Golgi organization, and transport flux require dystonin-a2-MAP1B interaction

    doi: 10.1083/jcb.201107096

    Figure Lengend Snippet: Impaired MT acetylation results in Golgi fragmentation after loss of dystonin-a2. (A) Silencing of dystonin-a2 in 293T cells results in decreased MT acetylation coupled with reorganization of the Golgi complex. cis-Golgi (GM130) tightly associates with Ac–α-tubulin (arrows vs. arrowheads). (B) 3D rendering of P15 primary sensory neurons from WT and dt 27J mice. dt 27J sensory neurons show reduced Ac–α-tubulin coupled to a decrease GM130 antigenic labeling in the cell soma. (C and D) Maintenance of MT acetylation status with TSA prevents Golgi fragmentation after depletion of dystonin-a2 (ANOVA posthoc Tukey; *, P

    Article Snippet: Primary antibodies used were rabbit polyclonal α-tubulin (1:2,000; Sigma-Aldrich), MAP1B (1:500; Sigma-Aldrich), mouse monoclonal anti–α-tubulin (IgG1; 1:2,000; Oncogene), anti–Ac–α-tubulin (IgG2B; 1:2,000; Sigma-Aldrich), anti-GM130 (1:500; BD), antipericentrin (1:1,000; Abcam), anti–c-myc (1:800; Santa Cruz Biotechnology, Inc.), and anti-VSVG (1:100; gift from X. Zha).

    Techniques: Mouse Assay, Labeling

    Maintaining Ac–α-tubulin status permits flux through the secretory pathway after dystonin depletion. (A) Western blot analysis of 293T cell lysate shows that TSA treatment maintains Ac–α-tubulin after depletion of dystonin-a2. (B) TSA promotes VSVG trafficking to the Golgi and PM after dystonin-a2 depletion relative to DMSO-treated control. Arrowheads identify aberrantly localized VSVG molecules, whereas arrows identify normal VSVG localization. (C and D) Quantification of TSA impact on VSVG accumulation in the Golgi (C) and at the PM (D; ANOVA posthoc Tukey; n = 3). (E and F) TSA promotes GLuc flux through the secretory pathway (time = 1 h) in P4 (E) and P15 (F) dt 27J sensory neurons (ANOVA posthoc Tukey; n = 6). Error bars show means ± SEM. Cont, control; Tub, tubulin. **, P

    Journal: The Journal of Cell Biology

    Article Title: Microtubule stability, Golgi organization, and transport flux require dystonin-a2-MAP1B interaction

    doi: 10.1083/jcb.201107096

    Figure Lengend Snippet: Maintaining Ac–α-tubulin status permits flux through the secretory pathway after dystonin depletion. (A) Western blot analysis of 293T cell lysate shows that TSA treatment maintains Ac–α-tubulin after depletion of dystonin-a2. (B) TSA promotes VSVG trafficking to the Golgi and PM after dystonin-a2 depletion relative to DMSO-treated control. Arrowheads identify aberrantly localized VSVG molecules, whereas arrows identify normal VSVG localization. (C and D) Quantification of TSA impact on VSVG accumulation in the Golgi (C) and at the PM (D; ANOVA posthoc Tukey; n = 3). (E and F) TSA promotes GLuc flux through the secretory pathway (time = 1 h) in P4 (E) and P15 (F) dt 27J sensory neurons (ANOVA posthoc Tukey; n = 6). Error bars show means ± SEM. Cont, control; Tub, tubulin. **, P

    Article Snippet: Primary antibodies used were rabbit polyclonal α-tubulin (1:2,000; Sigma-Aldrich), MAP1B (1:500; Sigma-Aldrich), mouse monoclonal anti–α-tubulin (IgG1; 1:2,000; Oncogene), anti–Ac–α-tubulin (IgG2B; 1:2,000; Sigma-Aldrich), anti-GM130 (1:500; BD), antipericentrin (1:1,000; Abcam), anti–c-myc (1:800; Santa Cruz Biotechnology, Inc.), and anti-VSVG (1:100; gift from X. Zha).

    Techniques: Western Blot

    Loss of dystonin-a2 reduces α-tubulin acetylation status. (A and B) Loss of either dystonin-a1 or dystonin-a2 in 293T cells results in disorganization of the MTs relative to the actin cortex (ANOVA posthoc Dunnett’s t test; *, P

    Journal: The Journal of Cell Biology

    Article Title: Microtubule stability, Golgi organization, and transport flux require dystonin-a2-MAP1B interaction

    doi: 10.1083/jcb.201107096

    Figure Lengend Snippet: Loss of dystonin-a2 reduces α-tubulin acetylation status. (A and B) Loss of either dystonin-a1 or dystonin-a2 in 293T cells results in disorganization of the MTs relative to the actin cortex (ANOVA posthoc Dunnett’s t test; *, P

    Article Snippet: Primary antibodies used were rabbit polyclonal α-tubulin (1:2,000; Sigma-Aldrich), MAP1B (1:500; Sigma-Aldrich), mouse monoclonal anti–α-tubulin (IgG1; 1:2,000; Oncogene), anti–Ac–α-tubulin (IgG2B; 1:2,000; Sigma-Aldrich), anti-GM130 (1:500; BD), antipericentrin (1:1,000; Abcam), anti–c-myc (1:800; Santa Cruz Biotechnology, Inc.), and anti-VSVG (1:100; gift from X. Zha).

    Techniques:

    MT acetylation is reduced in DRGs and primary sensory neurons of dt 27J mice. (A and B) P4 (A) and P15 (B) analysis of tubulin acetylation state. Decreased Ac–α-tubulin is evident in dt 27J sensory neurons relative to WT in the perinuclear region (arrows vs. arrowheads). (C–F) Western blot analysis of DRG tissue at P4 (C and D) and P15 (E and F) also shows decreased Ac–α-tubulin in dt 27J relative to WT samples. Each lane represents DRGs from one animal. (Student’s t test; *, P

    Journal: The Journal of Cell Biology

    Article Title: Microtubule stability, Golgi organization, and transport flux require dystonin-a2-MAP1B interaction

    doi: 10.1083/jcb.201107096

    Figure Lengend Snippet: MT acetylation is reduced in DRGs and primary sensory neurons of dt 27J mice. (A and B) P4 (A) and P15 (B) analysis of tubulin acetylation state. Decreased Ac–α-tubulin is evident in dt 27J sensory neurons relative to WT in the perinuclear region (arrows vs. arrowheads). (C–F) Western blot analysis of DRG tissue at P4 (C and D) and P15 (E and F) also shows decreased Ac–α-tubulin in dt 27J relative to WT samples. Each lane represents DRGs from one animal. (Student’s t test; *, P

    Article Snippet: Primary antibodies used were rabbit polyclonal α-tubulin (1:2,000; Sigma-Aldrich), MAP1B (1:500; Sigma-Aldrich), mouse monoclonal anti–α-tubulin (IgG1; 1:2,000; Oncogene), anti–Ac–α-tubulin (IgG2B; 1:2,000; Sigma-Aldrich), anti-GM130 (1:500; BD), antipericentrin (1:1,000; Abcam), anti–c-myc (1:800; Santa Cruz Biotechnology, Inc.), and anti-VSVG (1:100; gift from X. Zha).

    Techniques: Mouse Assay, Western Blot

    Western blot analysis for Nogo-A and NgR. Protein samples (10 μg for Nogo-A and 25 μg for NgR) were extracted from dissected samples from the ipsilateral cortex of sham- and brain-injured animals from 24 h to 7 days post-injury and resolved in polyacrylamide gels. Single bands emerged at approximately 200 kDa, corresponding to Nogo-A, and 80 kDa, corresponding to NgR, respectively. There were no significant changes between sham- and brain-injured animals. Alpha-tubulin was used as an internal control of protein load.

    Journal: Experimental neurology

    Article Title: Selective temporal and regional alterations of Nogo-A and small proline-rich repeat protein 1A (SPRR1A) but not Nogo-66 receptor (NgR) occur following traumatic brain injury in the rat

    doi: 10.1016/j.expneurol.2005.08.029

    Figure Lengend Snippet: Western blot analysis for Nogo-A and NgR. Protein samples (10 μg for Nogo-A and 25 μg for NgR) were extracted from dissected samples from the ipsilateral cortex of sham- and brain-injured animals from 24 h to 7 days post-injury and resolved in polyacrylamide gels. Single bands emerged at approximately 200 kDa, corresponding to Nogo-A, and 80 kDa, corresponding to NgR, respectively. There were no significant changes between sham- and brain-injured animals. Alpha-tubulin was used as an internal control of protein load.

    Article Snippet: For control of total protein load, a polyclonal rabbit anti alpha-tubulin antibody (Sigma, 1:2000) was added on stripped blots.

    Techniques: Western Blot

    Spontaneous differentiation of mESCs. EB s from all cell lines (A) and inmunofluorescence assays of differentiation markers in J1 and CMT cell lines at early and late passages (B). (a), (b) EBs in bacteriological dishes from J1 and E14 cell lines, respectively. (c) Cell migration and differentiation from attached EBs obtained from the CMT cell line (two images overlapped at 4× magnification). (B) CMT and J1 cell lines (chromosomally stable and unstable, respectively) maintained a functional pluripotency throughout the study. Both mESCs expressed markers of the three germ layers after at least 10 days of spontaneous differentiation. AFP : Alpha Fetoprotein, endodermal marker. SMA : alpha Smooth Muscle Actin, mesodermal marker. Nest : Nestin, ectodermal marker. A and B magnification:10×. Other magnifications are shown on the pictures. Images obtained from independent experiments.

    Journal: Scientific Reports

    Article Title: Chromosome Instability in mouse Embryonic Stem Cells

    doi: 10.1038/srep05324

    Figure Lengend Snippet: Spontaneous differentiation of mESCs. EB s from all cell lines (A) and inmunofluorescence assays of differentiation markers in J1 and CMT cell lines at early and late passages (B). (a), (b) EBs in bacteriological dishes from J1 and E14 cell lines, respectively. (c) Cell migration and differentiation from attached EBs obtained from the CMT cell line (two images overlapped at 4× magnification). (B) CMT and J1 cell lines (chromosomally stable and unstable, respectively) maintained a functional pluripotency throughout the study. Both mESCs expressed markers of the three germ layers after at least 10 days of spontaneous differentiation. AFP : Alpha Fetoprotein, endodermal marker. SMA : alpha Smooth Muscle Actin, mesodermal marker. Nest : Nestin, ectodermal marker. A and B magnification:10×. Other magnifications are shown on the pictures. Images obtained from independent experiments.

    Article Snippet: Applying the same protocol described for undifferentiated cells, the following primary antibodies were used to evaluate the differentiation potential: rabbit polyclonal anti-Nestin (1:250, Abcam; UK), rabbit polyclonal anti-Alpha Fetoprotein (AFP, 1:400, Dako; Denmark) and mouse monoclonal anti-Smooth Muscle Actin (SMA, 1:400, Sigma-Aldrich) as ectodermal, endodermal and mesodermal markers, respectively.

    Techniques: Migration, Functional Assay, Marker

    Detection of Golgi-derived MTs using the ice recovery assay. (A–F) Immunofluorescence images of RPE cells expressing Centrin-GFP (green) stained for alpha-tubulin (red) and the Golgi marker GM130 (blue). Maximum intensity projection of a confocal

    Journal: Methods in cell biology

    Article Title: Ice Recovery Assay for Detection of Golgi-Derived Microtubules

    doi: 10.1016/B978-0-12-417164-0.00024-0

    Figure Lengend Snippet: Detection of Golgi-derived MTs using the ice recovery assay. (A–F) Immunofluorescence images of RPE cells expressing Centrin-GFP (green) stained for alpha-tubulin (red) and the Golgi marker GM130 (blue). Maximum intensity projection of a confocal

    Article Snippet: Primary antibodies : Mouse monoclonal anti-GM130 antibody (used at 1:300 dilution, BD Biosciences), rabbit polyclonal anti-alpha tubulin (used at 1:1000 dilution, Abcam).

    Techniques: Derivative Assay, Immunofluorescence, Expressing, Staining, Marker