anti-goat antibodies Search Results


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  • 98
    Millipore antigoat
    Antigoat, supplied by Millipore, used in various techniques. Bioz Stars score: 98/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore goat anti apob antibody
    Liver-specific knockdown of <t>perilipin</t> 2 increases VLDL-TG secretion in Cideb −/− mice without affecting <t>apoB</t> synthesis and secretion. (A) Knockdown of perilipin 2 led to significant increases in VLDL-TAG secretion in wild-type and Cideb
    Goat Anti Apob Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Millipore goat anti apoe
    Western blot analysis of human <t>apoE</t> in forebrain lysates and concentrated astrocyte-conditioned media ( ACM ) from transgenic mice expressing apoE3 ( hE3 ; A ) or apoE4 ( hE4 ; B ). In forebrain samples, 50 μg of detergent-soluble protein was loaded per lane. Human apoE protein is detected with goat anti-human apoE antibody in the P14 and adult ( Ad ) brains of hE3 (lines 2 and 37) and hE4 (lines 11 and 22) transgenic mice but not in nontransgenic (−) apoE knock-out littermates. Astrocytes derived from transgenic (+) animals ( hE3 , line 37; hE4 , line 22) and from nontransgenic (−) apoE knock-out littermates were cultured until confluent and washed, and serum-free media were collected for 48 hr. Media were concentrated 50-fold, and 3 μl was loaded per lane. Human apoE is detected in the serum-free ACM from <t>GFAP-apoE3</t> line 37 and GFAP-apoE4 line 22 but not in the media of astrocytes derived from nontransgenic apoE knock-out littermates. Arrow indicates position of apoE.
    Goat Anti Apoe, supplied by Millipore, used in various techniques. Bioz Stars score: 89/100, based on 144 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Millipore goat anti rangap1 antibody
    Tpr regulates SUMOylation. 5A–5B. Analysis of the overall level of SUMOylation in cells transfected with Tpr, Nup153, SENP2 and mock siRNAs. 5A. Nuclear extracts of siRNAs treated cells were prepared and analyzed by western blotting using the antibodies as described on the left of the figure. RPB1 was used as loading control. 5B. Analysis of <t>RanGAP1</t> SUMOylation. Cytoplasmic extracts of Tpr, Nup153, SENP2 and mock siRNAs-treated cells were prepared and analyzed by western blotting using an anti-RanGAP1 antibody recognizing both SUMOylated and non-SUMOylated forms of RanGAP1 and with tubulin as loading control. 5C: SUMO-1 siRNA treatment overrides Tpr depletion-induced senescence in HeLa cells. HeLa cells were transfected with Tpr, SUMO-1, Tpr and SUMO-1 and mock siRNAs. After 6days, the cells were fixed and stained for SA-β-gal activity at pH 6.0. A representative picture of each condition is presented. Scale bar: 15 µm.
    Goat Anti Rangap1 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Millipore goat anti robo3
    Uncrossed inferior olivary axons cause ataxic gait. (A) <t>Ptf1a::cre;Robo3</t> lox/lox mice are severely ataxic, which is demonstrated by a very short latency to fall in the Rotarod test ( n = 7 mutants versus n = 9 for controls). (B) Image of a 4-mo-old Ptf1a::cre;Robo3 lox/lox mouse displaying an ataxic gait. (C) The step time of Ptf1a::cre;Robo3 lox/lox mice (light blue; n = 6) on the Erasmus Ladder is longer than in controls (violet and red; n = 4; p
    Goat Anti Robo3, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Millipore goat anti ephb2
    Neuropsin cleaves <t>EphB2</t> and regulates its expression both in vitro and in the amygdala after stress ( a, b ) EphB2-S band density in SH-SY5Y cells decreased upon 15 min neuropsin treatment (F (3,18) =11.24; p
    Goat Anti Ephb2, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore goat
    Neuropsin cleaves <t>EphB2</t> and regulates its expression both in vitro and in the amygdala after stress ( a, b ) EphB2-S band density in SH-SY5Y cells decreased upon 15 min neuropsin treatment (F (3,18) =11.24; p
    Goat, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 1304 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Agilent technologies anti goat antibody
    Neuropsin cleaves <t>EphB2</t> and regulates its expression both in vitro and in the amygdala after stress ( a, b ) EphB2-S band density in SH-SY5Y cells decreased upon 15 min neuropsin treatment (F (3,18) =11.24; p
    Anti Goat Antibody, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 91/100, based on 78 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Everest Biotech goat anti asna1 antibody
    Cell-based calpain cleavage assays. Lysates from MN9D cells overexpressing <t>T7-ASNA1-FLAG</t> ( A ), OPTN-GST ( B ), or T7-PERI-FLAG ( C ) were incubated for 2 h at 37 °C with the indicated calpains (5.55 unit m-calpain or 3.95 units μ-calpain) in
    Goat Anti Asna1 Antibody, supplied by Everest Biotech, used in various techniques. Bioz Stars score: 85/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Santa Cruz Biotechnology anti goat antibody
    Cell-based calpain cleavage assays. Lysates from MN9D cells overexpressing <t>T7-ASNA1-FLAG</t> ( A ), OPTN-GST ( B ), or T7-PERI-FLAG ( C ) were incubated for 2 h at 37 °C with the indicated calpains (5.55 unit m-calpain or 3.95 units μ-calpain) in
    Anti Goat Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 415 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GE Healthcare anti goat antibodies
    Cell-based calpain cleavage assays. Lysates from MN9D cells overexpressing <t>T7-ASNA1-FLAG</t> ( A ), OPTN-GST ( B ), or T7-PERI-FLAG ( C ) were incubated for 2 h at 37 °C with the indicated calpains (5.55 unit m-calpain or 3.95 units μ-calpain) in
    Anti Goat Antibodies, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 92/100, based on 102 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore goat anti sod1
    Immunohistochemistry for superoxide dismutase <t>(SOD1)</t> (a-c), manganese superoxide dismutase (SOD2) (d-f), catalase (CAT) (g-i) and glutathione peroxidase (GPx) (j-l) in the rat liver of the normal-(left column), ascorbic acid - (middle column) and Oenanthe javanica extract (OJE) - (right column) groups. Strong SOD1, SOD2, CAT, and GPx immunoreactive cells (arrows) are markedly increased in the OJE-group. Scale bar = 120 μ m.
    Goat Anti Sod1, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Millipore goat anti elmo1
    Activity-dependent expression of <t>ELMO1</t> regulates CGE-derived interneuron migration a. Expression of Dlx genes and ELMO1 at P5 in Dlx5/6-eGFP and Dlx5/6-Kir2.1 electroporated interneurons at e15.5. b. Expression of ELMO1 in GAD67-GFP transgenic mice at P2 and P5. Selective expression of ELMO1 in CGE-derived interneuron subtypes at P9. Quantification of ELMO1 expression in Re + , Cr + and VIP + interneurons (IN) at P9 (right). Mean percentage values (±SEM) were obtained from > 70 interneurons for each subtype. c. Quantification of DLX H and ELMO1 expression in Dlx5/6-eGFP (control) and Dlx5/6-Kir2.1 Re + e15.5-electroporated interneurons at P5. Mean percentage values (±SEM) were obtained from > 20 interneurons each in control and Kir2.1 electroporated mice for each quantification. d. Electroporation of Dlx5/6-Elmo1_TN558.FLAG plasmid at e15.5. FLAG immunoreactivity is detected in electroporated interneurons at P9 (inset). Neuronal morphology of a Re + interneuron and laminar distribution of electroporated interneurons at P9. Representative examples from 4 electroporated mice. e. Co-electroporation of Dlx5/6-Elmo1 and Dlx5/6-Kir2.1 plasmids at e15.5. ELMO1 expression in electroporated interneurons at P9. Morphological defects of an electroporated Re + interneuron and laminar distribution of electroporated interneurons. Representative examples from 6 electroporated mice. f. Quantification of the distribution of Re + interneurons across cortical layers at P9 upon expression of different plasmids. Mean percentage values (±SEM) were obtained from > 80 interneurons for each group. Values for control and Dlx5/6-Kir2.1 alone groups are repeated from Figure 2 to facilitate comparison between groups. The large bracket indicates comparison between the control and Dlx5/6-Elmo1_TN558.FLAG electroporated internerneurons. Paired t-test: *, P
    Goat Anti Elmo1, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore goat anti fos
    Medial <t>GFP+</t> neurons are active during locomotion. A , Few (~5%) GFP+ neurons express <t>Fos</t> when the animal is quiescent (top), but almost half express Fos following an overground locomotor task (bottom). B , When exposed to the same transmitters that elicit locomotion in the isolated spinal cord, these neurons recorded in slice fire tonically—they are not conditional bursting neurons.
    Goat Anti Fos, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Merck KGaA anti goat antibody
    Medial <t>GFP+</t> neurons are active during locomotion. A , Few (~5%) GFP+ neurons express <t>Fos</t> when the animal is quiescent (top), but almost half express Fos following an overground locomotor task (bottom). B , When exposed to the same transmitters that elicit locomotion in the isolated spinal cord, these neurons recorded in slice fire tonically—they are not conditional bursting neurons.
    Anti Goat Antibody, supplied by Merck KGaA, 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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    92
    Millipore goat anti sod2
    Immunohistochemistry for superoxide dismutase (SOD1) (a-c), manganese superoxide dismutase <t>(SOD2)</t> (d-f), catalase (CAT) (g-i) and glutathione peroxidase (GPx) (j-l) in the rat liver of the normal-(left column), ascorbic acid - (middle column) and Oenanthe javanica extract (OJE) - (right column) groups. Strong SOD1, SOD2, CAT, and GPx immunoreactive cells (arrows) are markedly increased in the OJE-group. Scale bar = 120 μ m.
    Goat Anti Sod2, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore goat anti choline acetyltransferase antibody
    Physiological and anatomical properties of OFF brisk-sustained ganglion cells (BSGCs). A : spikes in an OFF-BSGC, evoked by a dark spot flashed for 0.5 s. The stimulus timing is shown by the gray bar ( bottom ). The stimulus spot diameter (μm) is shown to the left of each response. Firing rates peaked within 85 ms of stimulus onset and were sustained for optimally sized spots, 75 and 150 μm in diameter. B : the mean spike count during the stimulus presentation is plotted vs. stimulus diameter (±SE; n = 141). The continuous line shows the best fit to a difference-of-Gaussians function, with a center size (2σ center Gaussian) of 130 ± 12 μm and a surround of 460 ± 36 μm. C : confocal z-projection of an OFF-BSGC filled with Alexa-594 hydrazide. Dendrites from an adjacent-filled BSGC are visible ( top ; original scale bar = 50 μm). Bottom ), were labeled with an antibody for choline <t>acetyltransferase</t> (gray; original scale bar = 25 μm).
    Goat Anti Choline Acetyltransferase Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Millipore antigoat igg antibody
    Physiological and anatomical properties of OFF brisk-sustained ganglion cells (BSGCs). A : spikes in an OFF-BSGC, evoked by a dark spot flashed for 0.5 s. The stimulus timing is shown by the gray bar ( bottom ). The stimulus spot diameter (μm) is shown to the left of each response. Firing rates peaked within 85 ms of stimulus onset and were sustained for optimally sized spots, 75 and 150 μm in diameter. B : the mean spike count during the stimulus presentation is plotted vs. stimulus diameter (±SE; n = 141). The continuous line shows the best fit to a difference-of-Gaussians function, with a center size (2σ center Gaussian) of 130 ± 12 μm and a surround of 460 ± 36 μm. C : confocal z-projection of an OFF-BSGC filled with Alexa-594 hydrazide. Dendrites from an adjacent-filled BSGC are visible ( top ; original scale bar = 50 μm). Bottom ), were labeled with an antibody for choline <t>acetyltransferase</t> (gray; original scale bar = 25 μm).
    Antigoat Igg Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    The Jackson Laboratory anti goat antibodies
    Physiological and anatomical properties of OFF brisk-sustained ganglion cells (BSGCs). A : spikes in an OFF-BSGC, evoked by a dark spot flashed for 0.5 s. The stimulus timing is shown by the gray bar ( bottom ). The stimulus spot diameter (μm) is shown to the left of each response. Firing rates peaked within 85 ms of stimulus onset and were sustained for optimally sized spots, 75 and 150 μm in diameter. B : the mean spike count during the stimulus presentation is plotted vs. stimulus diameter (±SE; n = 141). The continuous line shows the best fit to a difference-of-Gaussians function, with a center size (2σ center Gaussian) of 130 ± 12 μm and a surround of 460 ± 36 μm. C : confocal z-projection of an OFF-BSGC filled with Alexa-594 hydrazide. Dendrites from an adjacent-filled BSGC are visible ( top ; original scale bar = 50 μm). Bottom ), were labeled with an antibody for choline <t>acetyltransferase</t> (gray; original scale bar = 25 μm).
    Anti Goat Antibodies, supplied by The Jackson Laboratory, used in various techniques. Bioz Stars score: 91/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore goat anti biotin ab
    Physiological and anatomical properties of OFF brisk-sustained ganglion cells (BSGCs). A : spikes in an OFF-BSGC, evoked by a dark spot flashed for 0.5 s. The stimulus timing is shown by the gray bar ( bottom ). The stimulus spot diameter (μm) is shown to the left of each response. Firing rates peaked within 85 ms of stimulus onset and were sustained for optimally sized spots, 75 and 150 μm in diameter. B : the mean spike count during the stimulus presentation is plotted vs. stimulus diameter (±SE; n = 141). The continuous line shows the best fit to a difference-of-Gaussians function, with a center size (2σ center Gaussian) of 130 ± 12 μm and a surround of 460 ± 36 μm. C : confocal z-projection of an OFF-BSGC filled with Alexa-594 hydrazide. Dendrites from an adjacent-filled BSGC are visible ( top ; original scale bar = 50 μm). Bottom ), were labeled with an antibody for choline <t>acetyltransferase</t> (gray; original scale bar = 25 μm).
    Goat Anti Biotin Ab, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Millipore goat polyclonal anti timp1
    Increased levels of <t>TIMP1,</t> CD63 and β1-integrins in melanoma cells compared to primary melanocytes and association between Timp1 and β1- integrins only in human metastatic melanoma cells. mRNA levels of TIMP1 and CD63 were measured by real time PCR ( A and B , respectively). C. β1-integrin expression was analyzed in primary human melanocytes MP#2 and human metastatic melanoma cell lines (Mel2, Mel3, Mel4, Mel14, Mel25 and Mel28) by Western blotting using a specific <t>polyclonal</t> antibody. The same membrane was reprobed with anti-PCNA antibody as an internal control. D. Membrane-enriched protein extracts from MP#2, Mel2, Mel3 and Mel14 cell lines were immunoprecipitated with anti-TIMP1 and analyzed by Western blotting with anti-β1-integrin. E and F. Primary human MP#2 melanocytes and human metastatic melanomas Mel2, Mel3, Mel4, Mel11, Mel25, Mel28 and Mel33 (2x10 2 ) were incubated at 37°C on 60 mm plates and colony formation capacity was determined after 5 days. Spearman’s correlation (r) was used to correlate TIMP1 and clonogenic capacity ( G ), CD63 expression and clonogenic capacity ( H ), TIMP1 and CD63 expression ( I ), and combined TIMP1 and CD63 expression and clonogenic capacity ( J ). All analyses were performed using SPSS (v 18, Chicago, IL, USA). The significance level was set at 0.05.
    Goat Polyclonal Anti Timp1, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    R&D Systems anti goat antibody
    Increased levels of <t>TIMP1,</t> CD63 and β1-integrins in melanoma cells compared to primary melanocytes and association between Timp1 and β1- integrins only in human metastatic melanoma cells. mRNA levels of TIMP1 and CD63 were measured by real time PCR ( A and B , respectively). C. β1-integrin expression was analyzed in primary human melanocytes MP#2 and human metastatic melanoma cell lines (Mel2, Mel3, Mel4, Mel14, Mel25 and Mel28) by Western blotting using a specific <t>polyclonal</t> antibody. The same membrane was reprobed with anti-PCNA antibody as an internal control. D. Membrane-enriched protein extracts from MP#2, Mel2, Mel3 and Mel14 cell lines were immunoprecipitated with anti-TIMP1 and analyzed by Western blotting with anti-β1-integrin. E and F. Primary human MP#2 melanocytes and human metastatic melanomas Mel2, Mel3, Mel4, Mel11, Mel25, Mel28 and Mel33 (2x10 2 ) were incubated at 37°C on 60 mm plates and colony formation capacity was determined after 5 days. Spearman’s correlation (r) was used to correlate TIMP1 and clonogenic capacity ( G ), CD63 expression and clonogenic capacity ( H ), TIMP1 and CD63 expression ( I ), and combined TIMP1 and CD63 expression and clonogenic capacity ( J ). All analyses were performed using SPSS (v 18, Chicago, IL, USA). The significance level was set at 0.05.
    Anti Goat Antibody, supplied by R&D Systems, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher anti goat antibodies
    Impaired TNFα and IL-12 production in DGKζ-deficient mice after TLR-stimulation. (A and B) Measurement of serum cytokine concentrations by ELISA. WT and DGKζ −/− mice were intraperitoneally injected with 10 μg LPS or 5 μg STAg. 6 h after injection, sera were collected for measurement of IL-12 p40 and TNFα production by ELISA. Data shown are the mean ± SD for each group and are representative of three experiments. (C and D) Measurement of cytokine messengers by Northern blot. WT and DGKζ −/− mice were intraperitoneally injected with 10 μg LPS or 5 μg STAg. 2, 3, and 5 h after injection, total splenic RNA was isolated, and IL-12 p40 and TNFα transcripts were measured by Northern blot analysis. Data shown are representative of three experiments. (E) Increased resistance to LPS-induced shock in DGKζ −/− mice. Age- and sex-matched WT mice ( n = 10) and DGKζ −/− mice ( n = 6) were intraperitoneally injected with 20 mg d -galactosamine and 2 μg LPS per mouse, and their survival was observed at the indicated time points. Data shown are representative of two independent experiments. (F) Detection of IL-12 production by immunofluorescence. Spleens were removed from mice 6 h after injection of PBS, LPS, or STAg. Frozen sections of spleens were processed and stained with a goat anti–mouse IL-12 p40 antibody, followed by a double incubation with Alexa Fluor 488–conjugated anti–mouse CD11c and Alexa Fluor 594–conjugated <t>anti–goat</t> antibodies. The cells were counterstained for nuclei using DAPI. Images were captured using an Axiovert microscope with AxioVision software (see Materials and methods). Green, red, and blue represent CD11c + DCs, IL-12, and nuclei, respectively; yellow represents CD11c + IL-12 + after overlay. Bars, 50 μm.
    Anti Goat Antibodies, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 147 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Liver-specific knockdown of perilipin 2 increases VLDL-TG secretion in Cideb −/− mice without affecting apoB synthesis and secretion. (A) Knockdown of perilipin 2 led to significant increases in VLDL-TAG secretion in wild-type and Cideb

    Journal: Journal of Lipid Research

    Article Title: Opposing roles of cell death-inducing DFF45-like effector B and perilipin 2 in controlling hepatic VLDL lipidation [S]

    doi: 10.1194/jlr.M026591

    Figure Lengend Snippet: Liver-specific knockdown of perilipin 2 increases VLDL-TG secretion in Cideb −/− mice without affecting apoB synthesis and secretion. (A) Knockdown of perilipin 2 led to significant increases in VLDL-TAG secretion in wild-type and Cideb

    Article Snippet: The following antibodies were purchased from the indicated companies: guinea pig anti-perilipin 2 antibody (Fitzgerald Industries, USA), goat anti-apoB antibody (AB742, Chemicon, USA), mouse anti-MTP and mouse anti-GM130 antibodies (BD Biosciences, USA), mouse anti-Golgi 58K protein (p58K), mouse anti-β-actin and rabbit anti-calnexin (CNX) antibodies (Sigma-Aldrich, USA) and rat anti-GRP94 antibody (Santa Cruz Biotechnology, USA).

    Techniques: Mouse Assay

    Knockdown of perilipin 2 increases the levels of mature VLDL particles in the Golgi fractions from Cideb −/− livers. Density distribution of apoB-containing lipoproteins in the Golgi (A) and ER fractions (B) with or without perilipin 2

    Journal: Journal of Lipid Research

    Article Title: Opposing roles of cell death-inducing DFF45-like effector B and perilipin 2 in controlling hepatic VLDL lipidation [S]

    doi: 10.1194/jlr.M026591

    Figure Lengend Snippet: Knockdown of perilipin 2 increases the levels of mature VLDL particles in the Golgi fractions from Cideb −/− livers. Density distribution of apoB-containing lipoproteins in the Golgi (A) and ER fractions (B) with or without perilipin 2

    Article Snippet: The following antibodies were purchased from the indicated companies: guinea pig anti-perilipin 2 antibody (Fitzgerald Industries, USA), goat anti-apoB antibody (AB742, Chemicon, USA), mouse anti-MTP and mouse anti-GM130 antibodies (BD Biosciences, USA), mouse anti-Golgi 58K protein (p58K), mouse anti-β-actin and rabbit anti-calnexin (CNX) antibodies (Sigma-Aldrich, USA) and rat anti-GRP94 antibody (Santa Cruz Biotechnology, USA).

    Techniques:

    Distribution patterns of Cideb, perilipin 2, ApoB, and marker proteins in the Golgi and ER fractions. (A, B) Biochemical fractionation of liver microsomes was performed by sucrose density gradient ultracentrifugation using wild-type (+/+) (A) and Cideb

    Journal: Journal of Lipid Research

    Article Title: Opposing roles of cell death-inducing DFF45-like effector B and perilipin 2 in controlling hepatic VLDL lipidation [S]

    doi: 10.1194/jlr.M026591

    Figure Lengend Snippet: Distribution patterns of Cideb, perilipin 2, ApoB, and marker proteins in the Golgi and ER fractions. (A, B) Biochemical fractionation of liver microsomes was performed by sucrose density gradient ultracentrifugation using wild-type (+/+) (A) and Cideb

    Article Snippet: The following antibodies were purchased from the indicated companies: guinea pig anti-perilipin 2 antibody (Fitzgerald Industries, USA), goat anti-apoB antibody (AB742, Chemicon, USA), mouse anti-MTP and mouse anti-GM130 antibodies (BD Biosciences, USA), mouse anti-Golgi 58K protein (p58K), mouse anti-β-actin and rabbit anti-calnexin (CNX) antibodies (Sigma-Aldrich, USA) and rat anti-GRP94 antibody (Santa Cruz Biotechnology, USA).

    Techniques: Marker, Fractionation

    HCVcc secretion is impaired after clathrin and dynamin pharmacological inhibition. (A) HCV RNA levels after clathrin or dynamin inhibition in Huh7 Lunet N cells by 50 μM Pitstop 2 or 80 μM dynasore treatment, respectively. Data are presented relative to those for vehicle-treated control cells. Results are expressed as means and SEM for four experiments performed in triplicate. (B) Analysis of the possible effects of Pitstop 2 and dynasore carryover during titration of infective supernatants after inhibitor treatment. (C) Polarized hepatocyte-like localization of ZO-1 in Huh7 cells cultured on Matrigel. Red, ZO-1; blue (DAPI), nuclei. Bar, 25 μm. (D and E) Effects of Pitstop 2 (D) or dynasore (E) treatment on extracellular and intracellular HCV RNA levels in 3D-cultured, HCVcc-infected Huh7 cells. Results are presented relative to those for vehicle (dimethyl sulfoxide [DMSO])-treated control cells and are expressed as means and SEM for three experiments. (F and G) Western blot analyses of apoB and apoE levels in supernatants and cellular lysates obtained from 3D-cultured, HCVcc-infected Huh7 cells after clathrin (F) or dynamin (G) inhibition. α1AT and p53 were used as loading controls.

    Journal: Journal of Virology

    Article Title: Clathrin Mediates Infectious Hepatitis C Virus Particle Egress

    doi: 10.1128/JVI.03620-14

    Figure Lengend Snippet: HCVcc secretion is impaired after clathrin and dynamin pharmacological inhibition. (A) HCV RNA levels after clathrin or dynamin inhibition in Huh7 Lunet N cells by 50 μM Pitstop 2 or 80 μM dynasore treatment, respectively. Data are presented relative to those for vehicle-treated control cells. Results are expressed as means and SEM for four experiments performed in triplicate. (B) Analysis of the possible effects of Pitstop 2 and dynasore carryover during titration of infective supernatants after inhibitor treatment. (C) Polarized hepatocyte-like localization of ZO-1 in Huh7 cells cultured on Matrigel. Red, ZO-1; blue (DAPI), nuclei. Bar, 25 μm. (D and E) Effects of Pitstop 2 (D) or dynasore (E) treatment on extracellular and intracellular HCV RNA levels in 3D-cultured, HCVcc-infected Huh7 cells. Results are presented relative to those for vehicle (dimethyl sulfoxide [DMSO])-treated control cells and are expressed as means and SEM for three experiments. (F and G) Western blot analyses of apoB and apoE levels in supernatants and cellular lysates obtained from 3D-cultured, HCVcc-infected Huh7 cells after clathrin (F) or dynamin (G) inhibition. α1AT and p53 were used as loading controls.

    Article Snippet: Western blotting was carried out as previously described , using the following antibodies: rabbit anti-clathrin heavy chain, mouse anti-alpha 1 antitrypsin (anti-α1AT), rabbit anti-LAMP1 (Abcam), rabbit anti-EEA1 (Cell Signaling Technology), mouse anti-p53, mouse anti-HCV core (C7-50) (Santa Cruz Biotechnology), mouse anti-AP1G1, mouse anti-AP50 (AP-2 medium subunit) (BD Transduction Laboratories), goat anti-apoB, and goat anti-apoE (Calbiochem).

    Techniques: Inhibition, Titration, Cell Culture, Infection, Western Blot

    apoB and apoE secretion is not impaired by clathrin or AP-1 interference. (A and B) apoB and apoE levels were determined by ELISA (A) and Western blotting (B) after CHC or AP-1 knockdown in infected Huh7 cells. α1AT and p53 were used as loading controls. (C) apoB and apoE levels in the supernatants of infected Huh7 cells after 50 μM Pitstop 2 or 80 μM dynasore treatment were determined by ELISA. Results are expressed as means and SEM for two experiments performed in duplicate. (D and E) Kinetic analyses of secreted apoB, apoE, and α1AT (D) and of secreted HCV RNA (E) by Western blotting and real-time RT-PCR, respectively, after Pitstop 2 treatment of infected Huh7 cells. Results in panel E are presented relative to the maximum value and expressed as means and SEM for three experiments.

    Journal: Journal of Virology

    Article Title: Clathrin Mediates Infectious Hepatitis C Virus Particle Egress

    doi: 10.1128/JVI.03620-14

    Figure Lengend Snippet: apoB and apoE secretion is not impaired by clathrin or AP-1 interference. (A and B) apoB and apoE levels were determined by ELISA (A) and Western blotting (B) after CHC or AP-1 knockdown in infected Huh7 cells. α1AT and p53 were used as loading controls. (C) apoB and apoE levels in the supernatants of infected Huh7 cells after 50 μM Pitstop 2 or 80 μM dynasore treatment were determined by ELISA. Results are expressed as means and SEM for two experiments performed in duplicate. (D and E) Kinetic analyses of secreted apoB, apoE, and α1AT (D) and of secreted HCV RNA (E) by Western blotting and real-time RT-PCR, respectively, after Pitstop 2 treatment of infected Huh7 cells. Results in panel E are presented relative to the maximum value and expressed as means and SEM for three experiments.

    Article Snippet: Western blotting was carried out as previously described , using the following antibodies: rabbit anti-clathrin heavy chain, mouse anti-alpha 1 antitrypsin (anti-α1AT), rabbit anti-LAMP1 (Abcam), rabbit anti-EEA1 (Cell Signaling Technology), mouse anti-p53, mouse anti-HCV core (C7-50) (Santa Cruz Biotechnology), mouse anti-AP1G1, mouse anti-AP50 (AP-2 medium subunit) (BD Transduction Laboratories), goat anti-apoB, and goat anti-apoE (Calbiochem).

    Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Infection, Quantitative RT-PCR

    Intracellular HCVcc-apoE association in BFA-treated cells. (A) Immunoprecipitation (IP) from supernatants of HCVcc-infected cells, using control, anti-apoB, and anti-apoE antibodies. apoB and apoE levels were determined by Western blotting (WB). (B) Supernatant infectivity after immunoprecipitation was analyzed by titration on Huh7 cells and real-time RT-PCR. Results are presented relative to the infectivity of control Ig-immunoprecipitated supernatants and are expressed as means and SEM for three experiments. (C) Supernatant immunoprecipitation was carried out as described for panels A and B, and RNAs were extracted from both immunoprecipitates and postimmunoprecipitation supernatants. HCV RNA levels were assessed by real-time RT-PCR and presented as percentages of input HCV RNA. Results are expressed as means and SEM for three experiments. (D) Immunoprecipitates from detergent-free lysates of HCVcc-infected, siRNA-transfected cells were used for either Western blot analysis (inset; control siRNA-transfected cells) or HCV RNA quantification. (E) HCV RNA levels and infectivities after 1 μg/ml BFA treatment. (F) (Left) Immunoprecipitated HCV RNA levels after BFA treatment. (Right) Intracellular infectivities of BFA-treated cells after immunoprecipitation. (G) Intracellular infectivities of BFA-treated cells, assessed by titration on Huh7 cells in the presence of 5 μg/ml control or anti-apoE antibody. Results are expressed as means and SEM for at least three experiments.

    Journal: Journal of Virology

    Article Title: Clathrin Mediates Infectious Hepatitis C Virus Particle Egress

    doi: 10.1128/JVI.03620-14

    Figure Lengend Snippet: Intracellular HCVcc-apoE association in BFA-treated cells. (A) Immunoprecipitation (IP) from supernatants of HCVcc-infected cells, using control, anti-apoB, and anti-apoE antibodies. apoB and apoE levels were determined by Western blotting (WB). (B) Supernatant infectivity after immunoprecipitation was analyzed by titration on Huh7 cells and real-time RT-PCR. Results are presented relative to the infectivity of control Ig-immunoprecipitated supernatants and are expressed as means and SEM for three experiments. (C) Supernatant immunoprecipitation was carried out as described for panels A and B, and RNAs were extracted from both immunoprecipitates and postimmunoprecipitation supernatants. HCV RNA levels were assessed by real-time RT-PCR and presented as percentages of input HCV RNA. Results are expressed as means and SEM for three experiments. (D) Immunoprecipitates from detergent-free lysates of HCVcc-infected, siRNA-transfected cells were used for either Western blot analysis (inset; control siRNA-transfected cells) or HCV RNA quantification. (E) HCV RNA levels and infectivities after 1 μg/ml BFA treatment. (F) (Left) Immunoprecipitated HCV RNA levels after BFA treatment. (Right) Intracellular infectivities of BFA-treated cells after immunoprecipitation. (G) Intracellular infectivities of BFA-treated cells, assessed by titration on Huh7 cells in the presence of 5 μg/ml control or anti-apoE antibody. Results are expressed as means and SEM for at least three experiments.

    Article Snippet: Western blotting was carried out as previously described , using the following antibodies: rabbit anti-clathrin heavy chain, mouse anti-alpha 1 antitrypsin (anti-α1AT), rabbit anti-LAMP1 (Abcam), rabbit anti-EEA1 (Cell Signaling Technology), mouse anti-p53, mouse anti-HCV core (C7-50) (Santa Cruz Biotechnology), mouse anti-AP1G1, mouse anti-AP50 (AP-2 medium subunit) (BD Transduction Laboratories), goat anti-apoB, and goat anti-apoE (Calbiochem).

    Techniques: Immunoprecipitation, Infection, Western Blot, Titration, Quantitative RT-PCR, Transfection

    Western blot analysis of human apoE in forebrain lysates and concentrated astrocyte-conditioned media ( ACM ) from transgenic mice expressing apoE3 ( hE3 ; A ) or apoE4 ( hE4 ; B ). In forebrain samples, 50 μg of detergent-soluble protein was loaded per lane. Human apoE protein is detected with goat anti-human apoE antibody in the P14 and adult ( Ad ) brains of hE3 (lines 2 and 37) and hE4 (lines 11 and 22) transgenic mice but not in nontransgenic (−) apoE knock-out littermates. Astrocytes derived from transgenic (+) animals ( hE3 , line 37; hE4 , line 22) and from nontransgenic (−) apoE knock-out littermates were cultured until confluent and washed, and serum-free media were collected for 48 hr. Media were concentrated 50-fold, and 3 μl was loaded per lane. Human apoE is detected in the serum-free ACM from GFAP-apoE3 line 37 and GFAP-apoE4 line 22 but not in the media of astrocytes derived from nontransgenic apoE knock-out littermates. Arrow indicates position of apoE.

    Journal: The Journal of Neuroscience

    Article Title: Glial Fibrillary Acidic Protein–Apolipoprotein E (apoE) Transgenic Mice: Astrocyte-Specific Expression and Differing Biological Effects of Astrocyte-Secreted apoE3 and apoE4 Lipoproteins

    doi: 10.1523/JNEUROSCI.18-09-03261.1998

    Figure Lengend Snippet: Western blot analysis of human apoE in forebrain lysates and concentrated astrocyte-conditioned media ( ACM ) from transgenic mice expressing apoE3 ( hE3 ; A ) or apoE4 ( hE4 ; B ). In forebrain samples, 50 μg of detergent-soluble protein was loaded per lane. Human apoE protein is detected with goat anti-human apoE antibody in the P14 and adult ( Ad ) brains of hE3 (lines 2 and 37) and hE4 (lines 11 and 22) transgenic mice but not in nontransgenic (−) apoE knock-out littermates. Astrocytes derived from transgenic (+) animals ( hE3 , line 37; hE4 , line 22) and from nontransgenic (−) apoE knock-out littermates were cultured until confluent and washed, and serum-free media were collected for 48 hr. Media were concentrated 50-fold, and 3 μl was loaded per lane. Human apoE is detected in the serum-free ACM from GFAP-apoE3 line 37 and GFAP-apoE4 line 22 but not in the media of astrocytes derived from nontransgenic apoE knock-out littermates. Arrow indicates position of apoE.

    Article Snippet: For double-labeling immunofluorescence studies, goat anti-apoE and rat anti-GFAP were applied sequentially to sections, followed by coapplication of fluorescein-labeled anti-rat and indocarbocyanine-labeled anti-goat secondary antibodies as described ( ).

    Techniques: Western Blot, Transgenic Assay, Mouse Assay, Expressing, Knock-Out, Derivative Assay, Cell Culture

    GFAP-apoE construct used to generate transgenic mice expressing human apoE3 and apoE4 in the brain and Southern blot analysis for apoE in founder mice. A , Human apoE3 and apoE4 cDNAs ( hApoE ) were subcloned behind a human glial fibrillary acidic protein ( gfap . B , Southern blot analysis using a 32 P-labeled cDNA probe to mouse apoE reveals the presence of human (∼1 kb) and mouse apoE (∼3 kb) DNA in different apoE3 ( hE3 ) and apoE4 ( hE4 ) transgenic lines. Copy number is variable from line to line.

    Journal: The Journal of Neuroscience

    Article Title: Glial Fibrillary Acidic Protein–Apolipoprotein E (apoE) Transgenic Mice: Astrocyte-Specific Expression and Differing Biological Effects of Astrocyte-Secreted apoE3 and apoE4 Lipoproteins

    doi: 10.1523/JNEUROSCI.18-09-03261.1998

    Figure Lengend Snippet: GFAP-apoE construct used to generate transgenic mice expressing human apoE3 and apoE4 in the brain and Southern blot analysis for apoE in founder mice. A , Human apoE3 and apoE4 cDNAs ( hApoE ) were subcloned behind a human glial fibrillary acidic protein ( gfap . B , Southern blot analysis using a 32 P-labeled cDNA probe to mouse apoE reveals the presence of human (∼1 kb) and mouse apoE (∼3 kb) DNA in different apoE3 ( hE3 ) and apoE4 ( hE4 ) transgenic lines. Copy number is variable from line to line.

    Article Snippet: For double-labeling immunofluorescence studies, goat anti-apoE and rat anti-GFAP were applied sequentially to sections, followed by coapplication of fluorescein-labeled anti-rat and indocarbocyanine-labeled anti-goat secondary antibodies as described ( ).

    Techniques: Construct, Transgenic Assay, Mouse Assay, Expressing, Southern Blot, Labeling

    ApoE immunoreactivity is present in the brain of GFAP-apoE3 and GFAP-apoE4 mice. GFAP-apoE3 line 37 ( A, B ) and GFAP-apoE4 line 22 ( C, D ) mice on a mouse apoE−/− background were immunostained with a goat anti-apoE antibody. There was strong staining of cells, which by morphology appear to be astrocytes in the hippocampus in both P14 ( A, C ) and adult ( B, D ) mice. In addition to staining in glial cell bodies and their processes, apoE-IR also appears to be present in the neuropil. Qualitatively similar apoE-IR is seen in cells that appear to be glial in C57Bl6 apoE+/+ mice in both P14 ( E ) and adult animals ( F ). ApoE-IR is not observed in the hippocampus of an apoE−/− adult mouse ( G ). Scale bar, 130 μm.

    Journal: The Journal of Neuroscience

    Article Title: Glial Fibrillary Acidic Protein–Apolipoprotein E (apoE) Transgenic Mice: Astrocyte-Specific Expression and Differing Biological Effects of Astrocyte-Secreted apoE3 and apoE4 Lipoproteins

    doi: 10.1523/JNEUROSCI.18-09-03261.1998

    Figure Lengend Snippet: ApoE immunoreactivity is present in the brain of GFAP-apoE3 and GFAP-apoE4 mice. GFAP-apoE3 line 37 ( A, B ) and GFAP-apoE4 line 22 ( C, D ) mice on a mouse apoE−/− background were immunostained with a goat anti-apoE antibody. There was strong staining of cells, which by morphology appear to be astrocytes in the hippocampus in both P14 ( A, C ) and adult ( B, D ) mice. In addition to staining in glial cell bodies and their processes, apoE-IR also appears to be present in the neuropil. Qualitatively similar apoE-IR is seen in cells that appear to be glial in C57Bl6 apoE+/+ mice in both P14 ( E ) and adult animals ( F ). ApoE-IR is not observed in the hippocampus of an apoE−/− adult mouse ( G ). Scale bar, 130 μm.

    Article Snippet: For double-labeling immunofluorescence studies, goat anti-apoE and rat anti-GFAP were applied sequentially to sections, followed by coapplication of fluorescein-labeled anti-rat and indocarbocyanine-labeled anti-goat secondary antibodies as described ( ).

    Techniques: Mouse Assay, Staining

    GFAP and human apoE are co-localized, and apoE immunoreactivity is increased in regions of denervation in GFAP-apoE transgenic mice. A section through the hippocampus of an adult GFAP-apoE3 line 37 mouse was stained with a rat anti-GFAP antibody ( A, green ) and a goat anti-apoE antibody ( B, red ). Cells that are GFAP-immunoreactive are also immunoreactive for apoE ( arrows ). Three days after unilateral ( right ) entorhinal cortex lesion in an adult GFAP-apoE4 line 19 mouse, apoE-IR is clearly increased within astrocytes and the neuropil of the denervated outer molecular layer of the dentate gyrus ( C ). On the nonlesioned side ( D ), apoE-IR is present but is not upregulated. In C and D , the dorsal blade of the outer molecular layer is bracketed by arrowheads . Scale bars: A, B , 2 μm; C, D , 100 μm.

    Journal: The Journal of Neuroscience

    Article Title: Glial Fibrillary Acidic Protein–Apolipoprotein E (apoE) Transgenic Mice: Astrocyte-Specific Expression and Differing Biological Effects of Astrocyte-Secreted apoE3 and apoE4 Lipoproteins

    doi: 10.1523/JNEUROSCI.18-09-03261.1998

    Figure Lengend Snippet: GFAP and human apoE are co-localized, and apoE immunoreactivity is increased in regions of denervation in GFAP-apoE transgenic mice. A section through the hippocampus of an adult GFAP-apoE3 line 37 mouse was stained with a rat anti-GFAP antibody ( A, green ) and a goat anti-apoE antibody ( B, red ). Cells that are GFAP-immunoreactive are also immunoreactive for apoE ( arrows ). Three days after unilateral ( right ) entorhinal cortex lesion in an adult GFAP-apoE4 line 19 mouse, apoE-IR is clearly increased within astrocytes and the neuropil of the denervated outer molecular layer of the dentate gyrus ( C ). On the nonlesioned side ( D ), apoE-IR is present but is not upregulated. In C and D , the dorsal blade of the outer molecular layer is bracketed by arrowheads . Scale bars: A, B , 2 μm; C, D , 100 μm.

    Article Snippet: For double-labeling immunofluorescence studies, goat anti-apoE and rat anti-GFAP were applied sequentially to sections, followed by coapplication of fluorescein-labeled anti-rat and indocarbocyanine-labeled anti-goat secondary antibodies as described ( ).

    Techniques: Transgenic Assay, Mouse Assay, Staining

    The effects of apoE genotype on the levels of Aβ42, phosphorylated tau, VGluT1, and ApoER2 in α-syn expressing and α-syn–deficient female mice. Brains of apoE3 and apoE4 homozygous female mice that express normal levels of α-syn (left panel) or are α-syn–deficient (right panel) were subjected to histologic staining with anti-Aβ42, anti-AT8 mAb, which specifically recognizes the phosphorylated Ser202/Thr205 tau epitope, anti-VGluT1, and anti-ApoER2 Abs, as described in Section 2 . The results (mean ± SEM; n = 5–9 per group) of apoE3 mice (white bars) and apoE4 mice (black bars) were quantified by computerized image analysis and are presented relative to the apoE3 mice of the corresponding α-syn background. This revealed that in α-syn+/+ mice, unlike the α-syn−/− mice, no statistically significant difference between apoE3 and apoE4 mice was observed. Abbreviations: Abs, antibodies; apoE, apolipoprotein E; α-syn, α-synuclein; SEM, standard error of the mean. ∗ P

    Journal: Alzheimer's & Dementia : Diagnosis, Assessment & Disease Monitoring

    Article Title: The effects of apolipoprotein E genotype, α-synuclein deficiency, and sex on brain synaptic and Alzheimer's disease–related pathology

    doi: 10.1016/j.dadm.2017.08.003

    Figure Lengend Snippet: The effects of apoE genotype on the levels of Aβ42, phosphorylated tau, VGluT1, and ApoER2 in α-syn expressing and α-syn–deficient female mice. Brains of apoE3 and apoE4 homozygous female mice that express normal levels of α-syn (left panel) or are α-syn–deficient (right panel) were subjected to histologic staining with anti-Aβ42, anti-AT8 mAb, which specifically recognizes the phosphorylated Ser202/Thr205 tau epitope, anti-VGluT1, and anti-ApoER2 Abs, as described in Section 2 . The results (mean ± SEM; n = 5–9 per group) of apoE3 mice (white bars) and apoE4 mice (black bars) were quantified by computerized image analysis and are presented relative to the apoE3 mice of the corresponding α-syn background. This revealed that in α-syn+/+ mice, unlike the α-syn−/− mice, no statistically significant difference between apoE3 and apoE4 mice was observed. Abbreviations: Abs, antibodies; apoE, apolipoprotein E; α-syn, α-synuclein; SEM, standard error of the mean. ∗ P

    Article Snippet: Gels were then transferred to a nitrocellulose membrane and stained with goat anti-apoE Ab (1:10,000; Millipore) and rabbit anti–α-syn Ab (1:10,000; Abcam).

    Techniques: Expressing, Mouse Assay, Staining

    The effects of apoE genotype on performance of α-syn expressing and α-syn–deficient female mice in the novel object recognition test. ApoE3 and apoE4 homozygous female mice that are α-syn–deficient or express normal levels of α-syn were first exposed to two identical objects, followed by a delay of 24 hours, after which the mice were exposed to an old and a new object. The preference of the mice to the different objects was monitored, as described in Section 2 . The results obtained are depicted as the percent of visits made to the novel object out of the total number of visits to both familiar and novel objects. White bars correspond to apoE3 mice, whereas black bars correspond to apoE4 mice (mean ± SEM; n = 10 mice). As seen, the α-syn–deficient apoE4 mice are unable to discriminate between old and novel objects. Abbreviations: apoE, apolipoprotein E; α-syn, α-synuclein; SEM, standard error of the mean. ∗ P

    Journal: Alzheimer's & Dementia : Diagnosis, Assessment & Disease Monitoring

    Article Title: The effects of apolipoprotein E genotype, α-synuclein deficiency, and sex on brain synaptic and Alzheimer's disease–related pathology

    doi: 10.1016/j.dadm.2017.08.003

    Figure Lengend Snippet: The effects of apoE genotype on performance of α-syn expressing and α-syn–deficient female mice in the novel object recognition test. ApoE3 and apoE4 homozygous female mice that are α-syn–deficient or express normal levels of α-syn were first exposed to two identical objects, followed by a delay of 24 hours, after which the mice were exposed to an old and a new object. The preference of the mice to the different objects was monitored, as described in Section 2 . The results obtained are depicted as the percent of visits made to the novel object out of the total number of visits to both familiar and novel objects. White bars correspond to apoE3 mice, whereas black bars correspond to apoE4 mice (mean ± SEM; n = 10 mice). As seen, the α-syn–deficient apoE4 mice are unable to discriminate between old and novel objects. Abbreviations: apoE, apolipoprotein E; α-syn, α-synuclein; SEM, standard error of the mean. ∗ P

    Article Snippet: Gels were then transferred to a nitrocellulose membrane and stained with goat anti-apoE Ab (1:10,000; Millipore) and rabbit anti–α-syn Ab (1:10,000; Abcam).

    Techniques: Expressing, Mouse Assay

    The levels of apoE and α-syn in the hippocampus of apoE4 and apoE3-TR mice. Hippocampi of 4-month-old male and female apoE3 and apoE4 mice with or without α-syn were excised, homogenized, and subjected to apoE and α-syn immunoblotting and qRT-PCR, as described in Section 2 . Representative apoE, α-syn, and loading standard β-tubulin bands of α-syn–deficient or normal α-syn males (A) and females (B) apoE3 (white bars) and apoE4 (black bars) mice are presented. Quantitations of the apoE protein levels (mean ± SEM; n = 5 per group) are normalized relative to the α-syn−/− apoE3 mice of each sex. As can be seen, the levels of apoE were higher in the apoE3 than the apoE4 mice, regardless of α-syn status or sex. qRT-PCR measurement of α-syn and apoE in males (C) and females (D) show no difference in mRNA levels of α-syn and apoE between the different genotypes (mean ± SEM; n = 4 per group). Abbreviations: apoE, apolipoprotein E; α-syn, α-synuclein; mRNA, messenger RNA; SEM, standard error of the mean. ∗ P

    Journal: Alzheimer's & Dementia : Diagnosis, Assessment & Disease Monitoring

    Article Title: The effects of apolipoprotein E genotype, α-synuclein deficiency, and sex on brain synaptic and Alzheimer's disease–related pathology

    doi: 10.1016/j.dadm.2017.08.003

    Figure Lengend Snippet: The levels of apoE and α-syn in the hippocampus of apoE4 and apoE3-TR mice. Hippocampi of 4-month-old male and female apoE3 and apoE4 mice with or without α-syn were excised, homogenized, and subjected to apoE and α-syn immunoblotting and qRT-PCR, as described in Section 2 . Representative apoE, α-syn, and loading standard β-tubulin bands of α-syn–deficient or normal α-syn males (A) and females (B) apoE3 (white bars) and apoE4 (black bars) mice are presented. Quantitations of the apoE protein levels (mean ± SEM; n = 5 per group) are normalized relative to the α-syn−/− apoE3 mice of each sex. As can be seen, the levels of apoE were higher in the apoE3 than the apoE4 mice, regardless of α-syn status or sex. qRT-PCR measurement of α-syn and apoE in males (C) and females (D) show no difference in mRNA levels of α-syn and apoE between the different genotypes (mean ± SEM; n = 4 per group). Abbreviations: apoE, apolipoprotein E; α-syn, α-synuclein; mRNA, messenger RNA; SEM, standard error of the mean. ∗ P

    Article Snippet: Gels were then transferred to a nitrocellulose membrane and stained with goat anti-apoE Ab (1:10,000; Millipore) and rabbit anti–α-syn Ab (1:10,000; Abcam).

    Techniques: Mouse Assay, Quantitative RT-PCR

    The effects of apoE genotype and α-syn on the lipidation of apoE. Hippocampal homogenates of α-syn–deficient or normal α-syn apoE3 and apoE4 male (A) and female (B) mice were subjected to a blue native gel and stained with anti-apoE Ab as described in Section 2 . Representative immunoblots of three mice per group are presented. As can be seen, the apoE3 mice, both male and female, contain higher levels of high molecular weight apoE and this effect is maintained independently of the presence or absence of α-syn. Abbreviations: Ab, antibody; apoE, apolipoprotein E; α-syn, α-synuclein.

    Journal: Alzheimer's & Dementia : Diagnosis, Assessment & Disease Monitoring

    Article Title: The effects of apolipoprotein E genotype, α-synuclein deficiency, and sex on brain synaptic and Alzheimer's disease–related pathology

    doi: 10.1016/j.dadm.2017.08.003

    Figure Lengend Snippet: The effects of apoE genotype and α-syn on the lipidation of apoE. Hippocampal homogenates of α-syn–deficient or normal α-syn apoE3 and apoE4 male (A) and female (B) mice were subjected to a blue native gel and stained with anti-apoE Ab as described in Section 2 . Representative immunoblots of three mice per group are presented. As can be seen, the apoE3 mice, both male and female, contain higher levels of high molecular weight apoE and this effect is maintained independently of the presence or absence of α-syn. Abbreviations: Ab, antibody; apoE, apolipoprotein E; α-syn, α-synuclein.

    Article Snippet: Gels were then transferred to a nitrocellulose membrane and stained with goat anti-apoE Ab (1:10,000; Millipore) and rabbit anti–α-syn Ab (1:10,000; Abcam).

    Techniques: Mouse Assay, Staining, Western Blot, Molecular Weight

    The effects of apoE on the levels of Aβ42, phosphorylated tau, glial, and vascular marker genotype in α-syn–deficient female mice. Brains of apoE3, apoE4 homozygous, and apoE3/E4 heterozygous female mice were subjected to histologic staining with anti-Aβ42 (A), anti-AT8 mAb (B), which specifically recognizes the phosphorylated Ser202/Thr205 tau epitope, anti-GFAP (C), and anti-collagen IV (D) Abs. Representative images (10 ×magnification) of the CA3 hippocampal subfield are presented for Aβ42, AT8, and GFAP, whereas the collagen IV staining and analysis were performed in the stratum lacunosum molecular area of the hippocampus. The results (mean ± SEM; n = 7–10 per group) of apoE3 mice (white bars), apoE4 mice (black bars), and apoE3/E4 (checkered bars) were quantified by computerized image analysis as described in Section 2 . As seen, the AD-related phenotypes of the apoE4 mice, namely, high levels of Aβ42, tau phosphorylation, and the glial marker GFAP, are not present in the heterozygous mice. The results shown are normalized relative to control apoE3 mice. In contrast, the levels of the vascular marker collagen IV are reduced in both apoE4 and apoE3/E4 mice. Abbreviations: Abs, antibodies; apoE, apolipoprotein E; α-syn, α-synuclein; SEM, standard error of the mean. ∗ P ≤.05 for the effect of genotype on the levels of the markers.

    Journal: Alzheimer's & Dementia : Diagnosis, Assessment & Disease Monitoring

    Article Title: The effects of apolipoprotein E genotype, α-synuclein deficiency, and sex on brain synaptic and Alzheimer's disease–related pathology

    doi: 10.1016/j.dadm.2017.08.003

    Figure Lengend Snippet: The effects of apoE on the levels of Aβ42, phosphorylated tau, glial, and vascular marker genotype in α-syn–deficient female mice. Brains of apoE3, apoE4 homozygous, and apoE3/E4 heterozygous female mice were subjected to histologic staining with anti-Aβ42 (A), anti-AT8 mAb (B), which specifically recognizes the phosphorylated Ser202/Thr205 tau epitope, anti-GFAP (C), and anti-collagen IV (D) Abs. Representative images (10 ×magnification) of the CA3 hippocampal subfield are presented for Aβ42, AT8, and GFAP, whereas the collagen IV staining and analysis were performed in the stratum lacunosum molecular area of the hippocampus. The results (mean ± SEM; n = 7–10 per group) of apoE3 mice (white bars), apoE4 mice (black bars), and apoE3/E4 (checkered bars) were quantified by computerized image analysis as described in Section 2 . As seen, the AD-related phenotypes of the apoE4 mice, namely, high levels of Aβ42, tau phosphorylation, and the glial marker GFAP, are not present in the heterozygous mice. The results shown are normalized relative to control apoE3 mice. In contrast, the levels of the vascular marker collagen IV are reduced in both apoE4 and apoE3/E4 mice. Abbreviations: Abs, antibodies; apoE, apolipoprotein E; α-syn, α-synuclein; SEM, standard error of the mean. ∗ P ≤.05 for the effect of genotype on the levels of the markers.

    Article Snippet: Gels were then transferred to a nitrocellulose membrane and stained with goat anti-apoE Ab (1:10,000; Millipore) and rabbit anti–α-syn Ab (1:10,000; Abcam).

    Techniques: Marker, Mouse Assay, Staining

    The effects of apoE genotype on the levels of the synaptic marker synaptophysin, VGluT1, VGaT, and ApoER2 in α-syn–deficient female mice. Brains of apoE3, apoE4 homozygous, and apoE3/E4 heterozygous female mice were subjected to histologic staining with anti-synaptophysin (A), anti-VGluT1 (B), anti-VGaT (C), and anti-ApoER2 (D) Abs. Representative images (20 ×magnification) of the CA3 hippocampal subfield are presented in the upper part of each panel and show reduced levels in both apoE4 and apoE3/E4 mice compared with apoE3 mice. The results (mean ± SEM; n = 7–10 per group) of apoE3 mice (white bars), apoE4 mice (black bars), and apoE3/E4 (checkered bars) were quantified by computerized image analysis. qRT-PCR of the synaptic parameters (E) show no difference between apoE genotypes in the mRNA expression levels. The results shown were all normalized relative to control apoE3 mice (mean ± SEM; n = 4 per group). Abbreviations: Abs, antibodies; apoE, apolipoprotein E; α-syn, α-synuclein; mRNA, messenger RNA; SEM, standard error of the mean. ∗ P

    Journal: Alzheimer's & Dementia : Diagnosis, Assessment & Disease Monitoring

    Article Title: The effects of apolipoprotein E genotype, α-synuclein deficiency, and sex on brain synaptic and Alzheimer's disease–related pathology

    doi: 10.1016/j.dadm.2017.08.003

    Figure Lengend Snippet: The effects of apoE genotype on the levels of the synaptic marker synaptophysin, VGluT1, VGaT, and ApoER2 in α-syn–deficient female mice. Brains of apoE3, apoE4 homozygous, and apoE3/E4 heterozygous female mice were subjected to histologic staining with anti-synaptophysin (A), anti-VGluT1 (B), anti-VGaT (C), and anti-ApoER2 (D) Abs. Representative images (20 ×magnification) of the CA3 hippocampal subfield are presented in the upper part of each panel and show reduced levels in both apoE4 and apoE3/E4 mice compared with apoE3 mice. The results (mean ± SEM; n = 7–10 per group) of apoE3 mice (white bars), apoE4 mice (black bars), and apoE3/E4 (checkered bars) were quantified by computerized image analysis. qRT-PCR of the synaptic parameters (E) show no difference between apoE genotypes in the mRNA expression levels. The results shown were all normalized relative to control apoE3 mice (mean ± SEM; n = 4 per group). Abbreviations: Abs, antibodies; apoE, apolipoprotein E; α-syn, α-synuclein; mRNA, messenger RNA; SEM, standard error of the mean. ∗ P

    Article Snippet: Gels were then transferred to a nitrocellulose membrane and stained with goat anti-apoE Ab (1:10,000; Millipore) and rabbit anti–α-syn Ab (1:10,000; Abcam).

    Techniques: Marker, Mouse Assay, Staining, Quantitative RT-PCR, Expressing

    Tpr regulates SUMOylation. 5A–5B. Analysis of the overall level of SUMOylation in cells transfected with Tpr, Nup153, SENP2 and mock siRNAs. 5A. Nuclear extracts of siRNAs treated cells were prepared and analyzed by western blotting using the antibodies as described on the left of the figure. RPB1 was used as loading control. 5B. Analysis of RanGAP1 SUMOylation. Cytoplasmic extracts of Tpr, Nup153, SENP2 and mock siRNAs-treated cells were prepared and analyzed by western blotting using an anti-RanGAP1 antibody recognizing both SUMOylated and non-SUMOylated forms of RanGAP1 and with tubulin as loading control. 5C: SUMO-1 siRNA treatment overrides Tpr depletion-induced senescence in HeLa cells. HeLa cells were transfected with Tpr, SUMO-1, Tpr and SUMO-1 and mock siRNAs. After 6days, the cells were fixed and stained for SA-β-gal activity at pH 6.0. A representative picture of each condition is presented. Scale bar: 15 µm.

    Journal: PLoS ONE

    Article Title: Silencing Nuclear Pore Protein Tpr Elicits a Senescent-Like Phenotype in Cancer Cells

    doi: 10.1371/journal.pone.0022423

    Figure Lengend Snippet: Tpr regulates SUMOylation. 5A–5B. Analysis of the overall level of SUMOylation in cells transfected with Tpr, Nup153, SENP2 and mock siRNAs. 5A. Nuclear extracts of siRNAs treated cells were prepared and analyzed by western blotting using the antibodies as described on the left of the figure. RPB1 was used as loading control. 5B. Analysis of RanGAP1 SUMOylation. Cytoplasmic extracts of Tpr, Nup153, SENP2 and mock siRNAs-treated cells were prepared and analyzed by western blotting using an anti-RanGAP1 antibody recognizing both SUMOylated and non-SUMOylated forms of RanGAP1 and with tubulin as loading control. 5C: SUMO-1 siRNA treatment overrides Tpr depletion-induced senescence in HeLa cells. HeLa cells were transfected with Tpr, SUMO-1, Tpr and SUMO-1 and mock siRNAs. After 6days, the cells were fixed and stained for SA-β-gal activity at pH 6.0. A representative picture of each condition is presented. Scale bar: 15 µm.

    Article Snippet: Antibodies The following antibodies were used: mouse 203-37 (Oncogene research) and 3B9 (Santa Cruz) and rabbit (a gift from L. Gerace) anti-Tpr, mouse (DO1) and rabbit anti-p53, goat anti-Nup133, mouse anti-p16, goat anti-Crm1, rabbit anti-NFκB (Santa Cruz), mouse anti-Nup153 (Progen and SA-1, a gift from B. Burke), mouse anti-emerin (Novacastra), mouse anti-FG nucleoporins Mab414 (Eurogentec and Covance), goat anti-RanGap1 antibody (a gift from F. Melchior), mouse anti-p21 (Calbiochem), rabbit anti-SENP2 (Abgent and Calbiochem), mouse anti-GMP-1 (anti-SUMO-1, Invitrogen), mouse anti-Rb (BD Pharmingen), rabbit anti-TRF2 (Novus biologicals), rabbit anti-histone macroH2A1 (Active Motif), mouse anti-HP1γ and mouse anti-RPB1(Euromedex), mouse anti-actin AC-40 and anti-α tubulin DM1A (SIGMA).

    Techniques: Transfection, Western Blot, Staining, Activity Assay

    Uncrossed inferior olivary axons cause ataxic gait. (A) Ptf1a::cre;Robo3 lox/lox mice are severely ataxic, which is demonstrated by a very short latency to fall in the Rotarod test ( n = 7 mutants versus n = 9 for controls). (B) Image of a 4-mo-old Ptf1a::cre;Robo3 lox/lox mouse displaying an ataxic gait. (C) The step time of Ptf1a::cre;Robo3 lox/lox mice (light blue; n = 6) on the Erasmus Ladder is longer than in controls (violet and red; n = 4; p

    Journal: PLoS Biology

    Article Title: Genetic Dissection of the Function of Hindbrain Axonal Commissures

    doi: 10.1371/journal.pbio.1000325

    Figure Lengend Snippet: Uncrossed inferior olivary axons cause ataxic gait. (A) Ptf1a::cre;Robo3 lox/lox mice are severely ataxic, which is demonstrated by a very short latency to fall in the Rotarod test ( n = 7 mutants versus n = 9 for controls). (B) Image of a 4-mo-old Ptf1a::cre;Robo3 lox/lox mouse displaying an ataxic gait. (C) The step time of Ptf1a::cre;Robo3 lox/lox mice (light blue; n = 6) on the Erasmus Ladder is longer than in controls (violet and red; n = 4; p

    Article Snippet: Lee, Philadelphia, PA), mouse anti-Islet1 (1∶100, Developmental Studies Hybridoma Bank, University of Iowa), mouse anti-parvalbumin (1∶1,000, Swant), mouse anti-Robo3 (1∶200, R & D which recognizes the N-terminal domain of Robo3), guinea pig anti-VGLUT2 (1∶1,000, Millipore), rabbit anti-Hb9 (1∶1,000, Abcam), rabbit anti-CGRP (1∶1,000, Peninsula), rabbit anti-GFP (1∶300, Invitrogen), rabbit anti-Tag1 (1∶3,000, gift from Dr. Domna Karagogeos, University of Crete Medical School, Heraklion, Greece), rabbit anti-CaBP (1∶5,000, Swant), rabbit anti-βGal (1∶1,000, Cappel), goat anti-mouse Alcam (1∶200, R & D), goat anti-Brn3.2 (1∶200, Santa Cruz Biotechnology), goat anti-Robo3 (1∶300, R & D), goat anti-ChAT (1∶400, Millipore), and chicken anti-GFP (1∶800, Abcam).

    Techniques: Mouse Assay

    Rhombomere-specific deletion of Robo3. Whole-mount control (A, C, and E) or Krox20::cre;Robo3 lox/lox (B, D, and F) E12 embryos hybridized with a Robo3 riboprobe covering exons 12–14 (A and B) or immunostained with anti-Robo3 (C and D) or anti-neurofilament (E and F) antibodies. (B) In Krox20::cre;Robo3 lox/lox embryos, Robo3 exon12-14 transcripts are not expressed in rhombomeres 3 (r3) and 5 (r5). (C and D) Likewise, there is a severe reduction of Robo3 immunoreactive commissural axons in r3 and r5. (E and F) Anti-neurofilament immunostaining confirms the strong reduction of commissures in r3 and r5. The arrowheads in (F) indicate axons that abnormally follow the midline. Scale bars represent 100 µm.

    Journal: PLoS Biology

    Article Title: Genetic Dissection of the Function of Hindbrain Axonal Commissures

    doi: 10.1371/journal.pbio.1000325

    Figure Lengend Snippet: Rhombomere-specific deletion of Robo3. Whole-mount control (A, C, and E) or Krox20::cre;Robo3 lox/lox (B, D, and F) E12 embryos hybridized with a Robo3 riboprobe covering exons 12–14 (A and B) or immunostained with anti-Robo3 (C and D) or anti-neurofilament (E and F) antibodies. (B) In Krox20::cre;Robo3 lox/lox embryos, Robo3 exon12-14 transcripts are not expressed in rhombomeres 3 (r3) and 5 (r5). (C and D) Likewise, there is a severe reduction of Robo3 immunoreactive commissural axons in r3 and r5. (E and F) Anti-neurofilament immunostaining confirms the strong reduction of commissures in r3 and r5. The arrowheads in (F) indicate axons that abnormally follow the midline. Scale bars represent 100 µm.

    Article Snippet: Lee, Philadelphia, PA), mouse anti-Islet1 (1∶100, Developmental Studies Hybridoma Bank, University of Iowa), mouse anti-parvalbumin (1∶1,000, Swant), mouse anti-Robo3 (1∶200, R & D which recognizes the N-terminal domain of Robo3), guinea pig anti-VGLUT2 (1∶1,000, Millipore), rabbit anti-Hb9 (1∶1,000, Abcam), rabbit anti-CGRP (1∶1,000, Peninsula), rabbit anti-GFP (1∶300, Invitrogen), rabbit anti-Tag1 (1∶3,000, gift from Dr. Domna Karagogeos, University of Crete Medical School, Heraklion, Greece), rabbit anti-CaBP (1∶5,000, Swant), rabbit anti-βGal (1∶1,000, Cappel), goat anti-mouse Alcam (1∶200, R & D), goat anti-Brn3.2 (1∶200, Santa Cruz Biotechnology), goat anti-Robo3 (1∶300, R & D), goat anti-ChAT (1∶400, Millipore), and chicken anti-GFP (1∶800, Abcam).

    Techniques: Immunostaining

    Uncrossed aVCN-MNTB projections and abnormal ABRs in Krox20::cre;Robo3 lox/lox mice. (A to C) Robo3 is expressed by neurons of the cochlear nucleus (CN). Coronal sections of a E14 embryo (A) or side view of whole-mount E14 embryos (B and C) hybridized with a Robo3 probe (A and B) or labeled with anti-Robo3 antibodies (C). The arrowheads in (B) indicate migrating pontine neurons. The arrow in (C) points to cochlear axons. (D–G) Coronal sections of P12 mice injected with DiI in the cochlear nucleus (Hoechst counterstaining). In control (D and E), DiI-labeled axons end in the ipsilateral superior olive (SO) and the contralateral MNTB, whereas in Krox20::cre;Robo3 lox/lox mutant (F and G), all axons project ipsilaterally. The arrow in (D and F) indicates the midline. (E and G) are high magnification pictures of the MNTB showing DiI-labeled calyces of Held (arrows). (H) ABRs collected from the ipsilateral or contralateral mastoid electrode in response to 60 dB SPL clicks. Ipsilaterally, only three waves were observed in mutant instead of four in controls. Mutant wave III exhibited a mean latency of about 4.3 ms, much longer than that of control wave III (3.6 ms; n = 13 controls versus n = 12 mutants, average latency difference 0.77 ms for wave III; p

    Journal: PLoS Biology

    Article Title: Genetic Dissection of the Function of Hindbrain Axonal Commissures

    doi: 10.1371/journal.pbio.1000325

    Figure Lengend Snippet: Uncrossed aVCN-MNTB projections and abnormal ABRs in Krox20::cre;Robo3 lox/lox mice. (A to C) Robo3 is expressed by neurons of the cochlear nucleus (CN). Coronal sections of a E14 embryo (A) or side view of whole-mount E14 embryos (B and C) hybridized with a Robo3 probe (A and B) or labeled with anti-Robo3 antibodies (C). The arrowheads in (B) indicate migrating pontine neurons. The arrow in (C) points to cochlear axons. (D–G) Coronal sections of P12 mice injected with DiI in the cochlear nucleus (Hoechst counterstaining). In control (D and E), DiI-labeled axons end in the ipsilateral superior olive (SO) and the contralateral MNTB, whereas in Krox20::cre;Robo3 lox/lox mutant (F and G), all axons project ipsilaterally. The arrow in (D and F) indicates the midline. (E and G) are high magnification pictures of the MNTB showing DiI-labeled calyces of Held (arrows). (H) ABRs collected from the ipsilateral or contralateral mastoid electrode in response to 60 dB SPL clicks. Ipsilaterally, only three waves were observed in mutant instead of four in controls. Mutant wave III exhibited a mean latency of about 4.3 ms, much longer than that of control wave III (3.6 ms; n = 13 controls versus n = 12 mutants, average latency difference 0.77 ms for wave III; p

    Article Snippet: Lee, Philadelphia, PA), mouse anti-Islet1 (1∶100, Developmental Studies Hybridoma Bank, University of Iowa), mouse anti-parvalbumin (1∶1,000, Swant), mouse anti-Robo3 (1∶200, R & D which recognizes the N-terminal domain of Robo3), guinea pig anti-VGLUT2 (1∶1,000, Millipore), rabbit anti-Hb9 (1∶1,000, Abcam), rabbit anti-CGRP (1∶1,000, Peninsula), rabbit anti-GFP (1∶300, Invitrogen), rabbit anti-Tag1 (1∶3,000, gift from Dr. Domna Karagogeos, University of Crete Medical School, Heraklion, Greece), rabbit anti-CaBP (1∶5,000, Swant), rabbit anti-βGal (1∶1,000, Cappel), goat anti-mouse Alcam (1∶200, R & D), goat anti-Brn3.2 (1∶200, Santa Cruz Biotechnology), goat anti-Robo3 (1∶300, R & D), goat anti-ChAT (1∶400, Millipore), and chicken anti-GFP (1∶800, Abcam).

    Techniques: Mouse Assay, Labeling, Injection, Mutagenesis, Mass Spectrometry

    Impaired horizontal compensatory eye movements in Krox20::cre;Robo3 lox/lox mice. (A) During optokinetic stimulation horizontal gains are reduced most prominently at the lower frequencies in in Krox20::cre;Robo3 lox/lox mice ( p = 0.043 ANOVA; n = 6 versus n = 4 for controls; see Table S1 ). (B) At higher frequencies, the VOR is severely impaired ( p = 0.001 versus control mice curve; ANOVA for repeated measurements; Table S1 ), confirming the importance of the commissural connections in large-amplitude eye movements. (C) When horizontal visual and vestibular inputs are combined in the VVOR (visual vestibulo-ocular reflex), it results in lower gains over the entire range of frequencies tested ( p = 0.004 versus control mice curve; ANOVA for repeated measurements). (D) OKR deficits are strongly correlated to the amplitude of stimulation. (E–G) In marked contrast, in the vertical plane, no significant differences were observed in OKR (E), VOR (F), or VVOR (G), supporting the concept that primarily horizontal eye movements require the presence of commissural connections (see Table S1 for all statistics). Error bars indicate standard error of the mean. Results were obtained from four control and six Krox20::cre;Robo3 lox/lox mice .

    Journal: PLoS Biology

    Article Title: Genetic Dissection of the Function of Hindbrain Axonal Commissures

    doi: 10.1371/journal.pbio.1000325

    Figure Lengend Snippet: Impaired horizontal compensatory eye movements in Krox20::cre;Robo3 lox/lox mice. (A) During optokinetic stimulation horizontal gains are reduced most prominently at the lower frequencies in in Krox20::cre;Robo3 lox/lox mice ( p = 0.043 ANOVA; n = 6 versus n = 4 for controls; see Table S1 ). (B) At higher frequencies, the VOR is severely impaired ( p = 0.001 versus control mice curve; ANOVA for repeated measurements; Table S1 ), confirming the importance of the commissural connections in large-amplitude eye movements. (C) When horizontal visual and vestibular inputs are combined in the VVOR (visual vestibulo-ocular reflex), it results in lower gains over the entire range of frequencies tested ( p = 0.004 versus control mice curve; ANOVA for repeated measurements). (D) OKR deficits are strongly correlated to the amplitude of stimulation. (E–G) In marked contrast, in the vertical plane, no significant differences were observed in OKR (E), VOR (F), or VVOR (G), supporting the concept that primarily horizontal eye movements require the presence of commissural connections (see Table S1 for all statistics). Error bars indicate standard error of the mean. Results were obtained from four control and six Krox20::cre;Robo3 lox/lox mice .

    Article Snippet: Lee, Philadelphia, PA), mouse anti-Islet1 (1∶100, Developmental Studies Hybridoma Bank, University of Iowa), mouse anti-parvalbumin (1∶1,000, Swant), mouse anti-Robo3 (1∶200, R & D which recognizes the N-terminal domain of Robo3), guinea pig anti-VGLUT2 (1∶1,000, Millipore), rabbit anti-Hb9 (1∶1,000, Abcam), rabbit anti-CGRP (1∶1,000, Peninsula), rabbit anti-GFP (1∶300, Invitrogen), rabbit anti-Tag1 (1∶3,000, gift from Dr. Domna Karagogeos, University of Crete Medical School, Heraklion, Greece), rabbit anti-CaBP (1∶5,000, Swant), rabbit anti-βGal (1∶1,000, Cappel), goat anti-mouse Alcam (1∶200, R & D), goat anti-Brn3.2 (1∶200, Santa Cruz Biotechnology), goat anti-Robo3 (1∶300, R & D), goat anti-ChAT (1∶400, Millipore), and chicken anti-GFP (1∶800, Abcam).

    Techniques: Mouse Assay

    Analysis of Ptf1a::cre;Robo3 lox/lox ;Tau mGFP mice. (A and B) Coronal sections of the hindbrain at the level of inferior olive of E16 Ptf1a::cre;Tau mGFP embryo labeled with anti-βgal and Brn3.2 antibodies. Some Brn3.2-positive neurons in the inferior olive do not express the βgal (arrowheads and inset). (C–L) Coronal sections of the spinal cord at the level of the forelimbs in E13 Ptf1a::cre;Robo3 lox/+ ;Tau mGFP (C–E, K, and L) or Ptf1a::cre;Robo3 lox/lox ;Tau mGFP (F–H, I, and J) embryos labeled with anti-GFP and anti-Robo3 antibodies. Most GFP-positive axons are in the dorsal spinal cord, and only a small subset of GFP-positive axons (short arrows) cross the floor plate in Ptf1a::cre;Robo3 lox/+ ;Tau mGFP (C and K) but do not express Robo3 (E and L). This subset of GFP-positive commissural axons is still observed in Ptf1a::cre;Robo3 lox/lox ;Tau mGFP embryos (F and J). Scale bars represent 100 µm except in (B and I–L), where they indicate 50 µm.

    Journal: PLoS Biology

    Article Title: Genetic Dissection of the Function of Hindbrain Axonal Commissures

    doi: 10.1371/journal.pbio.1000325

    Figure Lengend Snippet: Analysis of Ptf1a::cre;Robo3 lox/lox ;Tau mGFP mice. (A and B) Coronal sections of the hindbrain at the level of inferior olive of E16 Ptf1a::cre;Tau mGFP embryo labeled with anti-βgal and Brn3.2 antibodies. Some Brn3.2-positive neurons in the inferior olive do not express the βgal (arrowheads and inset). (C–L) Coronal sections of the spinal cord at the level of the forelimbs in E13 Ptf1a::cre;Robo3 lox/+ ;Tau mGFP (C–E, K, and L) or Ptf1a::cre;Robo3 lox/lox ;Tau mGFP (F–H, I, and J) embryos labeled with anti-GFP and anti-Robo3 antibodies. Most GFP-positive axons are in the dorsal spinal cord, and only a small subset of GFP-positive axons (short arrows) cross the floor plate in Ptf1a::cre;Robo3 lox/+ ;Tau mGFP (C and K) but do not express Robo3 (E and L). This subset of GFP-positive commissural axons is still observed in Ptf1a::cre;Robo3 lox/lox ;Tau mGFP embryos (F and J). Scale bars represent 100 µm except in (B and I–L), where they indicate 50 µm.

    Article Snippet: Lee, Philadelphia, PA), mouse anti-Islet1 (1∶100, Developmental Studies Hybridoma Bank, University of Iowa), mouse anti-parvalbumin (1∶1,000, Swant), mouse anti-Robo3 (1∶200, R & D which recognizes the N-terminal domain of Robo3), guinea pig anti-VGLUT2 (1∶1,000, Millipore), rabbit anti-Hb9 (1∶1,000, Abcam), rabbit anti-CGRP (1∶1,000, Peninsula), rabbit anti-GFP (1∶300, Invitrogen), rabbit anti-Tag1 (1∶3,000, gift from Dr. Domna Karagogeos, University of Crete Medical School, Heraklion, Greece), rabbit anti-CaBP (1∶5,000, Swant), rabbit anti-βGal (1∶1,000, Cappel), goat anti-mouse Alcam (1∶200, R & D), goat anti-Brn3.2 (1∶200, Santa Cruz Biotechnology), goat anti-Robo3 (1∶300, R & D), goat anti-ChAT (1∶400, Millipore), and chicken anti-GFP (1∶800, Abcam).

    Techniques: Mouse Assay, Labeling

    Normal projection of abducens motor axons in Robo3-deficient mice. (A to I) show coronal hindbrain sections at the level of the abducens nucleus. (A and B) Robo3 transcripts are detected in the abducens nuclei of E12 (A) and E14 (B) embryos. The arrow in (B) points to the facial nerve. (C) abducens neurons coexpress BEN and GFP in Robo3 +/− E14 embryo. The floor plate (asterisk) also expresses BEN. (D) Hb9/GFP double labeling in E15 Robo3 +/− embryo. Note that some GFP+ cells (arrowhead) do not express Hb9. (E) abducens neurons (VI) and axons (arrow) express GFP in E14 Hb9::GFP transgenic embryo. (F–I) The abducens nucleus has an abnormal shape and is closer to the floor plate in Robo3 −/− (F) and Krox20::cre;Robo3 lox/lox (G) E14 embryos (compare with [E]). The arrow in (F) points to abducens axons. (H and I) This abnormal shape and position are also observed in adult animals with ChAT/Hb9 double immunostaining. (J to L) Coronal sections of P0 mouse head at eye cup (ec) level. GFP-positive abducens axons (arrows) still contact the lateral rectus muscle in Robo3 -null embryo (K) and Krox20::cre;Robo3 lox/lox mutant (L). Scale bars represent 100 µm, except in (D), where it indicates 50 µm.

    Journal: PLoS Biology

    Article Title: Genetic Dissection of the Function of Hindbrain Axonal Commissures

    doi: 10.1371/journal.pbio.1000325

    Figure Lengend Snippet: Normal projection of abducens motor axons in Robo3-deficient mice. (A to I) show coronal hindbrain sections at the level of the abducens nucleus. (A and B) Robo3 transcripts are detected in the abducens nuclei of E12 (A) and E14 (B) embryos. The arrow in (B) points to the facial nerve. (C) abducens neurons coexpress BEN and GFP in Robo3 +/− E14 embryo. The floor plate (asterisk) also expresses BEN. (D) Hb9/GFP double labeling in E15 Robo3 +/− embryo. Note that some GFP+ cells (arrowhead) do not express Hb9. (E) abducens neurons (VI) and axons (arrow) express GFP in E14 Hb9::GFP transgenic embryo. (F–I) The abducens nucleus has an abnormal shape and is closer to the floor plate in Robo3 −/− (F) and Krox20::cre;Robo3 lox/lox (G) E14 embryos (compare with [E]). The arrow in (F) points to abducens axons. (H and I) This abnormal shape and position are also observed in adult animals with ChAT/Hb9 double immunostaining. (J to L) Coronal sections of P0 mouse head at eye cup (ec) level. GFP-positive abducens axons (arrows) still contact the lateral rectus muscle in Robo3 -null embryo (K) and Krox20::cre;Robo3 lox/lox mutant (L). Scale bars represent 100 µm, except in (D), where it indicates 50 µm.

    Article Snippet: Lee, Philadelphia, PA), mouse anti-Islet1 (1∶100, Developmental Studies Hybridoma Bank, University of Iowa), mouse anti-parvalbumin (1∶1,000, Swant), mouse anti-Robo3 (1∶200, R & D which recognizes the N-terminal domain of Robo3), guinea pig anti-VGLUT2 (1∶1,000, Millipore), rabbit anti-Hb9 (1∶1,000, Abcam), rabbit anti-CGRP (1∶1,000, Peninsula), rabbit anti-GFP (1∶300, Invitrogen), rabbit anti-Tag1 (1∶3,000, gift from Dr. Domna Karagogeos, University of Crete Medical School, Heraklion, Greece), rabbit anti-CaBP (1∶5,000, Swant), rabbit anti-βGal (1∶1,000, Cappel), goat anti-mouse Alcam (1∶200, R & D), goat anti-Brn3.2 (1∶200, Santa Cruz Biotechnology), goat anti-Robo3 (1∶300, R & D), goat anti-ChAT (1∶400, Millipore), and chicken anti-GFP (1∶800, Abcam).

    Techniques: Mouse Assay, Labeling, Transgenic Assay, Double Immunostaining, Mutagenesis

    Rhombomere-specific deletion of commissures. Coronal sections at the level of r3, r5, and r6 of E11 control (A–C′) or Krox20::cre;Robo3 lox/lox (D–F′) embryos immunostained with antibodies against Robo3, neurofilament, and Hb9. The midline is indicated by an arrow, and the Hb9-positive abducens motor neurons by an asterisk. In r3 and r5, there is an almost complete absence of Robo3 and neurofilament-positive commissural axons in Krox20::cre;Robo3 lox/lox (D–E′) compared to controls (A–B′). The density of neurofilament-positive axons is also strongly reduced. By contrast, there is no obvious reduction of commissural axons or Robo3 expression in r6 (C, C′, F, and F′). Note that at E11, abducens shape and position are not altered in mutants compared to controls (see Figure 3 for later stages). VII, migrating facial neurons and facial nerve. Scale bars represent 60 µm.

    Journal: PLoS Biology

    Article Title: Genetic Dissection of the Function of Hindbrain Axonal Commissures

    doi: 10.1371/journal.pbio.1000325

    Figure Lengend Snippet: Rhombomere-specific deletion of commissures. Coronal sections at the level of r3, r5, and r6 of E11 control (A–C′) or Krox20::cre;Robo3 lox/lox (D–F′) embryos immunostained with antibodies against Robo3, neurofilament, and Hb9. The midline is indicated by an arrow, and the Hb9-positive abducens motor neurons by an asterisk. In r3 and r5, there is an almost complete absence of Robo3 and neurofilament-positive commissural axons in Krox20::cre;Robo3 lox/lox (D–E′) compared to controls (A–B′). The density of neurofilament-positive axons is also strongly reduced. By contrast, there is no obvious reduction of commissural axons or Robo3 expression in r6 (C, C′, F, and F′). Note that at E11, abducens shape and position are not altered in mutants compared to controls (see Figure 3 for later stages). VII, migrating facial neurons and facial nerve. Scale bars represent 60 µm.

    Article Snippet: Lee, Philadelphia, PA), mouse anti-Islet1 (1∶100, Developmental Studies Hybridoma Bank, University of Iowa), mouse anti-parvalbumin (1∶1,000, Swant), mouse anti-Robo3 (1∶200, R & D which recognizes the N-terminal domain of Robo3), guinea pig anti-VGLUT2 (1∶1,000, Millipore), rabbit anti-Hb9 (1∶1,000, Abcam), rabbit anti-CGRP (1∶1,000, Peninsula), rabbit anti-GFP (1∶300, Invitrogen), rabbit anti-Tag1 (1∶3,000, gift from Dr. Domna Karagogeos, University of Crete Medical School, Heraklion, Greece), rabbit anti-CaBP (1∶5,000, Swant), rabbit anti-βGal (1∶1,000, Cappel), goat anti-mouse Alcam (1∶200, R & D), goat anti-Brn3.2 (1∶200, Santa Cruz Biotechnology), goat anti-Robo3 (1∶300, R & D), goat anti-ChAT (1∶400, Millipore), and chicken anti-GFP (1∶800, Abcam).

    Techniques: Expressing

    Reduced internuclear commissure in Robo3 knockout mice. (A to I) show coronal hindbrain sections at the level of the abducens nucleus, visualized by Hb9 immunostaining (in A, D, E, F, and H). (A, B, and C) illustrate the projection of abducens axons (arrowheads) across the midline (dashed line) in Robo3 +/− E13 embryos. Some GFP+ axons originate from the abducens nucleus (VI) and are immunoreactive for Robo3 (B and C). (D) The internuclear commissure (arrowhead) is also observed in P0 controls, following DiI injection at the level of the oculomotor nucleus III. (E) This commissure is almost completely absent in P0 Krox20::cre;Robo3 lox/lox mice (arrowhead). (VIIn): Genu of facial nerve. (F to I) At E15, many neurofilament+ axons cross the midline at the VI level (arrowheads) in control embryo (F and G), whereas they are rare in Krox20::cre;Robo3 lox/lox embryo (H and I). Scale bars represent 100 µm, except in (G and I), where they indicate 50 µm.

    Journal: PLoS Biology

    Article Title: Genetic Dissection of the Function of Hindbrain Axonal Commissures

    doi: 10.1371/journal.pbio.1000325

    Figure Lengend Snippet: Reduced internuclear commissure in Robo3 knockout mice. (A to I) show coronal hindbrain sections at the level of the abducens nucleus, visualized by Hb9 immunostaining (in A, D, E, F, and H). (A, B, and C) illustrate the projection of abducens axons (arrowheads) across the midline (dashed line) in Robo3 +/− E13 embryos. Some GFP+ axons originate from the abducens nucleus (VI) and are immunoreactive for Robo3 (B and C). (D) The internuclear commissure (arrowhead) is also observed in P0 controls, following DiI injection at the level of the oculomotor nucleus III. (E) This commissure is almost completely absent in P0 Krox20::cre;Robo3 lox/lox mice (arrowhead). (VIIn): Genu of facial nerve. (F to I) At E15, many neurofilament+ axons cross the midline at the VI level (arrowheads) in control embryo (F and G), whereas they are rare in Krox20::cre;Robo3 lox/lox embryo (H and I). Scale bars represent 100 µm, except in (G and I), where they indicate 50 µm.

    Article Snippet: Lee, Philadelphia, PA), mouse anti-Islet1 (1∶100, Developmental Studies Hybridoma Bank, University of Iowa), mouse anti-parvalbumin (1∶1,000, Swant), mouse anti-Robo3 (1∶200, R & D which recognizes the N-terminal domain of Robo3), guinea pig anti-VGLUT2 (1∶1,000, Millipore), rabbit anti-Hb9 (1∶1,000, Abcam), rabbit anti-CGRP (1∶1,000, Peninsula), rabbit anti-GFP (1∶300, Invitrogen), rabbit anti-Tag1 (1∶3,000, gift from Dr. Domna Karagogeos, University of Crete Medical School, Heraklion, Greece), rabbit anti-CaBP (1∶5,000, Swant), rabbit anti-βGal (1∶1,000, Cappel), goat anti-mouse Alcam (1∶200, R & D), goat anti-Brn3.2 (1∶200, Santa Cruz Biotechnology), goat anti-Robo3 (1∶300, R & D), goat anti-ChAT (1∶400, Millipore), and chicken anti-GFP (1∶800, Abcam).

    Techniques: Knock-Out, Mouse Assay, Immunostaining, Injection

    Neuropsin cleaves EphB2 and regulates its expression both in vitro and in the amygdala after stress ( a, b ) EphB2-S band density in SH-SY5Y cells decreased upon 15 min neuropsin treatment (F (3,18) =11.24; p

    Journal: Nature

    Article Title: Neuropsin cleaves EphB2 in the amygdala to control anxiety

    doi: 10.1038/nature09938

    Figure Lengend Snippet: Neuropsin cleaves EphB2 and regulates its expression both in vitro and in the amygdala after stress ( a, b ) EphB2-S band density in SH-SY5Y cells decreased upon 15 min neuropsin treatment (F (3,18) =11.24; p

    Article Snippet: Slices were preincubated in PBS-T (PBS solution 0.5% bovine serum albumin, 0.02% Triton X-100 and blocking sera at 1:500) for 5 hours at room temperature, incubated with goat anti-EphB2 (1:300, R & D) or rabbit anti-neuropsin (1:200, Dr. Helena Castro; the antibody was preabsorbed on acetone powder prepared from neuropsin−/− brain for 1 hour at RT prior to use) antibodies, along with mouse anti-NeuN (1:200, Chemicon) and chicken anti-GFAP (1:1000, Dako) overnight at 4°C in PBS-T. Next, the slices were washed for 8-10 hours with PBS-T and incubated overnight with compatible FITC, Alexa Fluor 488, Alexa Fluor 546 or Alexa Fluor 647 secondary antibodies (1:500, Molecular Probes) in the same buffer.

    Techniques: Expressing, In Vitro

    Neuropsin and EphB2 colocalize in neurons of the basolateral complex of the amygdala ( a ) Double immunohistochemistry showed neuropsin (green) and EphB2 (red) co-localize in lateral amygdala neurons (arrows show EphB2-rich clusters at neuropsin detection sites). Cells were highlighted with TOTO-3 stain. ( b ) Triple immunohistochemistry confirmed the presence of neuropsin/EphB2-rich clusters on the neuronal surface and low degree of co-localization with cytoplasmic Fkbp51. ( c, d ) Western blotting revealed amydala neuropsin upregulation following 6 hour restraint stress (F (2, 7) =8.81; p

    Journal: Nature

    Article Title: Neuropsin cleaves EphB2 in the amygdala to control anxiety

    doi: 10.1038/nature09938

    Figure Lengend Snippet: Neuropsin and EphB2 colocalize in neurons of the basolateral complex of the amygdala ( a ) Double immunohistochemistry showed neuropsin (green) and EphB2 (red) co-localize in lateral amygdala neurons (arrows show EphB2-rich clusters at neuropsin detection sites). Cells were highlighted with TOTO-3 stain. ( b ) Triple immunohistochemistry confirmed the presence of neuropsin/EphB2-rich clusters on the neuronal surface and low degree of co-localization with cytoplasmic Fkbp51. ( c, d ) Western blotting revealed amydala neuropsin upregulation following 6 hour restraint stress (F (2, 7) =8.81; p

    Article Snippet: Slices were preincubated in PBS-T (PBS solution 0.5% bovine serum albumin, 0.02% Triton X-100 and blocking sera at 1:500) for 5 hours at room temperature, incubated with goat anti-EphB2 (1:300, R & D) or rabbit anti-neuropsin (1:200, Dr. Helena Castro; the antibody was preabsorbed on acetone powder prepared from neuropsin−/− brain for 1 hour at RT prior to use) antibodies, along with mouse anti-NeuN (1:200, Chemicon) and chicken anti-GFAP (1:1000, Dako) overnight at 4°C in PBS-T. Next, the slices were washed for 8-10 hours with PBS-T and incubated overnight with compatible FITC, Alexa Fluor 488, Alexa Fluor 546 or Alexa Fluor 647 secondary antibodies (1:500, Molecular Probes) in the same buffer.

    Techniques: Immunohistochemistry, Staining, Western Blot

    Neuropsin regulates the dynamics of EphB2/ NR1 interaction and controls the expression of Fkbp5 ( a, b ) EphB2 immunoprecipitation (before and after 15 minutes of restraint stress) from amygdalae revealed dissociation of EphB2/NR1 complexes in wild-type (F (3, 19) =4.2; p

    Journal: Nature

    Article Title: Neuropsin cleaves EphB2 in the amygdala to control anxiety

    doi: 10.1038/nature09938

    Figure Lengend Snippet: Neuropsin regulates the dynamics of EphB2/ NR1 interaction and controls the expression of Fkbp5 ( a, b ) EphB2 immunoprecipitation (before and after 15 minutes of restraint stress) from amygdalae revealed dissociation of EphB2/NR1 complexes in wild-type (F (3, 19) =4.2; p

    Article Snippet: Slices were preincubated in PBS-T (PBS solution 0.5% bovine serum albumin, 0.02% Triton X-100 and blocking sera at 1:500) for 5 hours at room temperature, incubated with goat anti-EphB2 (1:300, R & D) or rabbit anti-neuropsin (1:200, Dr. Helena Castro; the antibody was preabsorbed on acetone powder prepared from neuropsin−/− brain for 1 hour at RT prior to use) antibodies, along with mouse anti-NeuN (1:200, Chemicon) and chicken anti-GFAP (1:1000, Dako) overnight at 4°C in PBS-T. Next, the slices were washed for 8-10 hours with PBS-T and incubated overnight with compatible FITC, Alexa Fluor 488, Alexa Fluor 546 or Alexa Fluor 647 secondary antibodies (1:500, Molecular Probes) in the same buffer.

    Techniques: Expressing, Immunoprecipitation

    Neuropsin controls NMDA receptor current, E-LTP and stress-induced anxiety ( a-c ) Whole-cell recordings from basal nucleus neurons of neuropsin −/− animals demonstrated lower NMDA currents compared to wild-type mice. Induction of LTP in the lateral-basal pathway ( d ) using a strong ( e ) or weak ( f ) protocol revealed an impairment of E-LTP in neuropsin−/− mice. The elevated plus maze test following acute or chronic restraint stress demonstrated lack of anxiety in neuropsin −/− mice as indicated by the number of entries into open arms ( g ). General locomotor activity was similar in both genotypes ( h, i ). The behavioural phenotype was reversed by bilaterally injecting neuropsin back into the amygdala of neuropsin−/− mice ( j ). Stress-induced anxiety in wild-type animals was disrupted by blocking EphB2 ( k ) or silencing the Fkbp5 gene ( l ) in the amygdala. NP – neuropsin; LA-lateral amygdala; BLA – basal amygdala; CA – central amygdala; MeA – medial amygdala. Digits inside columns or near symbols indicate n number. 6hS – six hour stress, 21dS – 21 days of daily restraint. *p

    Journal: Nature

    Article Title: Neuropsin cleaves EphB2 in the amygdala to control anxiety

    doi: 10.1038/nature09938

    Figure Lengend Snippet: Neuropsin controls NMDA receptor current, E-LTP and stress-induced anxiety ( a-c ) Whole-cell recordings from basal nucleus neurons of neuropsin −/− animals demonstrated lower NMDA currents compared to wild-type mice. Induction of LTP in the lateral-basal pathway ( d ) using a strong ( e ) or weak ( f ) protocol revealed an impairment of E-LTP in neuropsin−/− mice. The elevated plus maze test following acute or chronic restraint stress demonstrated lack of anxiety in neuropsin −/− mice as indicated by the number of entries into open arms ( g ). General locomotor activity was similar in both genotypes ( h, i ). The behavioural phenotype was reversed by bilaterally injecting neuropsin back into the amygdala of neuropsin−/− mice ( j ). Stress-induced anxiety in wild-type animals was disrupted by blocking EphB2 ( k ) or silencing the Fkbp5 gene ( l ) in the amygdala. NP – neuropsin; LA-lateral amygdala; BLA – basal amygdala; CA – central amygdala; MeA – medial amygdala. Digits inside columns or near symbols indicate n number. 6hS – six hour stress, 21dS – 21 days of daily restraint. *p

    Article Snippet: Slices were preincubated in PBS-T (PBS solution 0.5% bovine serum albumin, 0.02% Triton X-100 and blocking sera at 1:500) for 5 hours at room temperature, incubated with goat anti-EphB2 (1:300, R & D) or rabbit anti-neuropsin (1:200, Dr. Helena Castro; the antibody was preabsorbed on acetone powder prepared from neuropsin−/− brain for 1 hour at RT prior to use) antibodies, along with mouse anti-NeuN (1:200, Chemicon) and chicken anti-GFAP (1:1000, Dako) overnight at 4°C in PBS-T. Next, the slices were washed for 8-10 hours with PBS-T and incubated overnight with compatible FITC, Alexa Fluor 488, Alexa Fluor 546 or Alexa Fluor 647 secondary antibodies (1:500, Molecular Probes) in the same buffer.

    Techniques: Mouse Assay, Activity Assay, Blocking Assay, Microelectrode Array

    Cell-based calpain cleavage assays. Lysates from MN9D cells overexpressing T7-ASNA1-FLAG ( A ), OPTN-GST ( B ), or T7-PERI-FLAG ( C ) were incubated for 2 h at 37 °C with the indicated calpains (5.55 unit m-calpain or 3.95 units μ-calpain) in

    Journal: The Journal of Biological Chemistry

    Article Title: Gel-based Protease Proteomics for Identifying the Novel Calpain Substrates in Dopaminergic Neuronal Cell *

    doi: 10.1074/jbc.M113.492876

    Figure Lengend Snippet: Cell-based calpain cleavage assays. Lysates from MN9D cells overexpressing T7-ASNA1-FLAG ( A ), OPTN-GST ( B ), or T7-PERI-FLAG ( C ) were incubated for 2 h at 37 °C with the indicated calpains (5.55 unit m-calpain or 3.95 units μ-calpain) in

    Article Snippet: The primary antibodies used were rabbit anti-Bax antibody (1:3,000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), goat anti-ASNA1 antibody (1:1,000; Everest Biotech, Oxfordshire, UK), rabbit anti-optineurin antibody (1:1,000; Cayman Chemical, Ann Arbor, MI), rabbit anti-peripherin antibody (1:1,000; Millipore Corp., Billerica, MA), mouse anti-fodrin antibody (1:4,000; Enzo Life Sciences Inc., Farmingdale, NY), mouse anti-T7 antibody (1:4,000; Novagen, Madison, WI), rabbit anti-GST antibody (1:1,000; Santa Cruz Biotechnology, Inc.), mouse anti-tyrosine hydroxylase antibody (TH; 1:7,500; Pel-Freez Biologicals, Rogers, AR), HRP-conjugated anti-FLAG antibody (Sigma), and HRP-conjugated anti-V5 antibody (Invitrogen).

    Techniques: Incubation

    In vitro calpain cleavage assays. Among the altered protein spots identified as putative calpain substrates, T7-ASNA1-FLAG ( A ), OPTN-GST ( B ), and PERI-Myc ( C ) were radiolabeled with [ 35 S]methionine using an in vitro transcription/translation kit. Reactants

    Journal: The Journal of Biological Chemistry

    Article Title: Gel-based Protease Proteomics for Identifying the Novel Calpain Substrates in Dopaminergic Neuronal Cell *

    doi: 10.1074/jbc.M113.492876

    Figure Lengend Snippet: In vitro calpain cleavage assays. Among the altered protein spots identified as putative calpain substrates, T7-ASNA1-FLAG ( A ), OPTN-GST ( B ), and PERI-Myc ( C ) were radiolabeled with [ 35 S]methionine using an in vitro transcription/translation kit. Reactants

    Article Snippet: The primary antibodies used were rabbit anti-Bax antibody (1:3,000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), goat anti-ASNA1 antibody (1:1,000; Everest Biotech, Oxfordshire, UK), rabbit anti-optineurin antibody (1:1,000; Cayman Chemical, Ann Arbor, MI), rabbit anti-peripherin antibody (1:1,000; Millipore Corp., Billerica, MA), mouse anti-fodrin antibody (1:4,000; Enzo Life Sciences Inc., Farmingdale, NY), mouse anti-T7 antibody (1:4,000; Novagen, Madison, WI), rabbit anti-GST antibody (1:1,000; Santa Cruz Biotechnology, Inc.), mouse anti-tyrosine hydroxylase antibody (TH; 1:7,500; Pel-Freez Biologicals, Rogers, AR), HRP-conjugated anti-FLAG antibody (Sigma), and HRP-conjugated anti-V5 antibody (Invitrogen).

    Techniques: In Vitro

    Overexpression of ASNA1, optineurin, or peripherin renders cell less vulnerable to MPP + treatment. A , cellular lysates obtained from MN9D cells transiently transfected with or without the vector containing T7-ASNA1-FLAG, OPTN-V5, PERI-Myc, or shRNA target

    Journal: The Journal of Biological Chemistry

    Article Title: Gel-based Protease Proteomics for Identifying the Novel Calpain Substrates in Dopaminergic Neuronal Cell *

    doi: 10.1074/jbc.M113.492876

    Figure Lengend Snippet: Overexpression of ASNA1, optineurin, or peripherin renders cell less vulnerable to MPP + treatment. A , cellular lysates obtained from MN9D cells transiently transfected with or without the vector containing T7-ASNA1-FLAG, OPTN-V5, PERI-Myc, or shRNA target

    Article Snippet: The primary antibodies used were rabbit anti-Bax antibody (1:3,000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), goat anti-ASNA1 antibody (1:1,000; Everest Biotech, Oxfordshire, UK), rabbit anti-optineurin antibody (1:1,000; Cayman Chemical, Ann Arbor, MI), rabbit anti-peripherin antibody (1:1,000; Millipore Corp., Billerica, MA), mouse anti-fodrin antibody (1:4,000; Enzo Life Sciences Inc., Farmingdale, NY), mouse anti-T7 antibody (1:4,000; Novagen, Madison, WI), rabbit anti-GST antibody (1:1,000; Santa Cruz Biotechnology, Inc.), mouse anti-tyrosine hydroxylase antibody (TH; 1:7,500; Pel-Freez Biologicals, Rogers, AR), HRP-conjugated anti-FLAG antibody (Sigma), and HRP-conjugated anti-V5 antibody (Invitrogen).

    Techniques: Over Expression, Transfection, Plasmid Preparation, shRNA

    Immunohistochemistry for superoxide dismutase (SOD1) (a-c), manganese superoxide dismutase (SOD2) (d-f), catalase (CAT) (g-i) and glutathione peroxidase (GPx) (j-l) in the rat liver of the normal-(left column), ascorbic acid - (middle column) and Oenanthe javanica extract (OJE) - (right column) groups. Strong SOD1, SOD2, CAT, and GPx immunoreactive cells (arrows) are markedly increased in the OJE-group. Scale bar = 120 μ m.

    Journal: Chinese Medical Journal

    Article Title: Effect of Oenanthe Javanica Extract on Antioxidant Enzyme in the Rat Liver

    doi: 10.4103/0366-6999.158363

    Figure Lengend Snippet: Immunohistochemistry for superoxide dismutase (SOD1) (a-c), manganese superoxide dismutase (SOD2) (d-f), catalase (CAT) (g-i) and glutathione peroxidase (GPx) (j-l) in the rat liver of the normal-(left column), ascorbic acid - (middle column) and Oenanthe javanica extract (OJE) - (right column) groups. Strong SOD1, SOD2, CAT, and GPx immunoreactive cells (arrows) are markedly increased in the OJE-group. Scale bar = 120 μ m.

    Article Snippet: To reduce background staining, the membranes were incubated with 5% nonfat dry milk in PBS containing 0.1% Tween 20 for 45 min, followed by incubation with goat anti-SOD1 (diluted 1:1000, Calbiochem, Darmstadt, Germany), goat anti-SOD2 (diluted 1:1000, Calbiochem), rabbit anti-CAT (diluted 1:1000, LabFrontier, Seoul, Korea) and sheep anti-GPx (diluted 1:1000, Chemicon International, Billerica, MA), peroxidase-conjugated donkey anti-goat IgG, goat anti-rabbit IgG or goat anti-sheep IgG (Santa Cruz, USA), and an ECL kit (Pierce Chemical, Rockford, USA).

    Techniques: Immunohistochemistry

    Activity-dependent expression of ELMO1 regulates CGE-derived interneuron migration a. Expression of Dlx genes and ELMO1 at P5 in Dlx5/6-eGFP and Dlx5/6-Kir2.1 electroporated interneurons at e15.5. b. Expression of ELMO1 in GAD67-GFP transgenic mice at P2 and P5. Selective expression of ELMO1 in CGE-derived interneuron subtypes at P9. Quantification of ELMO1 expression in Re + , Cr + and VIP + interneurons (IN) at P9 (right). Mean percentage values (±SEM) were obtained from > 70 interneurons for each subtype. c. Quantification of DLX H and ELMO1 expression in Dlx5/6-eGFP (control) and Dlx5/6-Kir2.1 Re + e15.5-electroporated interneurons at P5. Mean percentage values (±SEM) were obtained from > 20 interneurons each in control and Kir2.1 electroporated mice for each quantification. d. Electroporation of Dlx5/6-Elmo1_TN558.FLAG plasmid at e15.5. FLAG immunoreactivity is detected in electroporated interneurons at P9 (inset). Neuronal morphology of a Re + interneuron and laminar distribution of electroporated interneurons at P9. Representative examples from 4 electroporated mice. e. Co-electroporation of Dlx5/6-Elmo1 and Dlx5/6-Kir2.1 plasmids at e15.5. ELMO1 expression in electroporated interneurons at P9. Morphological defects of an electroporated Re + interneuron and laminar distribution of electroporated interneurons. Representative examples from 6 electroporated mice. f. Quantification of the distribution of Re + interneurons across cortical layers at P9 upon expression of different plasmids. Mean percentage values (±SEM) were obtained from > 80 interneurons for each group. Values for control and Dlx5/6-Kir2.1 alone groups are repeated from Figure 2 to facilitate comparison between groups. The large bracket indicates comparison between the control and Dlx5/6-Elmo1_TN558.FLAG electroporated internerneurons. Paired t-test: *, P

    Journal: Nature

    Article Title: Neuronal activity is required for the development of specific cortical interneuron subtypes

    doi: 10.1038/nature09865

    Figure Lengend Snippet: Activity-dependent expression of ELMO1 regulates CGE-derived interneuron migration a. Expression of Dlx genes and ELMO1 at P5 in Dlx5/6-eGFP and Dlx5/6-Kir2.1 electroporated interneurons at e15.5. b. Expression of ELMO1 in GAD67-GFP transgenic mice at P2 and P5. Selective expression of ELMO1 in CGE-derived interneuron subtypes at P9. Quantification of ELMO1 expression in Re + , Cr + and VIP + interneurons (IN) at P9 (right). Mean percentage values (±SEM) were obtained from > 70 interneurons for each subtype. c. Quantification of DLX H and ELMO1 expression in Dlx5/6-eGFP (control) and Dlx5/6-Kir2.1 Re + e15.5-electroporated interneurons at P5. Mean percentage values (±SEM) were obtained from > 20 interneurons each in control and Kir2.1 electroporated mice for each quantification. d. Electroporation of Dlx5/6-Elmo1_TN558.FLAG plasmid at e15.5. FLAG immunoreactivity is detected in electroporated interneurons at P9 (inset). Neuronal morphology of a Re + interneuron and laminar distribution of electroporated interneurons at P9. Representative examples from 4 electroporated mice. e. Co-electroporation of Dlx5/6-Elmo1 and Dlx5/6-Kir2.1 plasmids at e15.5. ELMO1 expression in electroporated interneurons at P9. Morphological defects of an electroporated Re + interneuron and laminar distribution of electroporated interneurons. Representative examples from 6 electroporated mice. f. Quantification of the distribution of Re + interneurons across cortical layers at P9 upon expression of different plasmids. Mean percentage values (±SEM) were obtained from > 80 interneurons for each group. Values for control and Dlx5/6-Kir2.1 alone groups are repeated from Figure 2 to facilitate comparison between groups. The large bracket indicates comparison between the control and Dlx5/6-Elmo1_TN558.FLAG electroporated internerneurons. Paired t-test: *, P

    Article Snippet: Primary antibodies used in the experiments include rat anti-GFP (1:2000; Nacalai Tesque), mouse anti-Reelin (CR50) (1:500; MBL), rabbit anti-VIP (1:1000, Immunostar), mouse anti-calretinin (1:1500; Millipore Bioscience Research Reagents), rabbit anti-Tbr1 (1:1000 Abcam), goat anti- Tbr1 (1:1000 Abcam), rabbit anti-Pan-DLX (a gift from Jhumku Kohtz), rabbit anti-NPAS1 (a gift from Miura Masayuki), goat anti-ELMO1 (1:250 Millipore) and mouse anti-FLAG (1:200 Sigma Aldrich).

    Techniques: Activity Assay, Expressing, Derivative Assay, Migration, Transgenic Assay, Mouse Assay, Electroporation, Plasmid Preparation

    Medial GFP+ neurons are active during locomotion. A , Few (~5%) GFP+ neurons express Fos when the animal is quiescent (top), but almost half express Fos following an overground locomotor task (bottom). B , When exposed to the same transmitters that elicit locomotion in the isolated spinal cord, these neurons recorded in slice fire tonically—they are not conditional bursting neurons.

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: Genetically Defined Inhibitory Neurons in the Mouse Spinal Cord Dorsal Horn: A Possible Source of Rhythmic Inhibition of Motoneurons during Fictive Locomotion

    doi: 10.1523/JNEUROSCI.1401-09.2010

    Figure Lengend Snippet: Medial GFP+ neurons are active during locomotion. A , Few (~5%) GFP+ neurons express Fos when the animal is quiescent (top), but almost half express Fos following an overground locomotor task (bottom). B , When exposed to the same transmitters that elicit locomotion in the isolated spinal cord, these neurons recorded in slice fire tonically—they are not conditional bursting neurons.

    Article Snippet: Fifty-micrometer spinal cord sections were cut and incubated, at room temperature for 16–24 h, in one or more of the following primary antibodies: mouse or sheep anti-GFP (1:500, Biogenesis), goat anti-Fos (1:1000, Millipore Bioscience Research Reagents), mouse anti-GAD65 or -GAD67 (1:500), guinea-pig anti-VGluT1 (1:2500), and/or goat anti-choline acetyltransferase (ChAT) (1: 100, all from Millipore Bioscience Research Reagents), and guinea-pig anti-GlyT2 (1:5000, Sigma) as previously described ( ).

    Techniques: Isolation

    Immunohistochemistry for superoxide dismutase (SOD1) (a-c), manganese superoxide dismutase (SOD2) (d-f), catalase (CAT) (g-i) and glutathione peroxidase (GPx) (j-l) in the rat liver of the normal-(left column), ascorbic acid - (middle column) and Oenanthe javanica extract (OJE) - (right column) groups. Strong SOD1, SOD2, CAT, and GPx immunoreactive cells (arrows) are markedly increased in the OJE-group. Scale bar = 120 μ m.

    Journal: Chinese Medical Journal

    Article Title: Effect of Oenanthe Javanica Extract on Antioxidant Enzyme in the Rat Liver

    doi: 10.4103/0366-6999.158363

    Figure Lengend Snippet: Immunohistochemistry for superoxide dismutase (SOD1) (a-c), manganese superoxide dismutase (SOD2) (d-f), catalase (CAT) (g-i) and glutathione peroxidase (GPx) (j-l) in the rat liver of the normal-(left column), ascorbic acid - (middle column) and Oenanthe javanica extract (OJE) - (right column) groups. Strong SOD1, SOD2, CAT, and GPx immunoreactive cells (arrows) are markedly increased in the OJE-group. Scale bar = 120 μ m.

    Article Snippet: The sections were next incubated with goat anti-SOD1 (diluted 1:500, Calbiochem, Darmstadt, Germany), goat anti-SOD2 (diluted 1:1000, Calbiochem), rabbit anti-CAT (diluted 1:1000, Lab Frontier, Seoul, Korea) and sheep anti-GPx (diluted 1:1000, Chemicon International, Billerica, MA).

    Techniques: Immunohistochemistry

    Physiological and anatomical properties of OFF brisk-sustained ganglion cells (BSGCs). A : spikes in an OFF-BSGC, evoked by a dark spot flashed for 0.5 s. The stimulus timing is shown by the gray bar ( bottom ). The stimulus spot diameter (μm) is shown to the left of each response. Firing rates peaked within 85 ms of stimulus onset and were sustained for optimally sized spots, 75 and 150 μm in diameter. B : the mean spike count during the stimulus presentation is plotted vs. stimulus diameter (±SE; n = 141). The continuous line shows the best fit to a difference-of-Gaussians function, with a center size (2σ center Gaussian) of 130 ± 12 μm and a surround of 460 ± 36 μm. C : confocal z-projection of an OFF-BSGC filled with Alexa-594 hydrazide. Dendrites from an adjacent-filled BSGC are visible ( top ; original scale bar = 50 μm). Bottom ), were labeled with an antibody for choline acetyltransferase (gray; original scale bar = 25 μm).

    Journal: Journal of Neurophysiology

    Article Title: Synaptic pathways that shape the excitatory drive in an OFF retinal ganglion cell

    doi: 10.1152/jn.00924.2011

    Figure Lengend Snippet: Physiological and anatomical properties of OFF brisk-sustained ganglion cells (BSGCs). A : spikes in an OFF-BSGC, evoked by a dark spot flashed for 0.5 s. The stimulus timing is shown by the gray bar ( bottom ). The stimulus spot diameter (μm) is shown to the left of each response. Firing rates peaked within 85 ms of stimulus onset and were sustained for optimally sized spots, 75 and 150 μm in diameter. B : the mean spike count during the stimulus presentation is plotted vs. stimulus diameter (±SE; n = 141). The continuous line shows the best fit to a difference-of-Gaussians function, with a center size (2σ center Gaussian) of 130 ± 12 μm and a surround of 460 ± 36 μm. C : confocal z-projection of an OFF-BSGC filled with Alexa-594 hydrazide. Dendrites from an adjacent-filled BSGC are visible ( top ; original scale bar = 50 μm). Bottom ), were labeled with an antibody for choline acetyltransferase (gray; original scale bar = 25 μm).

    Article Snippet: In experiments where dendritic stratification was confirmed, the retinas were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer for 45 min, rinsed, and then incubated for 7 days (25°C) in a goat anti-choline acetyltransferase antibody (Catalogue #AB144P; Millipore, Billerica, MA), diluted 1:200 in an incubation buffer containing 3% normal horse serum, 1% Triton X-100, and 0.025% NaN3 in 0.1 M PBS (pH 7.4).

    Techniques: Mass Spectrometry, Labeling

    Increased levels of TIMP1, CD63 and β1-integrins in melanoma cells compared to primary melanocytes and association between Timp1 and β1- integrins only in human metastatic melanoma cells. mRNA levels of TIMP1 and CD63 were measured by real time PCR ( A and B , respectively). C. β1-integrin expression was analyzed in primary human melanocytes MP#2 and human metastatic melanoma cell lines (Mel2, Mel3, Mel4, Mel14, Mel25 and Mel28) by Western blotting using a specific polyclonal antibody. The same membrane was reprobed with anti-PCNA antibody as an internal control. D. Membrane-enriched protein extracts from MP#2, Mel2, Mel3 and Mel14 cell lines were immunoprecipitated with anti-TIMP1 and analyzed by Western blotting with anti-β1-integrin. E and F. Primary human MP#2 melanocytes and human metastatic melanomas Mel2, Mel3, Mel4, Mel11, Mel25, Mel28 and Mel33 (2x10 2 ) were incubated at 37°C on 60 mm plates and colony formation capacity was determined after 5 days. Spearman’s correlation (r) was used to correlate TIMP1 and clonogenic capacity ( G ), CD63 expression and clonogenic capacity ( H ), TIMP1 and CD63 expression ( I ), and combined TIMP1 and CD63 expression and clonogenic capacity ( J ). All analyses were performed using SPSS (v 18, Chicago, IL, USA). The significance level was set at 0.05.

    Journal: Molecular Cancer

    Article Title: Timp1 interacts with beta-1 integrin and CD63 along melanoma genesis and confers anoikis resistance by activating PI3-K signaling pathway independently of Akt phosphorylation

    doi: 10.1186/1476-4598-12-22

    Figure Lengend Snippet: Increased levels of TIMP1, CD63 and β1-integrins in melanoma cells compared to primary melanocytes and association between Timp1 and β1- integrins only in human metastatic melanoma cells. mRNA levels of TIMP1 and CD63 were measured by real time PCR ( A and B , respectively). C. β1-integrin expression was analyzed in primary human melanocytes MP#2 and human metastatic melanoma cell lines (Mel2, Mel3, Mel4, Mel14, Mel25 and Mel28) by Western blotting using a specific polyclonal antibody. The same membrane was reprobed with anti-PCNA antibody as an internal control. D. Membrane-enriched protein extracts from MP#2, Mel2, Mel3 and Mel14 cell lines were immunoprecipitated with anti-TIMP1 and analyzed by Western blotting with anti-β1-integrin. E and F. Primary human MP#2 melanocytes and human metastatic melanomas Mel2, Mel3, Mel4, Mel11, Mel25, Mel28 and Mel33 (2x10 2 ) were incubated at 37°C on 60 mm plates and colony formation capacity was determined after 5 days. Spearman’s correlation (r) was used to correlate TIMP1 and clonogenic capacity ( G ), CD63 expression and clonogenic capacity ( H ), TIMP1 and CD63 expression ( I ), and combined TIMP1 and CD63 expression and clonogenic capacity ( J ). All analyses were performed using SPSS (v 18, Chicago, IL, USA). The significance level was set at 0.05.

    Article Snippet: After protein transfer, the membranes were blocked with 5% non-fat dry milk in PBS (10mM phosphate buffer pH 7.2, 150 mM NaCl), incubated with the indicated antibodies: goat polyclonal anti-Timp1 (R & D), rabbit polyclonal anti-β1-integrin (Calbiochem), rabbit polyclonal anti-CD63 (Santa Cruz), rabbit polyclonal anti-phospho Akt (Cell Signaling) and rabbit polyclonal Akt (Cell Signaling) overnight at 4°C, and the signal was detected using horseradish peroxidase–conjugated anti–immunoglobulin G antibody (KPL; Gaithersburg, MD) followed by development using chemiluminescence substrate (SuperSignal West Pico Chemiluminescent Substrate; Pierce Chemical, Rockford, IL).

    Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot, Immunoprecipitation, Incubation

    A tight Timp1/CD63/β1-integrin complex is formed only in tumorigenic cell lines. A. β1-integrin expression pattern was analyzed in murine melan-a, 4C, 4C11- and 4C11+ cell lines by Western blotting using a specific polyclonal antibody. Note the shift of β1-integrin eletrophoretic migration in the metastatic 4C11+ melanoma cell line. The same membrane was reprobed with anti-actin antibody as an endogenous control. B. CD63 protein expression was evaluated by flow cytometry using antibody specific. C. Membrane-enriched protein extracts from melan-a, 4C, 4C11-, and 4C11+ cell lines were immunoprecipitated with anti-CD63 and anti-β1-integrin and analyzed by Western blotting with anti-Timp1 and anti-β1-integrin. ma: non-tumorigenic melanocytes melan-a; 4C: pre-malignant melanocytes; 4C11-: non-metastatic melanoma cells; 4C11+: metastatic melanoma cells. *p

    Journal: Molecular Cancer

    Article Title: Timp1 interacts with beta-1 integrin and CD63 along melanoma genesis and confers anoikis resistance by activating PI3-K signaling pathway independently of Akt phosphorylation

    doi: 10.1186/1476-4598-12-22

    Figure Lengend Snippet: A tight Timp1/CD63/β1-integrin complex is formed only in tumorigenic cell lines. A. β1-integrin expression pattern was analyzed in murine melan-a, 4C, 4C11- and 4C11+ cell lines by Western blotting using a specific polyclonal antibody. Note the shift of β1-integrin eletrophoretic migration in the metastatic 4C11+ melanoma cell line. The same membrane was reprobed with anti-actin antibody as an endogenous control. B. CD63 protein expression was evaluated by flow cytometry using antibody specific. C. Membrane-enriched protein extracts from melan-a, 4C, 4C11-, and 4C11+ cell lines were immunoprecipitated with anti-CD63 and anti-β1-integrin and analyzed by Western blotting with anti-Timp1 and anti-β1-integrin. ma: non-tumorigenic melanocytes melan-a; 4C: pre-malignant melanocytes; 4C11-: non-metastatic melanoma cells; 4C11+: metastatic melanoma cells. *p

    Article Snippet: After protein transfer, the membranes were blocked with 5% non-fat dry milk in PBS (10mM phosphate buffer pH 7.2, 150 mM NaCl), incubated with the indicated antibodies: goat polyclonal anti-Timp1 (R & D), rabbit polyclonal anti-β1-integrin (Calbiochem), rabbit polyclonal anti-CD63 (Santa Cruz), rabbit polyclonal anti-phospho Akt (Cell Signaling) and rabbit polyclonal Akt (Cell Signaling) overnight at 4°C, and the signal was detected using horseradish peroxidase–conjugated anti–immunoglobulin G antibody (KPL; Gaithersburg, MD) followed by development using chemiluminescence substrate (SuperSignal West Pico Chemiluminescent Substrate; Pierce Chemical, Rockford, IL).

    Techniques: Expressing, Western Blot, Migration, Flow Cytometry, Cytometry, Immunoprecipitation

    Increased association of Timp1 on the cell surface along melanoma progression. A. mRNA levels of Timp1 were determined by real time PCR. B. Membrane-enriched extracts were separated on 15% polyacrylamide gel SDS/PAGE and transferred to a PVDF membrane. The membrane was incubated with polyclonal antibody specific to Timp1. The constitutive expression of β-actin was used as endogenous control. C. The supernatants of cell cultures were collected and lyophilized. The presence of soluble Timp1 was identified by Western Blotting. D. Conditioned media (10 μg) from melan-a, 4C, 4C11- and 4C11+ cell lines were evaluated for MMPs activity. E. The same cell lines were maintained in suspension for 96 hours and viable cells were estimated using MTT. Experiments were always performed in quadruplicates. ma: murine non-tumorigenic melanocyte lineage; 4C: pre-malignant melanocytes; 4C11-: non-metastatic melanoma cells; 4C11+: metastatic melanoma cells. **p

    Journal: Molecular Cancer

    Article Title: Timp1 interacts with beta-1 integrin and CD63 along melanoma genesis and confers anoikis resistance by activating PI3-K signaling pathway independently of Akt phosphorylation

    doi: 10.1186/1476-4598-12-22

    Figure Lengend Snippet: Increased association of Timp1 on the cell surface along melanoma progression. A. mRNA levels of Timp1 were determined by real time PCR. B. Membrane-enriched extracts were separated on 15% polyacrylamide gel SDS/PAGE and transferred to a PVDF membrane. The membrane was incubated with polyclonal antibody specific to Timp1. The constitutive expression of β-actin was used as endogenous control. C. The supernatants of cell cultures were collected and lyophilized. The presence of soluble Timp1 was identified by Western Blotting. D. Conditioned media (10 μg) from melan-a, 4C, 4C11- and 4C11+ cell lines were evaluated for MMPs activity. E. The same cell lines were maintained in suspension for 96 hours and viable cells were estimated using MTT. Experiments were always performed in quadruplicates. ma: murine non-tumorigenic melanocyte lineage; 4C: pre-malignant melanocytes; 4C11-: non-metastatic melanoma cells; 4C11+: metastatic melanoma cells. **p

    Article Snippet: After protein transfer, the membranes were blocked with 5% non-fat dry milk in PBS (10mM phosphate buffer pH 7.2, 150 mM NaCl), incubated with the indicated antibodies: goat polyclonal anti-Timp1 (R & D), rabbit polyclonal anti-β1-integrin (Calbiochem), rabbit polyclonal anti-CD63 (Santa Cruz), rabbit polyclonal anti-phospho Akt (Cell Signaling) and rabbit polyclonal Akt (Cell Signaling) overnight at 4°C, and the signal was detected using horseradish peroxidase–conjugated anti–immunoglobulin G antibody (KPL; Gaithersburg, MD) followed by development using chemiluminescence substrate (SuperSignal West Pico Chemiluminescent Substrate; Pierce Chemical, Rockford, IL).

    Techniques: Real-time Polymerase Chain Reaction, SDS Page, Incubation, Expressing, Western Blot, Activity Assay, MTT Assay

    Impaired TNFα and IL-12 production in DGKζ-deficient mice after TLR-stimulation. (A and B) Measurement of serum cytokine concentrations by ELISA. WT and DGKζ −/− mice were intraperitoneally injected with 10 μg LPS or 5 μg STAg. 6 h after injection, sera were collected for measurement of IL-12 p40 and TNFα production by ELISA. Data shown are the mean ± SD for each group and are representative of three experiments. (C and D) Measurement of cytokine messengers by Northern blot. WT and DGKζ −/− mice were intraperitoneally injected with 10 μg LPS or 5 μg STAg. 2, 3, and 5 h after injection, total splenic RNA was isolated, and IL-12 p40 and TNFα transcripts were measured by Northern blot analysis. Data shown are representative of three experiments. (E) Increased resistance to LPS-induced shock in DGKζ −/− mice. Age- and sex-matched WT mice ( n = 10) and DGKζ −/− mice ( n = 6) were intraperitoneally injected with 20 mg d -galactosamine and 2 μg LPS per mouse, and their survival was observed at the indicated time points. Data shown are representative of two independent experiments. (F) Detection of IL-12 production by immunofluorescence. Spleens were removed from mice 6 h after injection of PBS, LPS, or STAg. Frozen sections of spleens were processed and stained with a goat anti–mouse IL-12 p40 antibody, followed by a double incubation with Alexa Fluor 488–conjugated anti–mouse CD11c and Alexa Fluor 594–conjugated anti–goat antibodies. The cells were counterstained for nuclei using DAPI. Images were captured using an Axiovert microscope with AxioVision software (see Materials and methods). Green, red, and blue represent CD11c + DCs, IL-12, and nuclei, respectively; yellow represents CD11c + IL-12 + after overlay. Bars, 50 μm.

    Journal: The Journal of Experimental Medicine

    Article Title: Diacylglycerol kinase ? regulates microbial recognition and host resistance to Toxoplasma gondii

    doi: 10.1084/jem.20061856

    Figure Lengend Snippet: Impaired TNFα and IL-12 production in DGKζ-deficient mice after TLR-stimulation. (A and B) Measurement of serum cytokine concentrations by ELISA. WT and DGKζ −/− mice were intraperitoneally injected with 10 μg LPS or 5 μg STAg. 6 h after injection, sera were collected for measurement of IL-12 p40 and TNFα production by ELISA. Data shown are the mean ± SD for each group and are representative of three experiments. (C and D) Measurement of cytokine messengers by Northern blot. WT and DGKζ −/− mice were intraperitoneally injected with 10 μg LPS or 5 μg STAg. 2, 3, and 5 h after injection, total splenic RNA was isolated, and IL-12 p40 and TNFα transcripts were measured by Northern blot analysis. Data shown are representative of three experiments. (E) Increased resistance to LPS-induced shock in DGKζ −/− mice. Age- and sex-matched WT mice ( n = 10) and DGKζ −/− mice ( n = 6) were intraperitoneally injected with 20 mg d -galactosamine and 2 μg LPS per mouse, and their survival was observed at the indicated time points. Data shown are representative of two independent experiments. (F) Detection of IL-12 production by immunofluorescence. Spleens were removed from mice 6 h after injection of PBS, LPS, or STAg. Frozen sections of spleens were processed and stained with a goat anti–mouse IL-12 p40 antibody, followed by a double incubation with Alexa Fluor 488–conjugated anti–mouse CD11c and Alexa Fluor 594–conjugated anti–goat antibodies. The cells were counterstained for nuclei using DAPI. Images were captured using an Axiovert microscope with AxioVision software (see Materials and methods). Green, red, and blue represent CD11c + DCs, IL-12, and nuclei, respectively; yellow represents CD11c + IL-12 + after overlay. Bars, 50 μm.

    Article Snippet: Spleens were removed from mice before or 6 h after STAg or LPS stimulation, and frozen sections were processed and stained with a goat anti–mouse IL-12 p40 (BD Biosciences), followed by a double incubation with Alexa Fluor 488–conjugated anti–mouse CD11c and Alexa Fluor 594–conjugated anti–goat antibodies (Invitrogen).

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay, Injection, Northern Blot, Isolation, Immunofluorescence, Staining, Incubation, Microscopy, Software