in vitro antigen presenting cell assay twenty four h Search Results


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    Ipsen Group syntaxin 1a
    Co-localization between SNAP-25 and <t>Syntaxin</t> 1A, detected by antibodies or nanobodies in STED microscopy. ( a ) Co-staining for SNAP-25 and <t>Syntaxin</t> 1A in primary hippocampal neurons. A polyclonal rabbit antibody against SNAP-25 and a mouse monoclonal against Syntaxin 1A were used. Rabbit anti SNAP-25 was further detected with a secondary antibody conjugated to Abberior-Star580; Syntaxin 1A primary antibody was detected with an Atto647N-conjugated secondary antibody. Nanobody co-staining was performed with S25-Nb10 conjugated to Abberior-Star580 and with Stx1A-Nb6 conjugated to Atto647N. The scale bars represent 2 µm and 500 nm in the low and high zoom, respectively. ( b ) Pearson´s correlation coefficients (expressed as coefficients of determination, R 2 ) between the green and red signals within synapses were calculated from 17 independent experiments for the antibodies and from 9 independent experiments for the nanobodies (typically, 10 images per experiment were analyzed). The values are shown as box plots, with the median, 25th and 95th percentile shown in the graph, and with symbols showing outliers. The difference is significant (Wilcoxon rank sum test; p = 0.0311).
    Syntaxin 1a, supplied by Ipsen Group, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Roche dig rna labeling kit
    Expression of EFCAB2 in mouse testes. (A) Northern blot analysis of Efcab2 mRNA in adult mouse tissues. Total RNAs (10 μg each) prepared from the various tissues were electrophoresed, transferred to a nylon membrane, and then hybridized with a <t>DIG-labeled</t> cRNA probe. The arrow indicates Efcab2 mRNA. 28S and 18S <t>RNA</t> were stained as the loading controls. kb, kilobase. (B) Western blot analysis of total protein extracts from 293T cells transfected with control (P3xFLAG-CMV-14 plasmid) and EFCAB2-overexpression plasmids (OE). Immunoblotting with anti β-Actin as a loading control (left panel) or anti-EFCAB2 antiserum (right panel). The arrow indicates EFCAB2 protein band. (C) Western blot analysis of EFCAB2 protein in adult mouse testes. Total testis proteins were extracted, and 20 μg were separated by 12.5% SDS-PAGE. Left panel shows Coomassie Brilliant Blue-staining (CBB) as a loading control, while right panel shows the immunoblotted PVDF membrane with anti-EFCAB2 antiserum. The arrow indicates EFCAB2 protein band. Bovine serum albumin (BSA) was used as a negative control. M: Molecular marker (kilodalton; kDa).
    Dig Rna Labeling Kit, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    R&D Systems il 17
    Bleomycin-induced acute lung injury augments <t>IL-17</t> expression in alcohol-fed mice. Three-month-old wild-type mice were treated ± alcohol (20% v/v in drinking water for 8 weeks) before intratracheal administration of bleomycin (2.5 units/kg) or saline vehicle. Lungs were collected at 7 and 14 days following induction of injury and analyzed for IL-17 protein expression by Western blot. *p
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    86
    Roche t7 rna polymerase
    Bleomycin-induced acute lung injury augments <t>IL-17</t> expression in alcohol-fed mice. Three-month-old wild-type mice were treated ± alcohol (20% v/v in drinking water for 8 weeks) before intratracheal administration of bleomycin (2.5 units/kg) or saline vehicle. Lungs were collected at 7 and 14 days following induction of injury and analyzed for IL-17 protein expression by Western blot. *p
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    Image Search Results


    Co-localization between SNAP-25 and Syntaxin 1A, detected by antibodies or nanobodies in STED microscopy. ( a ) Co-staining for SNAP-25 and Syntaxin 1A in primary hippocampal neurons. A polyclonal rabbit antibody against SNAP-25 and a mouse monoclonal against Syntaxin 1A were used. Rabbit anti SNAP-25 was further detected with a secondary antibody conjugated to Abberior-Star580; Syntaxin 1A primary antibody was detected with an Atto647N-conjugated secondary antibody. Nanobody co-staining was performed with S25-Nb10 conjugated to Abberior-Star580 and with Stx1A-Nb6 conjugated to Atto647N. The scale bars represent 2 µm and 500 nm in the low and high zoom, respectively. ( b ) Pearson´s correlation coefficients (expressed as coefficients of determination, R 2 ) between the green and red signals within synapses were calculated from 17 independent experiments for the antibodies and from 9 independent experiments for the nanobodies (typically, 10 images per experiment were analyzed). The values are shown as box plots, with the median, 25th and 95th percentile shown in the graph, and with symbols showing outliers. The difference is significant (Wilcoxon rank sum test; p = 0.0311).

    Journal: mAbs

    Article Title: Nanobodies reveal an extra-synaptic population of SNAP-25 and Syntaxin 1A in hippocampal neurons

    doi: 10.1080/19420862.2018.1551675

    Figure Lengend Snippet: Co-localization between SNAP-25 and Syntaxin 1A, detected by antibodies or nanobodies in STED microscopy. ( a ) Co-staining for SNAP-25 and Syntaxin 1A in primary hippocampal neurons. A polyclonal rabbit antibody against SNAP-25 and a mouse monoclonal against Syntaxin 1A were used. Rabbit anti SNAP-25 was further detected with a secondary antibody conjugated to Abberior-Star580; Syntaxin 1A primary antibody was detected with an Atto647N-conjugated secondary antibody. Nanobody co-staining was performed with S25-Nb10 conjugated to Abberior-Star580 and with Stx1A-Nb6 conjugated to Atto647N. The scale bars represent 2 µm and 500 nm in the low and high zoom, respectively. ( b ) Pearson´s correlation coefficients (expressed as coefficients of determination, R 2 ) between the green and red signals within synapses were calculated from 17 independent experiments for the antibodies and from 9 independent experiments for the nanobodies (typically, 10 images per experiment were analyzed). The values are shown as box plots, with the median, 25th and 95th percentile shown in the graph, and with symbols showing outliers. The difference is significant (Wilcoxon rank sum test; p = 0.0311).

    Article Snippet: For this, we investigated by SDS-PAGE the following: Purified recombinant SNAP-25 or Syntaxin 1A produced in E. coli , lysates of HEK293 cell lines transiently expressing SNAP-25 or Syntaxin 1A, lysates of total rat brain and lysates of rat primary hippocampal cultures.

    Techniques: Microscopy, Staining

    Both nanobodies reveled epitopes that are not reported by antibodies in highly crowded regions of endogenously-expressing PC12 cells or over-expressing COS-7 cells. ( a ) PC12 cells were co-stained with conventional monoclonal antibodies and with our fluorescently labeled nanobodies. Monoclonal anti SNAP-25 and Syntaxin 1A (clone 71.1 and HPC-1) were detected with a secondary antibody conjugated to Abberior-Star580 (in green). S25-Nb10 or Stx1A-Nb6 were conjugated to a single Atto647N fluorophore (in red). Laser scanning confocal images of the nanobody and the antibody signals colocalize relatively good at the plasma membrane, but the nanobodies also reveal stronger signals in the perinuclear areas (especially evident for SNAP-25). The scale bar represents 10 µm. ( b ) Laser scanning confocal images of COS-7 cells transiently transfected with SNAP-25 or Syntaxin 1A fused to EGFP (in green) and co-stained with monoclonal primary and Cy3-fluorescently labeled secondary antibody (clone 71.1 and clone 78.2, in magenta) and nanobodies directly conjugated to Atto647N (in red). Scale bar represents 10 µm. ( c ) Line profiles of the white lines displayed in ( B ) are shown for all three channels, each channel was normalized to its maximum signal intensity on the picture. ( d ) The average fluorescence of full cells, black bars or at the perinuclear regions, light-grey bars in respect to their EGFP signals were calculated. For every selected region of interest, the averaged fluorescence signal of antibodies or nanobodies was calculated and normalized to the average EGFP-fluorescence in the respective region. Statistical analysis was done using unpaired t- test, error bars represent SEM, from three independent experiments analyzing a total of 37 cells for SNAP-25-EGFP and 36 cells for Stx1A-EGFP. n.s. = not significant.

    Journal: mAbs

    Article Title: Nanobodies reveal an extra-synaptic population of SNAP-25 and Syntaxin 1A in hippocampal neurons

    doi: 10.1080/19420862.2018.1551675

    Figure Lengend Snippet: Both nanobodies reveled epitopes that are not reported by antibodies in highly crowded regions of endogenously-expressing PC12 cells or over-expressing COS-7 cells. ( a ) PC12 cells were co-stained with conventional monoclonal antibodies and with our fluorescently labeled nanobodies. Monoclonal anti SNAP-25 and Syntaxin 1A (clone 71.1 and HPC-1) were detected with a secondary antibody conjugated to Abberior-Star580 (in green). S25-Nb10 or Stx1A-Nb6 were conjugated to a single Atto647N fluorophore (in red). Laser scanning confocal images of the nanobody and the antibody signals colocalize relatively good at the plasma membrane, but the nanobodies also reveal stronger signals in the perinuclear areas (especially evident for SNAP-25). The scale bar represents 10 µm. ( b ) Laser scanning confocal images of COS-7 cells transiently transfected with SNAP-25 or Syntaxin 1A fused to EGFP (in green) and co-stained with monoclonal primary and Cy3-fluorescently labeled secondary antibody (clone 71.1 and clone 78.2, in magenta) and nanobodies directly conjugated to Atto647N (in red). Scale bar represents 10 µm. ( c ) Line profiles of the white lines displayed in ( B ) are shown for all three channels, each channel was normalized to its maximum signal intensity on the picture. ( d ) The average fluorescence of full cells, black bars or at the perinuclear regions, light-grey bars in respect to their EGFP signals were calculated. For every selected region of interest, the averaged fluorescence signal of antibodies or nanobodies was calculated and normalized to the average EGFP-fluorescence in the respective region. Statistical analysis was done using unpaired t- test, error bars represent SEM, from three independent experiments analyzing a total of 37 cells for SNAP-25-EGFP and 36 cells for Stx1A-EGFP. n.s. = not significant.

    Article Snippet: For this, we investigated by SDS-PAGE the following: Purified recombinant SNAP-25 or Syntaxin 1A produced in E. coli , lysates of HEK293 cell lines transiently expressing SNAP-25 or Syntaxin 1A, lysates of total rat brain and lysates of rat primary hippocampal cultures.

    Techniques: Expressing, Staining, Labeling, Transfection, Fluorescence

    An analysis of the size and intensity of SNAP-25 and Syntaxin 1A clusters (spots) in hippocampal neurons. ( a ) The intensity of SNAP-25 spots is shown as intensity fold over single nanobody or antibody. The intensities were obtained by drawing line scans across 259 SNAP-25 clusters in synapses and 212 clusters outside of synapses for the antibody staining. For the nanobody staining, we analyzed 392 line-scans in synapses and 309 line-scans outside of synapses. Both within and outside of synapses, the antibody shows spots of a smaller intensity than the nanobody. The nanobody spots in synapses (green line in right panel) also show a large variability within the signal intensity. Bar graphs (insets) represent the mean of the intensity distributions with their associated SEM. ( b ) Same analysis as in ( a ) for Syntaxin 1A. We analyzed 348 line-scans in synapses and 227 outside synapses for antibodies. 378 line-scans were analyzed in synapses and 326 outside synapses for nanobodies. Interestingly, the nanobody spots in synapses (green line in right panel) show a far more limited variability than for SNAP-25 (note the difference in units for the X-axis of SNAP-25 and Syntaxin 1A analyses). ( c-d ) Analysis of spot size for SNAP-25 or Syntaxin 1A revealed by nanobody (red) or antibody (black), relative to the Synaptophysin intensity (very low signal is extra-synaptic, highest signal is the center of the synapse). The insets present the extra-synaptic areas at higher zoom in the regions of low Synaptophysin intensity. No obvious difference can be observed for SNAP-25 ( c ), but the Syntaxin 1A spots (clusters) are larger for the nanobody in extra-synaptic areas (p = 0.000082, paired t- test). The analysis was performed on the following number of automatically identified spots: 10,839 spots from seven independent experiments for SNAP-25 nanobodies; 8,546 spots from seven independent experiments for SNAP-25 antibodies; 35,609 spots from 20 independent experiments for Syntaxin 1A nanobodies; 12,468 spots from seven independent experiments for Syntaxin 1A antibodies. The graphs show bi-directional scatter plots, plus SEM, after binning the spots according to Synaptophysin intensity. Note that for the Synaptophysin staining the error bars in the horizontal direction are smaller than the symbol sizes.

    Journal: mAbs

    Article Title: Nanobodies reveal an extra-synaptic population of SNAP-25 and Syntaxin 1A in hippocampal neurons

    doi: 10.1080/19420862.2018.1551675

    Figure Lengend Snippet: An analysis of the size and intensity of SNAP-25 and Syntaxin 1A clusters (spots) in hippocampal neurons. ( a ) The intensity of SNAP-25 spots is shown as intensity fold over single nanobody or antibody. The intensities were obtained by drawing line scans across 259 SNAP-25 clusters in synapses and 212 clusters outside of synapses for the antibody staining. For the nanobody staining, we analyzed 392 line-scans in synapses and 309 line-scans outside of synapses. Both within and outside of synapses, the antibody shows spots of a smaller intensity than the nanobody. The nanobody spots in synapses (green line in right panel) also show a large variability within the signal intensity. Bar graphs (insets) represent the mean of the intensity distributions with their associated SEM. ( b ) Same analysis as in ( a ) for Syntaxin 1A. We analyzed 348 line-scans in synapses and 227 outside synapses for antibodies. 378 line-scans were analyzed in synapses and 326 outside synapses for nanobodies. Interestingly, the nanobody spots in synapses (green line in right panel) show a far more limited variability than for SNAP-25 (note the difference in units for the X-axis of SNAP-25 and Syntaxin 1A analyses). ( c-d ) Analysis of spot size for SNAP-25 or Syntaxin 1A revealed by nanobody (red) or antibody (black), relative to the Synaptophysin intensity (very low signal is extra-synaptic, highest signal is the center of the synapse). The insets present the extra-synaptic areas at higher zoom in the regions of low Synaptophysin intensity. No obvious difference can be observed for SNAP-25 ( c ), but the Syntaxin 1A spots (clusters) are larger for the nanobody in extra-synaptic areas (p = 0.000082, paired t- test). The analysis was performed on the following number of automatically identified spots: 10,839 spots from seven independent experiments for SNAP-25 nanobodies; 8,546 spots from seven independent experiments for SNAP-25 antibodies; 35,609 spots from 20 independent experiments for Syntaxin 1A nanobodies; 12,468 spots from seven independent experiments for Syntaxin 1A antibodies. The graphs show bi-directional scatter plots, plus SEM, after binning the spots according to Synaptophysin intensity. Note that for the Synaptophysin staining the error bars in the horizontal direction are smaller than the symbol sizes.

    Article Snippet: For this, we investigated by SDS-PAGE the following: Purified recombinant SNAP-25 or Syntaxin 1A produced in E. coli , lysates of HEK293 cell lines transiently expressing SNAP-25 or Syntaxin 1A, lysates of total rat brain and lysates of rat primary hippocampal cultures.

    Techniques: Staining

    Schematics of the proteins involved in this study. ( a ) Molecular models of SNAP-25 (based on PDB: 1KIL, in red) and Syntaxin 1A (based on PDBs: 1HVV 1BR0, in green) residing in the plasma membrane (in yellow). On the right we show a nanobody (PDB: 1I3V, in purple) bearing a single Atto647N at the C-terminus, and a complex of a primary and a randomly-labeled secondary antibody with Atto647N on lysines (PDB: 1IGY in blue and light-blue). All molecular models are displayed in the same scale and the bar represents 2 nm. ( b ) Schematic view of antigens used for the immunization and their wild-type forms. Important functional domains are marked in gray (TM = trans-membrane domain) and the amino acids positions are denoted below. For immunization (injected), all four cysteine residues of SNAP-25 were mutated to serines. For injection of Syntaxin 1A, only the cytosolic portion of the molecule was used to facilitate it expression in E. coli.

    Journal: mAbs

    Article Title: Nanobodies reveal an extra-synaptic population of SNAP-25 and Syntaxin 1A in hippocampal neurons

    doi: 10.1080/19420862.2018.1551675

    Figure Lengend Snippet: Schematics of the proteins involved in this study. ( a ) Molecular models of SNAP-25 (based on PDB: 1KIL, in red) and Syntaxin 1A (based on PDBs: 1HVV 1BR0, in green) residing in the plasma membrane (in yellow). On the right we show a nanobody (PDB: 1I3V, in purple) bearing a single Atto647N at the C-terminus, and a complex of a primary and a randomly-labeled secondary antibody with Atto647N on lysines (PDB: 1IGY in blue and light-blue). All molecular models are displayed in the same scale and the bar represents 2 nm. ( b ) Schematic view of antigens used for the immunization and their wild-type forms. Important functional domains are marked in gray (TM = trans-membrane domain) and the amino acids positions are denoted below. For immunization (injected), all four cysteine residues of SNAP-25 were mutated to serines. For injection of Syntaxin 1A, only the cytosolic portion of the molecule was used to facilitate it expression in E. coli.

    Article Snippet: For this, we investigated by SDS-PAGE the following: Purified recombinant SNAP-25 or Syntaxin 1A produced in E. coli , lysates of HEK293 cell lines transiently expressing SNAP-25 or Syntaxin 1A, lysates of total rat brain and lysates of rat primary hippocampal cultures.

    Techniques: Labeling, Functional Assay, Injection, Expressing

    Specificity test for the selected nanobodies in immunofluorescence and Western Blots. ( a b ) COS-7 cells transiently transfected with SNAP-25-EGFP or Syntaxin 1A-EGFP were stained with S25-Nb10 or Stx1A-Nb6 conjugated with a single Atto647N fluorophore. The nanobody signal correlates with the EGFP signal and shows no staining in untransfected cells (revealed by Hoechst-nuclear staining; white arrowheads). The scale bar represents 10 µm. Note that Syntaxin 1A accumulates in the ER-Golgi region due to an impaired export caused by the lack of neuronal cofactors, as it was found in the past 30 ( c d ) The following samples were loaded in a denaturating SDS-PAGE and were blotted on a nitrocellulose membrane (2 µg of each purified protein, and 20 µg of total protein for the cell or brain lysates): E. coli- purified full length SNAP-25 including a Twin-Strep-Tag (tst; 27.8 kDa), or Syntaxin 1A fused to a SUMO domain and a Twin-Strep-Tag (44.7 kDa); lysates from HEK293 cells transiently transfected with full length Syntaxin 1A-tst (HEK-Stx1A, 37.5 kDa) or SNAP-25-tst (HEK-SNAP-25, 27.8 kDa); whole rat brain and rat primary hippocampal neurons (15 days in vitro ). Detection was performed using S25-Nb10 ( c ) or Stx1A-Nb6 ( d ) conjugated to a single Atto647N. Both candidates specifically detect the bands at the expected molecular weights (displayed in the protein schematics), while showing no cross-reactivity to the opposite antigen or to any other proteins present in the lysates.

    Journal: mAbs

    Article Title: Nanobodies reveal an extra-synaptic population of SNAP-25 and Syntaxin 1A in hippocampal neurons

    doi: 10.1080/19420862.2018.1551675

    Figure Lengend Snippet: Specificity test for the selected nanobodies in immunofluorescence and Western Blots. ( a b ) COS-7 cells transiently transfected with SNAP-25-EGFP or Syntaxin 1A-EGFP were stained with S25-Nb10 or Stx1A-Nb6 conjugated with a single Atto647N fluorophore. The nanobody signal correlates with the EGFP signal and shows no staining in untransfected cells (revealed by Hoechst-nuclear staining; white arrowheads). The scale bar represents 10 µm. Note that Syntaxin 1A accumulates in the ER-Golgi region due to an impaired export caused by the lack of neuronal cofactors, as it was found in the past 30 ( c d ) The following samples were loaded in a denaturating SDS-PAGE and were blotted on a nitrocellulose membrane (2 µg of each purified protein, and 20 µg of total protein for the cell or brain lysates): E. coli- purified full length SNAP-25 including a Twin-Strep-Tag (tst; 27.8 kDa), or Syntaxin 1A fused to a SUMO domain and a Twin-Strep-Tag (44.7 kDa); lysates from HEK293 cells transiently transfected with full length Syntaxin 1A-tst (HEK-Stx1A, 37.5 kDa) or SNAP-25-tst (HEK-SNAP-25, 27.8 kDa); whole rat brain and rat primary hippocampal neurons (15 days in vitro ). Detection was performed using S25-Nb10 ( c ) or Stx1A-Nb6 ( d ) conjugated to a single Atto647N. Both candidates specifically detect the bands at the expected molecular weights (displayed in the protein schematics), while showing no cross-reactivity to the opposite antigen or to any other proteins present in the lysates.

    Article Snippet: For this, we investigated by SDS-PAGE the following: Purified recombinant SNAP-25 or Syntaxin 1A produced in E. coli , lysates of HEK293 cell lines transiently expressing SNAP-25 or Syntaxin 1A, lysates of total rat brain and lysates of rat primary hippocampal cultures.

    Techniques: Immunofluorescence, Western Blot, Transfection, Staining, SDS Page, Purification, Strep-tag, In Vitro

    Investigation of the extra-synaptic populations of SNAP-25 and Syntaxin 1A in hippocampal neurons upon electrical stimulation. Cultured neurons were co-stained with nanobodies directly coupled to Atto647N and with anti-Synaptophysin antibodies (detected with a secondary coupled to Alexa488) to identify synaptic regions. ( a ) STED images of the nanobody immunostainings were analyzed. The distribution of extra-synaptic SNAP-25 remains unchanged upon stimulation at 20 Hertz for 60 seconds, resembling the pattern previously observed in Figure 5 . For control, stimulation and recovery conditions, 356, 388 and 424 synapse line profiles were analyzed from three independent experiments. ( b ) Upon stimulation, the population of Syntaxin 1A that is outside of synapses relocates into neighboring synapses. This population recovers if the neurons are further incubated for five minutes at 37 °C after stimulation. For control, stimulation and recovery conditions, 288, 144 and 280 synapses were analyzed from three independent experiments. The exemplary images show the nanobody signals in regions centered on synapses (determined by Synaptophysin staining). The scale bars represent 1 µm.

    Journal: mAbs

    Article Title: Nanobodies reveal an extra-synaptic population of SNAP-25 and Syntaxin 1A in hippocampal neurons

    doi: 10.1080/19420862.2018.1551675

    Figure Lengend Snippet: Investigation of the extra-synaptic populations of SNAP-25 and Syntaxin 1A in hippocampal neurons upon electrical stimulation. Cultured neurons were co-stained with nanobodies directly coupled to Atto647N and with anti-Synaptophysin antibodies (detected with a secondary coupled to Alexa488) to identify synaptic regions. ( a ) STED images of the nanobody immunostainings were analyzed. The distribution of extra-synaptic SNAP-25 remains unchanged upon stimulation at 20 Hertz for 60 seconds, resembling the pattern previously observed in Figure 5 . For control, stimulation and recovery conditions, 356, 388 and 424 synapse line profiles were analyzed from three independent experiments. ( b ) Upon stimulation, the population of Syntaxin 1A that is outside of synapses relocates into neighboring synapses. This population recovers if the neurons are further incubated for five minutes at 37 °C after stimulation. For control, stimulation and recovery conditions, 288, 144 and 280 synapses were analyzed from three independent experiments. The exemplary images show the nanobody signals in regions centered on synapses (determined by Synaptophysin staining). The scale bars represent 1 µm.

    Article Snippet: For this, we investigated by SDS-PAGE the following: Purified recombinant SNAP-25 or Syntaxin 1A produced in E. coli , lysates of HEK293 cell lines transiently expressing SNAP-25 or Syntaxin 1A, lysates of total rat brain and lysates of rat primary hippocampal cultures.

    Techniques: Cell Culture, Staining, Incubation

    STED microscopy shows that nanobodies detect an extra-synaptic population of SNAP-25 and Syntaxin 1A in primary hippocampal neurons. ( a b ) Cultured neurons (14–16 days in vitro ) were co-stained with SNAP-25 or Syntaxin 1A monoclonal antibodies (clone 71.1 or 78.2, respectively) or with the nanobodies bearing Atto647N fluorophores (in red) and with the pre-synaptic marker antibodies against Synaptophysin 1 (Syp1) and the post-synaptic marker Homer1. The merge panels display the synaptic markers and the other co-stained protein in red. Zoomed region of synapses suggest that antibodies and nanobodies are enriched at synapses, but the signals from the nanobodies are also present in extra-synaptic areas. The scale bars represent 2 µm and 500 nm in the low and high zoom, respectively. ( c ) For two-color STED microscopy, primary neurons were co-stained using the same monoclonal antibodies as in ( a ), detected using a secondary antibody conjugated to Abberior-Star580 and with the nanobodies coupled to Atto647N. Synapses were located in confocal mode with Synaptophysin antibodies as in ( a ). The merge panel only includes the two STED channels for simplicity. The zoomed areas show each channel and the overlap of both antibody and nanobody STED signals. The scale bars represent 2 µm and 500 nm in the low and high zoom, respectively. ( d e ) Analysis of the signal distribution up to 1 µm from the center of a synapse (determined by the center of mass of the Synaptophysin staining). We analyzed 176 synapse line scans from six independent experiments for SNAP-25 and 632 synapse line scans from six independent experiments for Syntaxin 1A. The nanobody signals are significantly higher than the antibody signals outside synapses for both experiments (Wilcoxon rank sum tests; p = 0.0016 for SNAP-25; p

    Journal: mAbs

    Article Title: Nanobodies reveal an extra-synaptic population of SNAP-25 and Syntaxin 1A in hippocampal neurons

    doi: 10.1080/19420862.2018.1551675

    Figure Lengend Snippet: STED microscopy shows that nanobodies detect an extra-synaptic population of SNAP-25 and Syntaxin 1A in primary hippocampal neurons. ( a b ) Cultured neurons (14–16 days in vitro ) were co-stained with SNAP-25 or Syntaxin 1A monoclonal antibodies (clone 71.1 or 78.2, respectively) or with the nanobodies bearing Atto647N fluorophores (in red) and with the pre-synaptic marker antibodies against Synaptophysin 1 (Syp1) and the post-synaptic marker Homer1. The merge panels display the synaptic markers and the other co-stained protein in red. Zoomed region of synapses suggest that antibodies and nanobodies are enriched at synapses, but the signals from the nanobodies are also present in extra-synaptic areas. The scale bars represent 2 µm and 500 nm in the low and high zoom, respectively. ( c ) For two-color STED microscopy, primary neurons were co-stained using the same monoclonal antibodies as in ( a ), detected using a secondary antibody conjugated to Abberior-Star580 and with the nanobodies coupled to Atto647N. Synapses were located in confocal mode with Synaptophysin antibodies as in ( a ). The merge panel only includes the two STED channels for simplicity. The zoomed areas show each channel and the overlap of both antibody and nanobody STED signals. The scale bars represent 2 µm and 500 nm in the low and high zoom, respectively. ( d e ) Analysis of the signal distribution up to 1 µm from the center of a synapse (determined by the center of mass of the Synaptophysin staining). We analyzed 176 synapse line scans from six independent experiments for SNAP-25 and 632 synapse line scans from six independent experiments for Syntaxin 1A. The nanobody signals are significantly higher than the antibody signals outside synapses for both experiments (Wilcoxon rank sum tests; p = 0.0016 for SNAP-25; p

    Article Snippet: For this, we investigated by SDS-PAGE the following: Purified recombinant SNAP-25 or Syntaxin 1A produced in E. coli , lysates of HEK293 cell lines transiently expressing SNAP-25 or Syntaxin 1A, lysates of total rat brain and lysates of rat primary hippocampal cultures.

    Techniques: Microscopy, Cell Culture, In Vitro, Staining, Marker

    Expression of EFCAB2 in mouse testes. (A) Northern blot analysis of Efcab2 mRNA in adult mouse tissues. Total RNAs (10 μg each) prepared from the various tissues were electrophoresed, transferred to a nylon membrane, and then hybridized with a DIG-labeled cRNA probe. The arrow indicates Efcab2 mRNA. 28S and 18S RNA were stained as the loading controls. kb, kilobase. (B) Western blot analysis of total protein extracts from 293T cells transfected with control (P3xFLAG-CMV-14 plasmid) and EFCAB2-overexpression plasmids (OE). Immunoblotting with anti β-Actin as a loading control (left panel) or anti-EFCAB2 antiserum (right panel). The arrow indicates EFCAB2 protein band. (C) Western blot analysis of EFCAB2 protein in adult mouse testes. Total testis proteins were extracted, and 20 μg were separated by 12.5% SDS-PAGE. Left panel shows Coomassie Brilliant Blue-staining (CBB) as a loading control, while right panel shows the immunoblotted PVDF membrane with anti-EFCAB2 antiserum. The arrow indicates EFCAB2 protein band. Bovine serum albumin (BSA) was used as a negative control. M: Molecular marker (kilodalton; kDa).

    Journal: PLoS ONE

    Article Title: EFCAB2 is a novel calcium-binding protein in mouse testis and sperm

    doi: 10.1371/journal.pone.0214687

    Figure Lengend Snippet: Expression of EFCAB2 in mouse testes. (A) Northern blot analysis of Efcab2 mRNA in adult mouse tissues. Total RNAs (10 μg each) prepared from the various tissues were electrophoresed, transferred to a nylon membrane, and then hybridized with a DIG-labeled cRNA probe. The arrow indicates Efcab2 mRNA. 28S and 18S RNA were stained as the loading controls. kb, kilobase. (B) Western blot analysis of total protein extracts from 293T cells transfected with control (P3xFLAG-CMV-14 plasmid) and EFCAB2-overexpression plasmids (OE). Immunoblotting with anti β-Actin as a loading control (left panel) or anti-EFCAB2 antiserum (right panel). The arrow indicates EFCAB2 protein band. (C) Western blot analysis of EFCAB2 protein in adult mouse testes. Total testis proteins were extracted, and 20 μg were separated by 12.5% SDS-PAGE. Left panel shows Coomassie Brilliant Blue-staining (CBB) as a loading control, while right panel shows the immunoblotted PVDF membrane with anti-EFCAB2 antiserum. The arrow indicates EFCAB2 protein band. Bovine serum albumin (BSA) was used as a negative control. M: Molecular marker (kilodalton; kDa).

    Article Snippet: After linearization by restriction enzymes, sense and antisense cRNA probes were synthesized by in vitro transcription with T7 or SP6 RNA polymerase (DIG RNA Labeling Kit; Roche Applied Science).

    Techniques: Expressing, Northern Blot, Labeling, Staining, Western Blot, Transfection, Plasmid Preparation, Over Expression, SDS Page, Negative Control, Marker

    Bleomycin-induced acute lung injury augments IL-17 expression in alcohol-fed mice. Three-month-old wild-type mice were treated ± alcohol (20% v/v in drinking water for 8 weeks) before intratracheal administration of bleomycin (2.5 units/kg) or saline vehicle. Lungs were collected at 7 and 14 days following induction of injury and analyzed for IL-17 protein expression by Western blot. *p

    Journal: Alcoholism, clinical and experimental research

    Article Title: Alcohol-induced interleukin-17 expression causes murine lung fibroblast-to-myofibroblast transdifferentiation via Thy-1 downregulation

    doi: 10.1111/acer.14110

    Figure Lengend Snippet: Bleomycin-induced acute lung injury augments IL-17 expression in alcohol-fed mice. Three-month-old wild-type mice were treated ± alcohol (20% v/v in drinking water for 8 weeks) before intratracheal administration of bleomycin (2.5 units/kg) or saline vehicle. Lungs were collected at 7 and 14 days following induction of injury and analyzed for IL-17 protein expression by Western blot. *p

    Article Snippet: Cells were treated with either alcohol (60 mM), TGFβ1 (2 ng/ml; R & D Systems, Minneapolis, MN), or IL-17 (10 ng/ml; R & D Systems).

    Techniques: Expressing, Mouse Assay, Western Blot

    Chronic alcohol ingestion increases the systemic Th17 immune response. CD4 + T cells were harvested from the spleen and lymph nodes of three-month-old control-fed and alcohol-fed wild-type mice (20% v/v in drinking water for 8 weeks). ( A ) Cells were analyzed for IL-17 gene expression by quantitative PCR. CD4 + T cells harvested from control-fed and alcohol-fed animals were activated ex vivo with anti-CD3 (5 μg/ml) and anti-CD28 (1 μg/ml) for 72 hours. ( B ) Cell culture supernatant was analyzed for IL-17 protein expression by ELISA. ( C ) Lastly, FACS analysis was performed on freshly isolated peripheral CD4 + T cells to determine the percentage of Th17 cells (CD4 + IL-17 + ). The right panel shows representative scattered dot plots. *p

    Journal: Alcoholism, clinical and experimental research

    Article Title: Alcohol-induced interleukin-17 expression causes murine lung fibroblast-to-myofibroblast transdifferentiation via Thy-1 downregulation

    doi: 10.1111/acer.14110

    Figure Lengend Snippet: Chronic alcohol ingestion increases the systemic Th17 immune response. CD4 + T cells were harvested from the spleen and lymph nodes of three-month-old control-fed and alcohol-fed wild-type mice (20% v/v in drinking water for 8 weeks). ( A ) Cells were analyzed for IL-17 gene expression by quantitative PCR. CD4 + T cells harvested from control-fed and alcohol-fed animals were activated ex vivo with anti-CD3 (5 μg/ml) and anti-CD28 (1 μg/ml) for 72 hours. ( B ) Cell culture supernatant was analyzed for IL-17 protein expression by ELISA. ( C ) Lastly, FACS analysis was performed on freshly isolated peripheral CD4 + T cells to determine the percentage of Th17 cells (CD4 + IL-17 + ). The right panel shows representative scattered dot plots. *p

    Article Snippet: Cells were treated with either alcohol (60 mM), TGFβ1 (2 ng/ml; R & D Systems, Minneapolis, MN), or IL-17 (10 ng/ml; R & D Systems).

    Techniques: Mouse Assay, Expressing, Real-time Polymerase Chain Reaction, Ex Vivo, Cell Culture, Enzyme-linked Immunosorbent Assay, FACS, Isolation

    Chronic alcohol ingestion did not alter the CD4 + Th17 population in the lung. CD4 + T cells were isolated from the lung of three-month-old control-fed and alcohol-fed wild-type mice. FACS analysis was performed on freshly isolated peripheral CD4 + T cells to determine the percentage of Th17 cells (CD4 + IL-17 + ). The right panel shows representative scattered dot plots. N = 3 per group. Data are presented as mean ± SEM.

    Journal: Alcoholism, clinical and experimental research

    Article Title: Alcohol-induced interleukin-17 expression causes murine lung fibroblast-to-myofibroblast transdifferentiation via Thy-1 downregulation

    doi: 10.1111/acer.14110

    Figure Lengend Snippet: Chronic alcohol ingestion did not alter the CD4 + Th17 population in the lung. CD4 + T cells were isolated from the lung of three-month-old control-fed and alcohol-fed wild-type mice. FACS analysis was performed on freshly isolated peripheral CD4 + T cells to determine the percentage of Th17 cells (CD4 + IL-17 + ). The right panel shows representative scattered dot plots. N = 3 per group. Data are presented as mean ± SEM.

    Article Snippet: Cells were treated with either alcohol (60 mM), TGFβ1 (2 ng/ml; R & D Systems, Minneapolis, MN), or IL-17 (10 ng/ml; R & D Systems).

    Techniques: Isolation, Mouse Assay, FACS

    Alcohol exposure induces Th17 differentiation and enhances IL-17 production in both naïve CD4 + T helper cells (Th0) and Th17 cells in vitro . CD4 + T cells were harvested from the spleen and lymph nodes of three-month-old wild-type mice. Naïve CD4 + T helper cells were activated ex vivo with anti-CD3 (5 μg/ml) and anti-CD28 (1 μg/ml) for 72 hours (Th0). A subgroup of Th0 cells were activated and polarized with recombinant IL-6 (20 ng/ml) and recombinant TGFβ1 (5 ng/ml) for 72 hours to generate Th17 cells. Th0 and Th17 CD4 + T cells were cultured ± alcohol (60 mM) for 72 hours. The cell culture supernatant was collected and analyzed for IL-17 production by ELISA. *p

    Journal: Alcoholism, clinical and experimental research

    Article Title: Alcohol-induced interleukin-17 expression causes murine lung fibroblast-to-myofibroblast transdifferentiation via Thy-1 downregulation

    doi: 10.1111/acer.14110

    Figure Lengend Snippet: Alcohol exposure induces Th17 differentiation and enhances IL-17 production in both naïve CD4 + T helper cells (Th0) and Th17 cells in vitro . CD4 + T cells were harvested from the spleen and lymph nodes of three-month-old wild-type mice. Naïve CD4 + T helper cells were activated ex vivo with anti-CD3 (5 μg/ml) and anti-CD28 (1 μg/ml) for 72 hours (Th0). A subgroup of Th0 cells were activated and polarized with recombinant IL-6 (20 ng/ml) and recombinant TGFβ1 (5 ng/ml) for 72 hours to generate Th17 cells. Th0 and Th17 CD4 + T cells were cultured ± alcohol (60 mM) for 72 hours. The cell culture supernatant was collected and analyzed for IL-17 production by ELISA. *p

    Article Snippet: Cells were treated with either alcohol (60 mM), TGFβ1 (2 ng/ml; R & D Systems, Minneapolis, MN), or IL-17 (10 ng/ml; R & D Systems).

    Techniques: In Vitro, Mouse Assay, Ex Vivo, Recombinant, Cell Culture, Enzyme-linked Immunosorbent Assay

    IL-17 and TGFβ1 independently and additively inhibit lung fibroblast Thy-1 expression. Primary lung fibroblasts (PLFs from passage 3–8) were isolated from wild-type mice and cultured ± IL-17 (10 ng/ml) ± TGFβ1 (2 ng/ml) for 72 hours prior to analysis for Thy-1 by FACS. *p

    Journal: Alcoholism, clinical and experimental research

    Article Title: Alcohol-induced interleukin-17 expression causes murine lung fibroblast-to-myofibroblast transdifferentiation via Thy-1 downregulation

    doi: 10.1111/acer.14110

    Figure Lengend Snippet: IL-17 and TGFβ1 independently and additively inhibit lung fibroblast Thy-1 expression. Primary lung fibroblasts (PLFs from passage 3–8) were isolated from wild-type mice and cultured ± IL-17 (10 ng/ml) ± TGFβ1 (2 ng/ml) for 72 hours prior to analysis for Thy-1 by FACS. *p

    Article Snippet: Cells were treated with either alcohol (60 mM), TGFβ1 (2 ng/ml; R & D Systems, Minneapolis, MN), or IL-17 (10 ng/ml; R & D Systems).

    Techniques: Expressing, Isolation, Mouse Assay, Cell Culture, FACS

    Thy-1 mediates the effects of IL-17 on α-SMA. Thy-1 subpopulations were enriched by immunomagnetic separation using the Miltenyi magnetic bead system. Thy-1 + and Thy-1 − PLFs were exposed to IL-17 (10 ng/ml) for 72 hours. Thy1 − PLFs (white bar (untreated) and dark gray bar (treated)) and Thy-1 + PLFs (black bar (untreated) and light gray bar (treated)) were harvested and then analyzed for α-SMA protein expression by Western blot. The top panel shows representative immunoblots. *p

    Journal: Alcoholism, clinical and experimental research

    Article Title: Alcohol-induced interleukin-17 expression causes murine lung fibroblast-to-myofibroblast transdifferentiation via Thy-1 downregulation

    doi: 10.1111/acer.14110

    Figure Lengend Snippet: Thy-1 mediates the effects of IL-17 on α-SMA. Thy-1 subpopulations were enriched by immunomagnetic separation using the Miltenyi magnetic bead system. Thy-1 + and Thy-1 − PLFs were exposed to IL-17 (10 ng/ml) for 72 hours. Thy1 − PLFs (white bar (untreated) and dark gray bar (treated)) and Thy-1 + PLFs (black bar (untreated) and light gray bar (treated)) were harvested and then analyzed for α-SMA protein expression by Western blot. The top panel shows representative immunoblots. *p

    Article Snippet: Cells were treated with either alcohol (60 mM), TGFβ1 (2 ng/ml; R & D Systems, Minneapolis, MN), or IL-17 (10 ng/ml; R & D Systems).

    Techniques: Immunomagnetic Separation, Expressing, Western Blot

    IL-17 selectively induces myofibroblast stress fiber development in Thy-1 negative (Thy-1 − ) lung fibroblasts. Thy-1 + and Thy-1 − mouse PLFs were separately treated with IL-17 (10 ng/ml) or TGFβ1 (5 ng/ml) for 96 hours. Myofibroblast transdifferentiation was evaluated using immunofluorescent staining for α-SMA (green). DAPI (blue) was used for nuclear staining. Panels ( A - B ) show untreated Thy-1 + and Thy-1 − cells, respectively, ( C - D ) show Thy-1 + and Thy-1 − cells treated with IL-17, respectively, and ( E and F ) show Thy-1 + and Thy-1 − cells treated with TGFβ1, respectively. ( G ) Four random high-power fields from each sample were analyzed for stress fiber formation. Scale bar = 100 μm. *p

    Journal: Alcoholism, clinical and experimental research

    Article Title: Alcohol-induced interleukin-17 expression causes murine lung fibroblast-to-myofibroblast transdifferentiation via Thy-1 downregulation

    doi: 10.1111/acer.14110

    Figure Lengend Snippet: IL-17 selectively induces myofibroblast stress fiber development in Thy-1 negative (Thy-1 − ) lung fibroblasts. Thy-1 + and Thy-1 − mouse PLFs were separately treated with IL-17 (10 ng/ml) or TGFβ1 (5 ng/ml) for 96 hours. Myofibroblast transdifferentiation was evaluated using immunofluorescent staining for α-SMA (green). DAPI (blue) was used for nuclear staining. Panels ( A - B ) show untreated Thy-1 + and Thy-1 − cells, respectively, ( C - D ) show Thy-1 + and Thy-1 − cells treated with IL-17, respectively, and ( E and F ) show Thy-1 + and Thy-1 − cells treated with TGFβ1, respectively. ( G ) Four random high-power fields from each sample were analyzed for stress fiber formation. Scale bar = 100 μm. *p

    Article Snippet: Cells were treated with either alcohol (60 mM), TGFβ1 (2 ng/ml; R & D Systems, Minneapolis, MN), or IL-17 (10 ng/ml; R & D Systems).

    Techniques: Staining

    IL-17 and alcohol upregulate α-SMA in lung fibroblasts. Total lung fibroblasts (Thy-1 + and Thy-1 − subpopulations) were exposed to either alcohol (60 mM) or IL-17 (10 ng/ml) for 72 hours. PLFs were harvested and then analyzed for α-SMA protein expression by Western blot. Untreated cells are represented by the white bar, alcohol-treated cells by the black bar, and IL-17-treated cells by the gray bar. The top panel shows a representative immunoblot. *p

    Journal: Alcoholism, clinical and experimental research

    Article Title: Alcohol-induced interleukin-17 expression causes murine lung fibroblast-to-myofibroblast transdifferentiation via Thy-1 downregulation

    doi: 10.1111/acer.14110

    Figure Lengend Snippet: IL-17 and alcohol upregulate α-SMA in lung fibroblasts. Total lung fibroblasts (Thy-1 + and Thy-1 − subpopulations) were exposed to either alcohol (60 mM) or IL-17 (10 ng/ml) for 72 hours. PLFs were harvested and then analyzed for α-SMA protein expression by Western blot. Untreated cells are represented by the white bar, alcohol-treated cells by the black bar, and IL-17-treated cells by the gray bar. The top panel shows a representative immunoblot. *p

    Article Snippet: Cells were treated with either alcohol (60 mM), TGFβ1 (2 ng/ml; R & D Systems, Minneapolis, MN), or IL-17 (10 ng/ml; R & D Systems).

    Techniques: Expressing, Western Blot

    Hypothesis schematic showing the effects of IL-17 on myofibroblast development in chronic alcohol ingestion. In the current study, we showed that in the otherwise healthy animals, chronic alcohol ingestion increased systemic Th17 immune response. Acute injury caused a persistent increase in IL-17 in the lung. Alcohol, IL-17, and TGFβ1 independently inhibited Thy-1 expression by lung fibroblasts and IL-17 and TGFβ1 additively decreased Thy-1 expression leading to myofibroblast differentiation. We believe this sequence of events is one of the mechanisms by which alcohol induces fibroproliferative disrepair following acute lung injury.

    Journal: Alcoholism, clinical and experimental research

    Article Title: Alcohol-induced interleukin-17 expression causes murine lung fibroblast-to-myofibroblast transdifferentiation via Thy-1 downregulation

    doi: 10.1111/acer.14110

    Figure Lengend Snippet: Hypothesis schematic showing the effects of IL-17 on myofibroblast development in chronic alcohol ingestion. In the current study, we showed that in the otherwise healthy animals, chronic alcohol ingestion increased systemic Th17 immune response. Acute injury caused a persistent increase in IL-17 in the lung. Alcohol, IL-17, and TGFβ1 independently inhibited Thy-1 expression by lung fibroblasts and IL-17 and TGFβ1 additively decreased Thy-1 expression leading to myofibroblast differentiation. We believe this sequence of events is one of the mechanisms by which alcohol induces fibroproliferative disrepair following acute lung injury.

    Article Snippet: Cells were treated with either alcohol (60 mM), TGFβ1 (2 ng/ml; R & D Systems, Minneapolis, MN), or IL-17 (10 ng/ml; R & D Systems).

    Techniques: Expressing, Sequencing