anti polyglutamine expansion diseases marker antibody  (Millipore)


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    Structured Review

    Millipore anti polyglutamine expansion diseases marker antibody
    Staufen1 protein but not mRNA steady-state levels are increased in neurodegenerative disease cells and tissues. Western blot analysis of SCA2- FBs ( a ) and LBCs ( b ) show increased STAU1 levels compared with normal controls. DDX6 levels are unchanged. HD and SCA3 patient <t>(polyQ</t> expanded) FBs were used as additional controls. Four normal and five SCA2 FBs, and two normal and three SCA2 LBCs were used. c , d Western blot analyses of ATXN2 Q127 ( c ) and BAC-Q72 ( d ) mouse cerebellar extracts (24 weeks of age) showing increased Stau1 levels compared with wild-type or BAC-Q22 controls ( n = 2–3 animals per group). e Western blot of FB extracts from an ALS patient with the TDP-43 G298S mutation show increased STAU1 levels. β-Actin was used as loading control and representative blots of three independent experiments are shown. f – h STAU1 RNA levels are unaltered in SCA2 and ALS cells and SCA2 mice. qRT-PCR analyses of STAU1 mRNA in SCA2 FBs and ALS FB with TDP-43 G298S mutation ( f ) or SCA2 LBCs ( g ). h qRT-PCR analyses of cerebellar RNAs from ATXN2 Q127 and BAC-Q72 mice compared to wild-type littermates (24 weeks of age; n = animals per group). Gene expression levels were normalized to Actb
    Anti Polyglutamine Expansion Diseases Marker Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 948 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Staufen1 links RNA stress granules and autophagy in a model of neurodegeneration"

    Article Title: Staufen1 links RNA stress granules and autophagy in a model of neurodegeneration

    Journal: Nature Communications

    doi: 10.1038/s41467-018-06041-3

    Staufen1 protein but not mRNA steady-state levels are increased in neurodegenerative disease cells and tissues. Western blot analysis of SCA2- FBs ( a ) and LBCs ( b ) show increased STAU1 levels compared with normal controls. DDX6 levels are unchanged. HD and SCA3 patient (polyQ expanded) FBs were used as additional controls. Four normal and five SCA2 FBs, and two normal and three SCA2 LBCs were used. c , d Western blot analyses of ATXN2 Q127 ( c ) and BAC-Q72 ( d ) mouse cerebellar extracts (24 weeks of age) showing increased Stau1 levels compared with wild-type or BAC-Q22 controls ( n = 2–3 animals per group). e Western blot of FB extracts from an ALS patient with the TDP-43 G298S mutation show increased STAU1 levels. β-Actin was used as loading control and representative blots of three independent experiments are shown. f – h STAU1 RNA levels are unaltered in SCA2 and ALS cells and SCA2 mice. qRT-PCR analyses of STAU1 mRNA in SCA2 FBs and ALS FB with TDP-43 G298S mutation ( f ) or SCA2 LBCs ( g ). h qRT-PCR analyses of cerebellar RNAs from ATXN2 Q127 and BAC-Q72 mice compared to wild-type littermates (24 weeks of age; n = animals per group). Gene expression levels were normalized to Actb
    Figure Legend Snippet: Staufen1 protein but not mRNA steady-state levels are increased in neurodegenerative disease cells and tissues. Western blot analysis of SCA2- FBs ( a ) and LBCs ( b ) show increased STAU1 levels compared with normal controls. DDX6 levels are unchanged. HD and SCA3 patient (polyQ expanded) FBs were used as additional controls. Four normal and five SCA2 FBs, and two normal and three SCA2 LBCs were used. c , d Western blot analyses of ATXN2 Q127 ( c ) and BAC-Q72 ( d ) mouse cerebellar extracts (24 weeks of age) showing increased Stau1 levels compared with wild-type or BAC-Q22 controls ( n = 2–3 animals per group). e Western blot of FB extracts from an ALS patient with the TDP-43 G298S mutation show increased STAU1 levels. β-Actin was used as loading control and representative blots of three independent experiments are shown. f – h STAU1 RNA levels are unaltered in SCA2 and ALS cells and SCA2 mice. qRT-PCR analyses of STAU1 mRNA in SCA2 FBs and ALS FB with TDP-43 G298S mutation ( f ) or SCA2 LBCs ( g ). h qRT-PCR analyses of cerebellar RNAs from ATXN2 Q127 and BAC-Q72 mice compared to wild-type littermates (24 weeks of age; n = animals per group). Gene expression levels were normalized to Actb

    Techniques Used: Western Blot, BAC Assay, Mutagenesis, Mouse Assay, Quantitative RT-PCR, Expressing

    2) Product Images from "ClC-2 knockdown prevents cerebrovascular remodeling via inhibition of the Wnt/β-catenin signaling pathway"

    Article Title: ClC-2 knockdown prevents cerebrovascular remodeling via inhibition of the Wnt/β-catenin signaling pathway

    Journal: Cellular & Molecular Biology Letters

    doi: 10.1186/s11658-018-0095-z

    Lack of ClC-2 reduced AngII-induced HBVSMC proliferation. a and b Cells were transfected with ClC-2 siRNA (siClC-2; 20 nM) or negative siRNA (negative; 20 nM) for 48 h in prior to angiotensin II (AngII) treatment (10 − 7 M) for another 48 h. Cell proliferation was determined using the CCK-8 assay ( a ) and BrdU incorporation ( b ). c and d The protein expressions of PCNA ( c ) and Ki67 ( d ) were detected using western blotting. ** p
    Figure Legend Snippet: Lack of ClC-2 reduced AngII-induced HBVSMC proliferation. a and b Cells were transfected with ClC-2 siRNA (siClC-2; 20 nM) or negative siRNA (negative; 20 nM) for 48 h in prior to angiotensin II (AngII) treatment (10 − 7 M) for another 48 h. Cell proliferation was determined using the CCK-8 assay ( a ) and BrdU incorporation ( b ). c and d The protein expressions of PCNA ( c ) and Ki67 ( d ) were detected using western blotting. ** p

    Techniques Used: Transfection, CCK-8 Assay, BrdU Incorporation Assay, Western Blot

    3) Product Images from "Filamin A Phosphorylation at Serine 2152 by the Serine/Threonine Kinase Ndr2 Controls TCR-Induced LFA-1 Activation in T Cells"

    Article Title: Filamin A Phosphorylation at Serine 2152 by the Serine/Threonine Kinase Ndr2 Controls TCR-Induced LFA-1 Activation in T Cells

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02852

    Ndr2-deficiency in murine CD4 + T cells attenuates TCR-induced FLNa phosphorylation at S2152, T-cell adhesion and LFA-1-dependent upregulation of CD69 in vitro . (A) Purified splenic wild type (WT) and Ndr2 −/− CD4 + T cells were left untreated or stimulated with anti-CD3 antibodies for the indicated time points. Lysates were prepared and analyzed by Western blotting using the indicated antibodies. Densitrometric quantification of FLNa phosphorylation at Serine 2152 (pFLNa) normalized to total FLNa (tFLNa) ( n = 3). (B) Purified splenic WT and Ndr2 −/− CD4 + T cells were left untreated (non) or stimulated with anti-CD3 antibodies (CD3) and subsequently analyzed for their ability to bind plate-bound Fc-ICAM-1. Adherent cells were counted and calculated as percentage of input ( n = 3). (C) Purified splenic CD4 + T cells from WT and Ndr2 mice were cultured with plate-bound anti-CD3 antibodies (CD3) in the absence or presence of Fc-ICAM-1 (ICAM-1) with or without blocking LFA-1 antibodies (LFA-1) for 12 h. The upregulation of the activation marker CD69 of unstimulated (0 h) or activated T cells (12 h) were assessed by flow cytometry to determine the mean fluorescence intensity (MFI) ( n = 3). (mean ± SEM; * p ≤ 0.05, ** p ≤ 0.01).
    Figure Legend Snippet: Ndr2-deficiency in murine CD4 + T cells attenuates TCR-induced FLNa phosphorylation at S2152, T-cell adhesion and LFA-1-dependent upregulation of CD69 in vitro . (A) Purified splenic wild type (WT) and Ndr2 −/− CD4 + T cells were left untreated or stimulated with anti-CD3 antibodies for the indicated time points. Lysates were prepared and analyzed by Western blotting using the indicated antibodies. Densitrometric quantification of FLNa phosphorylation at Serine 2152 (pFLNa) normalized to total FLNa (tFLNa) ( n = 3). (B) Purified splenic WT and Ndr2 −/− CD4 + T cells were left untreated (non) or stimulated with anti-CD3 antibodies (CD3) and subsequently analyzed for their ability to bind plate-bound Fc-ICAM-1. Adherent cells were counted and calculated as percentage of input ( n = 3). (C) Purified splenic CD4 + T cells from WT and Ndr2 mice were cultured with plate-bound anti-CD3 antibodies (CD3) in the absence or presence of Fc-ICAM-1 (ICAM-1) with or without blocking LFA-1 antibodies (LFA-1) for 12 h. The upregulation of the activation marker CD69 of unstimulated (0 h) or activated T cells (12 h) were assessed by flow cytometry to determine the mean fluorescence intensity (MFI) ( n = 3). (mean ± SEM; * p ≤ 0.05, ** p ≤ 0.01).

    Techniques Used: In Vitro, Purification, Western Blot, Mouse Assay, Cell Culture, Blocking Assay, Activation Assay, Marker, Flow Cytometry, Cytometry, Fluorescence

    Activated Ndr2 releases FLNa binding from LFA-1. (A) Jurkat T cells were transfected with constructs that suppress endogenous Ndr2 (shNdr2) and re-express a FLAG-tagged shRNA-resistant wild type (WT Ndr2) or a kinase-dead mutant of Ndr2 (K119A Ndr2). 48 h after transfection, whole-cell extracts were prepared and analyzed by Western blotting using the indicated antibodies. Numbers represent the reduction and re-expression of Ndr2 and its mutant after normalization to the Ndr2 expression level of shC-tranfected control cells. (B,C) Cells left untreated or stimulated for the indicated time points with CD3 antibodies. Lysates were used for immunoprecipitation of LFA-1 using anti-CD11a antibodies. Precipitates were divided and analyzed by Western blotting for FLNa, Talin and Kindlin-3 association. Densitrometric analyses of FLNa, Talin, or Kindlin-3 associated to LFA-1 are depicted in Figure S8 .
    Figure Legend Snippet: Activated Ndr2 releases FLNa binding from LFA-1. (A) Jurkat T cells were transfected with constructs that suppress endogenous Ndr2 (shNdr2) and re-express a FLAG-tagged shRNA-resistant wild type (WT Ndr2) or a kinase-dead mutant of Ndr2 (K119A Ndr2). 48 h after transfection, whole-cell extracts were prepared and analyzed by Western blotting using the indicated antibodies. Numbers represent the reduction and re-expression of Ndr2 and its mutant after normalization to the Ndr2 expression level of shC-tranfected control cells. (B,C) Cells left untreated or stimulated for the indicated time points with CD3 antibodies. Lysates were used for immunoprecipitation of LFA-1 using anti-CD11a antibodies. Precipitates were divided and analyzed by Western blotting for FLNa, Talin and Kindlin-3 association. Densitrometric analyses of FLNa, Talin, or Kindlin-3 associated to LFA-1 are depicted in Figure S8 .

    Techniques Used: Binding Assay, Transfection, Construct, shRNA, Mutagenesis, Western Blot, Expressing, Immunoprecipitation

    Kinase activity of Ndr2 controls TCR-mediated adhesion, interaction of T cells with APCs and LFA-1 activation. (A) Schematic representation of the suppression/re-expression plasmids for Ndr2 used in this study. (B) Jurkat T cells were transfected with suppression/re-expression plasmids which do not suppress endogenous Ndr2 (shC), reduce the endogenous protein level of Ndr2 (shNdr2), re-express a FLAG-tagged shRNA-resistant wild type Ndr2 (WT Ndr2) or re-express its kinase dead mutant (K119A Ndr2). 48 h after transfection, lysates were analyzed by Western blotting for Ndr2, Ndr1, FLAG, and β-actin (loading control). Numbers represent the reduction and re-expression of Ndr2 and its mutant after normalization to the Ndr2 expression level of the shC-tranfected control cells, which were set to 1 ( n = 4; right graph). (C) Transfected Jurkat T cells as described in (B) were analyzed for their ability to adhere to ICAM-1-coated wells in a resting state or stimulated for 30 min with CD3 antibodies. Adherent cells were counted and calculated as percentage of input ( n = 4). (D) Cells were transfected as described in (B) and analyzed for their ability to form conjugates with DDAO-SE (red)-stained Raji B cells that were pulsed without (non) or with superantigen (SA) for 30 min. The percentage of conjugates was defined as the number of double positive events in the upper right quadrant ( n = 4). (E) Jurkat T cells transfected as described in (B) were left untreated (non) or stimulated with CD3 antibodies (CD3), followed by staining with the anti-LFA-1 antibody mAb24 which recognizes the high affinity conformation of LFA-1. mAb24 epitope expression was assessed by flow cytometry within the GFP gate and data are normalized against LFA-1 expression detected by MEM48 ( n = 4). (mean ± SEM; * p ≤ 0.05; *** p ≤ 0.001).
    Figure Legend Snippet: Kinase activity of Ndr2 controls TCR-mediated adhesion, interaction of T cells with APCs and LFA-1 activation. (A) Schematic representation of the suppression/re-expression plasmids for Ndr2 used in this study. (B) Jurkat T cells were transfected with suppression/re-expression plasmids which do not suppress endogenous Ndr2 (shC), reduce the endogenous protein level of Ndr2 (shNdr2), re-express a FLAG-tagged shRNA-resistant wild type Ndr2 (WT Ndr2) or re-express its kinase dead mutant (K119A Ndr2). 48 h after transfection, lysates were analyzed by Western blotting for Ndr2, Ndr1, FLAG, and β-actin (loading control). Numbers represent the reduction and re-expression of Ndr2 and its mutant after normalization to the Ndr2 expression level of the shC-tranfected control cells, which were set to 1 ( n = 4; right graph). (C) Transfected Jurkat T cells as described in (B) were analyzed for their ability to adhere to ICAM-1-coated wells in a resting state or stimulated for 30 min with CD3 antibodies. Adherent cells were counted and calculated as percentage of input ( n = 4). (D) Cells were transfected as described in (B) and analyzed for their ability to form conjugates with DDAO-SE (red)-stained Raji B cells that were pulsed without (non) or with superantigen (SA) for 30 min. The percentage of conjugates was defined as the number of double positive events in the upper right quadrant ( n = 4). (E) Jurkat T cells transfected as described in (B) were left untreated (non) or stimulated with CD3 antibodies (CD3), followed by staining with the anti-LFA-1 antibody mAb24 which recognizes the high affinity conformation of LFA-1. mAb24 epitope expression was assessed by flow cytometry within the GFP gate and data are normalized against LFA-1 expression detected by MEM48 ( n = 4). (mean ± SEM; * p ≤ 0.05; *** p ≤ 0.001).

    Techniques Used: Activity Assay, Activation Assay, Expressing, Transfection, shRNA, Mutagenesis, Western Blot, Staining, Flow Cytometry, Cytometry

    Ndr2 phosphorylates FLNa at S2152 in vitro . (A) Purified WT Ndr2/Mob2 heterodimer was used to phosphorylate a positional scanning peptide library using radiolabeled ATP. The degree of phosphorylation of each component of the library, harboring the indicated amino acid residue at the indicated position relative to the phosphorylation site, is shown at left. Quantified data were normalized, log 2 transformed, and used to generate a heat map shown at right ( n = 2). (B) HEK 293T cells were transfected with either empty pEFBOS vector (vector) or plasmids encoding FLAG-tagged wild type Ndr2 (FNdr2) and a kinase dead (K119A) mutant of Ndr2 (FNdr2K119A). Cells were left untreated or treated with okadaic acid (OA), lysed and Ndr2 was immunoprecipitated using FLAG antibodies. A GST-FLNa fragment (19–24 repeats) was used as substrate for an in vitro kinase assay. Reactions were analyzed by Western blotting with the indicated antibodies ( n = 3). (C) Jurkat T cells were left untreated or stimulated for the indicated time points with CD3 antibodies. Cells were lysed and analyzed by Western Blotting with the indicated antibodies. Aliquots of whole-cell extracts were analyzed for the phosphorylation status of ERK1/2 to verify successful stimulation of T cells. Densitrometric analysis of the FLNa phosphorylation status at Serine 2152 (pFLNa) normalized to total FLNa (tFLNa) ( n = 4) (mean ± SEM; ** p ≤ 0.01).
    Figure Legend Snippet: Ndr2 phosphorylates FLNa at S2152 in vitro . (A) Purified WT Ndr2/Mob2 heterodimer was used to phosphorylate a positional scanning peptide library using radiolabeled ATP. The degree of phosphorylation of each component of the library, harboring the indicated amino acid residue at the indicated position relative to the phosphorylation site, is shown at left. Quantified data were normalized, log 2 transformed, and used to generate a heat map shown at right ( n = 2). (B) HEK 293T cells were transfected with either empty pEFBOS vector (vector) or plasmids encoding FLAG-tagged wild type Ndr2 (FNdr2) and a kinase dead (K119A) mutant of Ndr2 (FNdr2K119A). Cells were left untreated or treated with okadaic acid (OA), lysed and Ndr2 was immunoprecipitated using FLAG antibodies. A GST-FLNa fragment (19–24 repeats) was used as substrate for an in vitro kinase assay. Reactions were analyzed by Western blotting with the indicated antibodies ( n = 3). (C) Jurkat T cells were left untreated or stimulated for the indicated time points with CD3 antibodies. Cells were lysed and analyzed by Western Blotting with the indicated antibodies. Aliquots of whole-cell extracts were analyzed for the phosphorylation status of ERK1/2 to verify successful stimulation of T cells. Densitrometric analysis of the FLNa phosphorylation status at Serine 2152 (pFLNa) normalized to total FLNa (tFLNa) ( n = 4) (mean ± SEM; ** p ≤ 0.01).

    Techniques Used: In Vitro, Purification, Transformation Assay, Transfection, Plasmid Preparation, Mutagenesis, Immunoprecipitation, Kinase Assay, Western Blot

    Ndr2 phosphorylates FLNa at S2152 in Jurkat T cells in vivo . (A) Jurkat T cells were transfected with suppression/re-expression constructs which suppress endogenous Ndr2 (shNdr2) and re-express a FLAG-tagged shRNA-resistant wild type (WT Ndr2) or a kinase-dead mutant of Ndr2 (K119A Ndr2). Numbers represent the reduction and re-expression of Ndr2 and its mutant after normalization to the Ndr2 expression level of the shC-tranfected control cells. (B) At 48 h post-transfection, cells left untreated or stimulated for the indicated time points with CD3 antibodies. Cells were lysed and analyzed by Western Blotting with the indicated antibodies. Densitrometric quantification of FLNa phosphorylation at Serine 2152 (pFLNa) normalized to total FLNa (tFLNa) ( n = 3). (mean ± SEM; ** p ≤ 0.05).
    Figure Legend Snippet: Ndr2 phosphorylates FLNa at S2152 in Jurkat T cells in vivo . (A) Jurkat T cells were transfected with suppression/re-expression constructs which suppress endogenous Ndr2 (shNdr2) and re-express a FLAG-tagged shRNA-resistant wild type (WT Ndr2) or a kinase-dead mutant of Ndr2 (K119A Ndr2). Numbers represent the reduction and re-expression of Ndr2 and its mutant after normalization to the Ndr2 expression level of the shC-tranfected control cells. (B) At 48 h post-transfection, cells left untreated or stimulated for the indicated time points with CD3 antibodies. Cells were lysed and analyzed by Western Blotting with the indicated antibodies. Densitrometric quantification of FLNa phosphorylation at Serine 2152 (pFLNa) normalized to total FLNa (tFLNa) ( n = 3). (mean ± SEM; ** p ≤ 0.05).

    Techniques Used: In Vivo, Transfection, Expressing, Construct, shRNA, Mutagenesis, Western Blot

    Expression profile, activation status and localization of Ndr2 in primary lymphocytes and lymphocyte-derived cell lines. (A) Total cell lysates of primary human and murine lymphocytes, Jurkat T cells, Raji B cells and HEK 293T cells were analyzed by Western blotting for expression of Ndr2 and Ndr1. β-actin staining served as loading control. Densitrometric analysis was performed to determine the Ndr2/Ndr1 ratio ( n = 3; right graph). (B) Jurkat T cells were stimulated with CD3 antibodies for the indicated time points. Cells were lysed and Ndr2 was immunoprecipitated using Ndr2 rabbit antibody. Ndr2-precipitates were divided and one half of the precipitates was used to assess Ndr2 kinase activity by an in vitro kinase assay (IVK) using the myelin basic protein (MBP) as substrate. Phosphorylation of MBP was visualized with autoradiography. Densitrometric analysis were performed to determine the intensity of all MBP bands and values of MBP intensities from time point 0 min were set to 1 ( n = 2; right graph). The second half of precipitates was used to detect Ndr2 by Western blotting. Aliquots of whole-cell extracts were analyzed for the phosphorylation status of ERK1/2 to verify successful stimulation of T cells (Input/lower panel). (C) Splenic B cells were loaded with OVA-peptide and co-incubated with purified T cells derived from OVA-TCR transgenic DO11.10 mice for 30 min. Cells were fixed, permeabilized and stained with an anti-Ndr2 Abs in combination with anti-rabbit IgG-FITC (green). F-actin was visualized with TRITC-Phalloidin (red) (upper panel). T/B cell conjugates were stained with Cy3-labeled anti-CD3 mAbs (red) and for Ndr2 (green; as described above; lower panel). Cells were imaged by confocal microscopy. Representative conjugates are shown. Each study was repeated at least three times and more than 25 conjugates were examined per condition. Scale bars define 5 μm. (mean ± SEM).
    Figure Legend Snippet: Expression profile, activation status and localization of Ndr2 in primary lymphocytes and lymphocyte-derived cell lines. (A) Total cell lysates of primary human and murine lymphocytes, Jurkat T cells, Raji B cells and HEK 293T cells were analyzed by Western blotting for expression of Ndr2 and Ndr1. β-actin staining served as loading control. Densitrometric analysis was performed to determine the Ndr2/Ndr1 ratio ( n = 3; right graph). (B) Jurkat T cells were stimulated with CD3 antibodies for the indicated time points. Cells were lysed and Ndr2 was immunoprecipitated using Ndr2 rabbit antibody. Ndr2-precipitates were divided and one half of the precipitates was used to assess Ndr2 kinase activity by an in vitro kinase assay (IVK) using the myelin basic protein (MBP) as substrate. Phosphorylation of MBP was visualized with autoradiography. Densitrometric analysis were performed to determine the intensity of all MBP bands and values of MBP intensities from time point 0 min were set to 1 ( n = 2; right graph). The second half of precipitates was used to detect Ndr2 by Western blotting. Aliquots of whole-cell extracts were analyzed for the phosphorylation status of ERK1/2 to verify successful stimulation of T cells (Input/lower panel). (C) Splenic B cells were loaded with OVA-peptide and co-incubated with purified T cells derived from OVA-TCR transgenic DO11.10 mice for 30 min. Cells were fixed, permeabilized and stained with an anti-Ndr2 Abs in combination with anti-rabbit IgG-FITC (green). F-actin was visualized with TRITC-Phalloidin (red) (upper panel). T/B cell conjugates were stained with Cy3-labeled anti-CD3 mAbs (red) and for Ndr2 (green; as described above; lower panel). Cells were imaged by confocal microscopy. Representative conjugates are shown. Each study was repeated at least three times and more than 25 conjugates were examined per condition. Scale bars define 5 μm. (mean ± SEM).

    Techniques Used: Expressing, Activation Assay, Derivative Assay, Western Blot, Staining, Immunoprecipitation, Activity Assay, In Vitro, Kinase Assay, Autoradiography, Incubation, Purification, Transgenic Assay, Mouse Assay, Labeling, Confocal Microscopy

    4) Product Images from "Human ex vivo 3D bone model recapitulates osteocyte response to metastatic prostate cancer"

    Article Title: Human ex vivo 3D bone model recapitulates osteocyte response to metastatic prostate cancer

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-36424-x

    Histology sections of the engineered 3D bone tissues. Representative H E staining of vertical 3D tissue sections ( a) −PCa cells (control cultures without PCa cells), showing an intact endosteal layer (black arrows) and ( b ) +PCa cells (co-cultured with PCa cells) showing compromised tissue (black arrows). ( c ) Sections were stained with pan-cytokeratin to identify PCa cells (green). ( d ) Representative image showing the atypically rounded morphology of osteocytes throughout the tissue when cultured with PCa cells. ( e ) Quantification of active caspase-3 immunofluorescence staining (*p
    Figure Legend Snippet: Histology sections of the engineered 3D bone tissues. Representative H E staining of vertical 3D tissue sections ( a) −PCa cells (control cultures without PCa cells), showing an intact endosteal layer (black arrows) and ( b ) +PCa cells (co-cultured with PCa cells) showing compromised tissue (black arrows). ( c ) Sections were stained with pan-cytokeratin to identify PCa cells (green). ( d ) Representative image showing the atypically rounded morphology of osteocytes throughout the tissue when cultured with PCa cells. ( e ) Quantification of active caspase-3 immunofluorescence staining (*p

    Techniques Used: Staining, Cell Culture, Immunofluorescence

    5) Product Images from "Cryo-EM structure of the bacteria killing type IV secretion system core complex from Xanthomonas citri"

    Article Title: Cryo-EM structure of the bacteria killing type IV secretion system core complex from Xanthomonas citri

    Journal: Nature microbiology

    doi: 10.1038/s41564-018-0262-z

    Effect of specific mutations in the core complex on T4S system-mediated cell lysis of neighboring E. coli cells and VirB10 localization. a , Left panel: Bacterial killing assay measuring the ability of X. citri to use the T4S system to lyse neighboring E. coli cells. The results obtained for wild-type X. citri cells (dashed grey line) are compared to the assay performed using the X. citri virB10-msfGFP strain (solid black line). The dashed black horizontal line corresponds to the no lysis baseline after subtracting the E. coli only background signal from the data. Data are mean ± s.d. (n=9 for X. citri virB10-msfGFP and n=27 for wild-type X. citri ). Central panel: Representative image of the X. citri virB10-msfGFP strain as obtained by epifluorescence microscopy displaying discrete msfGFP foci that indicate the presence of T4S systems. Image shows msfGFP intensity levels of a 0.5-µm region containing the focal plane of the cells. The inset shows the superposition of the locations of fluorescent VirB10-msfGFP foci obtained from 100 individual X. citri virB10-msfGFP cells. Right panel: An enhanced image obtained by deconvolution of the obtained Z-slices (not used for quantification) more clearly showing discrete foci (see Methods ). Scale bar: 5 μm. The X. citri virB10-msfGFP strain was imaged and analyzed at least 6 times independently with similar results. b , Representative epifluorescence microscopy images (as described in a ) for a selected series of X. citri virB10-msfGFP mutant strains in VirB7, VirB9 or VirB10. Note that ΔVirB7 and VirB10 C206S cells are mostly devoid of fluorescence. Other strains, such as VirB7 W34A , present more diffuse fluorescence and lack clear foci. Also note that the few foci shown in the cell contour insets of mutants severely deficient in killing (see below) are due to occasional background detection. Scale bar: 5 μm. All X. citri virB10-msfGFP mutant strains were imaged and analyzed together on two separate occasions independently with similar results. c , Bacterial killing assays of the X. citri virB10-msfGFP strains shown in b . Each X. citri virB10-msfGFP mutant was compared to the X. citri virB10-msfGFP strain (solid black line). Data are mean ± s.d. (n=9 for X. citri virB10-msfGFP and n=4 for each mutant).
    Figure Legend Snippet: Effect of specific mutations in the core complex on T4S system-mediated cell lysis of neighboring E. coli cells and VirB10 localization. a , Left panel: Bacterial killing assay measuring the ability of X. citri to use the T4S system to lyse neighboring E. coli cells. The results obtained for wild-type X. citri cells (dashed grey line) are compared to the assay performed using the X. citri virB10-msfGFP strain (solid black line). The dashed black horizontal line corresponds to the no lysis baseline after subtracting the E. coli only background signal from the data. Data are mean ± s.d. (n=9 for X. citri virB10-msfGFP and n=27 for wild-type X. citri ). Central panel: Representative image of the X. citri virB10-msfGFP strain as obtained by epifluorescence microscopy displaying discrete msfGFP foci that indicate the presence of T4S systems. Image shows msfGFP intensity levels of a 0.5-µm region containing the focal plane of the cells. The inset shows the superposition of the locations of fluorescent VirB10-msfGFP foci obtained from 100 individual X. citri virB10-msfGFP cells. Right panel: An enhanced image obtained by deconvolution of the obtained Z-slices (not used for quantification) more clearly showing discrete foci (see Methods ). Scale bar: 5 μm. The X. citri virB10-msfGFP strain was imaged and analyzed at least 6 times independently with similar results. b , Representative epifluorescence microscopy images (as described in a ) for a selected series of X. citri virB10-msfGFP mutant strains in VirB7, VirB9 or VirB10. Note that ΔVirB7 and VirB10 C206S cells are mostly devoid of fluorescence. Other strains, such as VirB7 W34A , present more diffuse fluorescence and lack clear foci. Also note that the few foci shown in the cell contour insets of mutants severely deficient in killing (see below) are due to occasional background detection. Scale bar: 5 μm. All X. citri virB10-msfGFP mutant strains were imaged and analyzed together on two separate occasions independently with similar results. c , Bacterial killing assays of the X. citri virB10-msfGFP strains shown in b . Each X. citri virB10-msfGFP mutant was compared to the X. citri virB10-msfGFP strain (solid black line). Data are mean ± s.d. (n=9 for X. citri virB10-msfGFP and n=4 for each mutant).

    Techniques Used: Lysis, Epifluorescence Microscopy, Mutagenesis, Fluorescence

    Structure of the X. citri T4S system core complex. a , Top view of the structure in ribbon representation with one VirB7-VirB9-VirB10 heterotrimer shown in red, green and blue, respectively (coloring maintained in parts b - e ). The heterotrimer numbering used in this study is indicated, with the colored heterotrimer serving as reference and therefore numbered 0. b and c , Top view ( b ) and side view ( c, upper panel ) of the structure in surface representation. c, lower panel : cut-away side view of the model. External and internal dimensions of specific structural features are indicated in b and c . The 80-Å opening at the bottom of the I-chamber is similar to that observed for the 8.5-Å cryo-EM structure of the elastase-digested pKM101 core complex 17 . d and e , Side view of the structure in surface representation as in c but rotated 60 degrees counter-clockwise to show heterotrimer 0 more clearly. e is a zoomed in view of the region delimited by the dashed line in d .
    Figure Legend Snippet: Structure of the X. citri T4S system core complex. a , Top view of the structure in ribbon representation with one VirB7-VirB9-VirB10 heterotrimer shown in red, green and blue, respectively (coloring maintained in parts b - e ). The heterotrimer numbering used in this study is indicated, with the colored heterotrimer serving as reference and therefore numbered 0. b and c , Top view ( b ) and side view ( c, upper panel ) of the structure in surface representation. c, lower panel : cut-away side view of the model. External and internal dimensions of specific structural features are indicated in b and c . The 80-Å opening at the bottom of the I-chamber is similar to that observed for the 8.5-Å cryo-EM structure of the elastase-digested pKM101 core complex 17 . d and e , Side view of the structure in surface representation as in c but rotated 60 degrees counter-clockwise to show heterotrimer 0 more clearly. e is a zoomed in view of the region delimited by the dashed line in d .

    Techniques Used:

    The heterotrimer of the X. citri T4S system core complex structure and comparison with that of pKM101 from E. coli . The X. citri VirB7-VirB9-VirB10 heterotrimer. a , Proteins are shown in ribbon representation and color-coded as in Figure 2 . Specific domains are labeled, as also are the secondary structures in each protein. The linkers between VirB9 NTD and CTD, the two α-helices of the VirB10 “antennae” and the CTD of VirB10 and VirB10 NTD_150-161 helix are shown in dashed green and blue lines, respectively, as they are disordered in the structure. The orientation is as in Figure 2a . b , Same as in a , but rotated 90 degrees to correspond to the side view shown in Figure 2c . The box delimits the central compact core. c , Superposition of the structure of the X. citri heterotrimer (in red, green and blue) with that of the pKM101 complex (grey) composed of TraN/VirB7, TraO CTD /VirB9 CTD , and TraF CTD /VirB10 CTD .
    Figure Legend Snippet: The heterotrimer of the X. citri T4S system core complex structure and comparison with that of pKM101 from E. coli . The X. citri VirB7-VirB9-VirB10 heterotrimer. a , Proteins are shown in ribbon representation and color-coded as in Figure 2 . Specific domains are labeled, as also are the secondary structures in each protein. The linkers between VirB9 NTD and CTD, the two α-helices of the VirB10 “antennae” and the CTD of VirB10 and VirB10 NTD_150-161 helix are shown in dashed green and blue lines, respectively, as they are disordered in the structure. The orientation is as in Figure 2a . b , Same as in a , but rotated 90 degrees to correspond to the side view shown in Figure 2c . The box delimits the central compact core. c , Superposition of the structure of the X. citri heterotrimer (in red, green and blue) with that of the pKM101 complex (grey) composed of TraN/VirB7, TraO CTD /VirB9 CTD , and TraF CTD /VirB10 CTD .

    Techniques Used: Labeling

    Interactions between heterotrimers in the X. citri T4S system core complex. a , Overall structure of the core complex with 3 heterotrimers highlighted. These are labeled 0, -1, and -2 and color-coded as in Figure 2 with different shades of the respective colors for each heterotrimer. b , Interactions between the VirB10 CTD s of heterotrimer 0 (0VirB10 CTD ) and -1 (-1VirB10 CTD ). The β1 strand is labeled “Lever arm” due to its correspondence to part of the same structural feature observed in pKM101 core complex O-layer 15 . c , Interactions between the VirB9 CTD of heterotrimer 0 (0VirB9 CTD ) and VirB7 of heterotrimer 0 (0VirB7) and -1 (-1VirB7). d , Interactions between adjacent VirB9 NTD s. The two VirB9 NTD s shown are from heterotrimer 0 (0VirB9 NTD ) and -1 (-1VirB9 NTD ). e , Interactions between the VirB9 NTD of heterotrimer 0 (0VirB9 NTD ) and VirB10 CTD of heterotrimer -1 (-1VirB10 CTD ) and -2 (-2VirB10 CTD ). f , Interactions of a helical structure belonging to VirB10 NTD (VirB10 NTD_150-161 ) with two adjacent VirB9 NTD s. Left panel: surface representation of the lumen of the X. citri core complex structure, color-coded by proteins with VirB7, VirB9, and VirB10 in red, green and blue, respectively. Upper-right panel: zoomed view of the region contained in the black box shown at left. VirB9 NTD s and the VirB10 NTD_150-161 helix are shown in ribbon representation. The electron density for the VirB10 NTD_150-161 is also shown (grey) contoured at 4 σ level. Lower-right panel: stereo figure of residue specific interactions between the VirB10 NTD_150-161 helix and two adjacent VirB9 NTD s. The side chains of interfacing residues are in stick representation with nitrogen and oxygen atoms colored in blue and red, respectively. Carbon atoms are in the color of the ribbon they emanate from. Two shades of green were used to distinguish residues from two different VirB9 chains.
    Figure Legend Snippet: Interactions between heterotrimers in the X. citri T4S system core complex. a , Overall structure of the core complex with 3 heterotrimers highlighted. These are labeled 0, -1, and -2 and color-coded as in Figure 2 with different shades of the respective colors for each heterotrimer. b , Interactions between the VirB10 CTD s of heterotrimer 0 (0VirB10 CTD ) and -1 (-1VirB10 CTD ). The β1 strand is labeled “Lever arm” due to its correspondence to part of the same structural feature observed in pKM101 core complex O-layer 15 . c , Interactions between the VirB9 CTD of heterotrimer 0 (0VirB9 CTD ) and VirB7 of heterotrimer 0 (0VirB7) and -1 (-1VirB7). d , Interactions between adjacent VirB9 NTD s. The two VirB9 NTD s shown are from heterotrimer 0 (0VirB9 NTD ) and -1 (-1VirB9 NTD ). e , Interactions between the VirB9 NTD of heterotrimer 0 (0VirB9 NTD ) and VirB10 CTD of heterotrimer -1 (-1VirB10 CTD ) and -2 (-2VirB10 CTD ). f , Interactions of a helical structure belonging to VirB10 NTD (VirB10 NTD_150-161 ) with two adjacent VirB9 NTD s. Left panel: surface representation of the lumen of the X. citri core complex structure, color-coded by proteins with VirB7, VirB9, and VirB10 in red, green and blue, respectively. Upper-right panel: zoomed view of the region contained in the black box shown at left. VirB9 NTD s and the VirB10 NTD_150-161 helix are shown in ribbon representation. The electron density for the VirB10 NTD_150-161 is also shown (grey) contoured at 4 σ level. Lower-right panel: stereo figure of residue specific interactions between the VirB10 NTD_150-161 helix and two adjacent VirB9 NTD s. The side chains of interfacing residues are in stick representation with nitrogen and oxygen atoms colored in blue and red, respectively. Carbon atoms are in the color of the ribbon they emanate from. Two shades of green were used to distinguish residues from two different VirB9 chains.

    Techniques Used: Labeling

    Killing efficiency is correlated with T4S system assembly in X. citri . a , Top panel: The relative number of VirB10-msfGFP foci per cell is plotted for each strain. The distribution of cells according to the number of foci per cell is represented by the randomly placed shaded circles in each bin. Tukey box and whisker plots depict the data: black central line (median), box (first and third quartiles), whiskers (data within 1.5 IQR), green triangles (mean) (see Supplementary Table 4 ). The sample size (n) of cells of each strain analyzed by fluorescence microscopy (from two independent experiments) is listed at the top. Bottom panel: The mean (± s.d.) relative capacity of each mutant to lyse E. coli cells in a X. citri / E. coli co-culture is shown (as in Figure 5 and Supplementary Table 4 ). Red dots represent mutants produced in the VirB10-msfGFP background and black dots represent mutants produced in the non-GFP background. In competitions using GFP background strains the n=4 (except for VirB9 A142-S147 (n=3), VirB10 T325-T335 (n=3), ΔVirB7 (n=6), and VirB10-msfGFP (n=9). In competitions using non-GFP background strains the n=3. b , Western blot assays using polyclonal antibodies (Ab) against specific T4S system components or against msfGFP in different X. citri virB10-msfGFP mutant strains (see Methods ). The first lane contains total protein extract from E. coli BL21(DE3) expressing the X. citri core complex with normal length VirB10 as does that for the VirB9 V29D mutant (red asterisks), which was obtained only in non-GFP genomic background. Experiments were repeated 3 times with similar results. † indicates mutations in the VirB9 Linker and ‡ indicates mutations in the VirB10 linker for VirB10 ΔQ175-D182 or the VirB10 antenna for VirB10 ΔT325-T335 . Colored diamonds denote the values of the relative killing efficiency according to the code: Less than 0.3 (red), 0.3 to 0.7 (yellow), greater than 0.7 (green) (see a and Supplementary Table 4 ). WT=wild-type. Theoretical molecular weight (in kDa) for each mature protein is shown at right. c , Western blot detection of VirB10 in mutant strains produced in the non-GFP genomic background. Annotations are the same as in b . Experiments were repeated twice with similar results. Full western blots are presented in Supplementary Figure 9b-g .
    Figure Legend Snippet: Killing efficiency is correlated with T4S system assembly in X. citri . a , Top panel: The relative number of VirB10-msfGFP foci per cell is plotted for each strain. The distribution of cells according to the number of foci per cell is represented by the randomly placed shaded circles in each bin. Tukey box and whisker plots depict the data: black central line (median), box (first and third quartiles), whiskers (data within 1.5 IQR), green triangles (mean) (see Supplementary Table 4 ). The sample size (n) of cells of each strain analyzed by fluorescence microscopy (from two independent experiments) is listed at the top. Bottom panel: The mean (± s.d.) relative capacity of each mutant to lyse E. coli cells in a X. citri / E. coli co-culture is shown (as in Figure 5 and Supplementary Table 4 ). Red dots represent mutants produced in the VirB10-msfGFP background and black dots represent mutants produced in the non-GFP background. In competitions using GFP background strains the n=4 (except for VirB9 A142-S147 (n=3), VirB10 T325-T335 (n=3), ΔVirB7 (n=6), and VirB10-msfGFP (n=9). In competitions using non-GFP background strains the n=3. b , Western blot assays using polyclonal antibodies (Ab) against specific T4S system components or against msfGFP in different X. citri virB10-msfGFP mutant strains (see Methods ). The first lane contains total protein extract from E. coli BL21(DE3) expressing the X. citri core complex with normal length VirB10 as does that for the VirB9 V29D mutant (red asterisks), which was obtained only in non-GFP genomic background. Experiments were repeated 3 times with similar results. † indicates mutations in the VirB9 Linker and ‡ indicates mutations in the VirB10 linker for VirB10 ΔQ175-D182 or the VirB10 antenna for VirB10 ΔT325-T335 . Colored diamonds denote the values of the relative killing efficiency according to the code: Less than 0.3 (red), 0.3 to 0.7 (yellow), greater than 0.7 (green) (see a and Supplementary Table 4 ). WT=wild-type. Theoretical molecular weight (in kDa) for each mature protein is shown at right. c , Western blot detection of VirB10 in mutant strains produced in the non-GFP genomic background. Annotations are the same as in b . Experiments were repeated twice with similar results. Full western blots are presented in Supplementary Figure 9b-g .

    Techniques Used: Whisker Assay, Fluorescence, Microscopy, Mutagenesis, Co-Culture Assay, Produced, Western Blot, Expressing, Molecular Weight

    Biochemistry and electron microscopy map and model of the X. citri T4S system core complex. a , SDS-PAGE of the VirB7-VirB9-VirB10 core complex. Left lane labeled “Marker”: molecular weight markers, with the molecular weight for each band shown at left. Right lane labeled “CC”: purified core complex, with the three components labeled at right. Purification assays were repeated more than ten times with similar results (see also Supplementary Figure 9a ). b , Electron micrograph of the X. citri core complex, with some particles highlighted in blue circles. Scale bar: 50 nm. Experiments were repeated at least 8 times showing similar results. c , Representative of top, tilt, and side view 2D class averages obtained using RELION 2.0 (see the Methods section for more information). d , Overview of the 3.3-Å electron density, contoured at 4 σ level. e , Two representative regions of the electron density of the X. citri core complex. Electron density map contoured at a 4 σ level is shown in chicken wire representation, color-coded in grey-blue. The final model built into the map is shown in a ribbon and stick representation color-coded dark blue, red, yellow, and light blue for nitrogen, oxygen, sulfur and carbon atoms, respectively. Secondary structures are labeled. The regions depicted are both from the VirB10 CTD (left panel for α-helices and right panel for β-strands). f , Topology secondary structure diagrams of VirB7 (red), VirB9 (green), and VirB10 (blue). β-strands and α-helices are represented as arrows and cylinders, respectively. Regions for which no electron density was observed are indicated by dashed lines. Note that the 182 N-terminal residues of VirB10 were disordered and could not be traced except for a small helix corresponding to residues 150-161. This is not unexpected considering that the 101 amino acid stretch (residues 84-184) between the inner membrane spanning helix and the globular VirB10 CTD is rich in proline residues ( Supplementary Fig. 2 ).
    Figure Legend Snippet: Biochemistry and electron microscopy map and model of the X. citri T4S system core complex. a , SDS-PAGE of the VirB7-VirB9-VirB10 core complex. Left lane labeled “Marker”: molecular weight markers, with the molecular weight for each band shown at left. Right lane labeled “CC”: purified core complex, with the three components labeled at right. Purification assays were repeated more than ten times with similar results (see also Supplementary Figure 9a ). b , Electron micrograph of the X. citri core complex, with some particles highlighted in blue circles. Scale bar: 50 nm. Experiments were repeated at least 8 times showing similar results. c , Representative of top, tilt, and side view 2D class averages obtained using RELION 2.0 (see the Methods section for more information). d , Overview of the 3.3-Å electron density, contoured at 4 σ level. e , Two representative regions of the electron density of the X. citri core complex. Electron density map contoured at a 4 σ level is shown in chicken wire representation, color-coded in grey-blue. The final model built into the map is shown in a ribbon and stick representation color-coded dark blue, red, yellow, and light blue for nitrogen, oxygen, sulfur and carbon atoms, respectively. Secondary structures are labeled. The regions depicted are both from the VirB10 CTD (left panel for α-helices and right panel for β-strands). f , Topology secondary structure diagrams of VirB7 (red), VirB9 (green), and VirB10 (blue). β-strands and α-helices are represented as arrows and cylinders, respectively. Regions for which no electron density was observed are indicated by dashed lines. Note that the 182 N-terminal residues of VirB10 were disordered and could not be traced except for a small helix corresponding to residues 150-161. This is not unexpected considering that the 101 amino acid stretch (residues 84-184) between the inner membrane spanning helix and the globular VirB10 CTD is rich in proline residues ( Supplementary Fig. 2 ).

    Techniques Used: Electron Microscopy, SDS Page, Labeling, Molecular Weight, Purification

    6) Product Images from "Epitope Mapping Immunoassay Analysis of the Interaction between β-Amyloid and Fibrinogen"

    Article Title: Epitope Mapping Immunoassay Analysis of the Interaction between β-Amyloid and Fibrinogen

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms20030496

    Each anti-Fbg, pAb and mAb, was tested using the indirect ELISA system. ( A ) At an Fbg-coated plate, the pAb had good affinity while the mAb had poor affinity. ( B ) At an Fbg fragment E-coated plate, the pAb had good affinity while the mAb had no affinity. ( C ) At an Fbg fragment D-coated plate, both the pAb and the mAb had good affinity.
    Figure Legend Snippet: Each anti-Fbg, pAb and mAb, was tested using the indirect ELISA system. ( A ) At an Fbg-coated plate, the pAb had good affinity while the mAb had poor affinity. ( B ) At an Fbg fragment E-coated plate, the pAb had good affinity while the mAb had no affinity. ( C ) At an Fbg fragment D-coated plate, both the pAb and the mAb had good affinity.

    Techniques Used: Indirect ELISA

    7) Product Images from "Deletion of Mtu1 (Trmu) in zebrafish revealed the essential role of tRNA modification in mitochondrial biogenesis and hearing function"

    Article Title: Deletion of Mtu1 (Trmu) in zebrafish revealed the essential role of tRNA modification in mitochondrial biogenesis and hearing function

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky758

    Mitochondrial defects in hair cells. ( A ) Assessment of mitochondrial function in hair cells by enzyme histochemistry (EHC) staining for SDH and COX in the frozen-sections of posterior macula of mtu1 −/− , mtu1 +/− and mtu1 +/+ zebrafish at 5dpf. Loss of EHC signal is indicated by arrows (magnification X400). V, ventral; L, lateral; Ov, otic vesicle; Hc, hair cell; Sc, supporting cell. ( B ) Mitochondrial networks from hair cells of inner electron microscopy. Ultrathin sections were visualized with 5000×, 10 000×, 20 000× and 60 000× magnifications. ( C ) Quantification of mitochondrial numbers of hair cells from the mtu1 −/− , mtu1 +/− mutant and mtu1 +/+ zebrafish. The calculations were based on 50 different hair cells of mtu1 −/− , mtu1 +/− mutant and mtu1 +/+ zebrafish, respectively. Graph details and symbols are explained in the legend to Figure 3 .
    Figure Legend Snippet: Mitochondrial defects in hair cells. ( A ) Assessment of mitochondrial function in hair cells by enzyme histochemistry (EHC) staining for SDH and COX in the frozen-sections of posterior macula of mtu1 −/− , mtu1 +/− and mtu1 +/+ zebrafish at 5dpf. Loss of EHC signal is indicated by arrows (magnification X400). V, ventral; L, lateral; Ov, otic vesicle; Hc, hair cell; Sc, supporting cell. ( B ) Mitochondrial networks from hair cells of inner electron microscopy. Ultrathin sections were visualized with 5000×, 10 000×, 20 000× and 60 000× magnifications. ( C ) Quantification of mitochondrial numbers of hair cells from the mtu1 −/− , mtu1 +/− mutant and mtu1 +/+ zebrafish. The calculations were based on 50 different hair cells of mtu1 −/− , mtu1 +/− mutant and mtu1 +/+ zebrafish, respectively. Graph details and symbols are explained in the legend to Figure 3 .

    Techniques Used: Staining, Electron Microscopy, Mutagenesis

    The expression patterns of Zebrafish Mtu1 in the sensory organs. ( A ) Whole-mount in situ hybridization on wild type larval zebrafish at various ages (4–72 hpf). ( B ) Lateral views with anterior to the left show mtu1 expression in otic vesicle (Ov), liver (Lv), esophagus (Eso), swimming bladder (Sb) and intestine (In) at 120 hpf. ( C, D ) Transverse frozen sections through dashed line C’ and D’ in B were used for the assessment of mtu1 expression in neuromast (Nm), posterior macula (Pm) and anterior macula (Am) hair cells, respectively. Insets show higher magnifications of Nm and Pm. Scale bars: 100 μm. V, ventral; L, lateral; B: brain; E: eye; Pf: pectoral fin; Ec: epidermal cells; C: cristae; Nc: notochord.
    Figure Legend Snippet: The expression patterns of Zebrafish Mtu1 in the sensory organs. ( A ) Whole-mount in situ hybridization on wild type larval zebrafish at various ages (4–72 hpf). ( B ) Lateral views with anterior to the left show mtu1 expression in otic vesicle (Ov), liver (Lv), esophagus (Eso), swimming bladder (Sb) and intestine (In) at 120 hpf. ( C, D ) Transverse frozen sections through dashed line C’ and D’ in B were used for the assessment of mtu1 expression in neuromast (Nm), posterior macula (Pm) and anterior macula (Am) hair cells, respectively. Insets show higher magnifications of Nm and Pm. Scale bars: 100 μm. V, ventral; L, lateral; B: brain; E: eye; Pf: pectoral fin; Ec: epidermal cells; C: cristae; Nc: notochord.

    Techniques Used: Expressing, In Situ Hybridization

    Northern blot analysis of mitochondrial tRNAs. ( A, C ). Five μg of total cellular RNA samples from mutant and wild type zebrafish were electrophoresed through a denaturing polyacrylamide gel, electroblotted and hybridized with DIG-labeled oligonucleotide probes specific for mitochondrial tRNA Lys , tRNA Glu , tRNA Gln , tRNA Met , tRNA Trp , tRNA His , tRNA Leu(UUR) , cytoplasmic tRNA Glu , tRNA Ala and tRNA Gly as well as 5S rRNA probe, respectively. ( B, D ) Quantification of the levels of tRNAs. Average relative levels tRNA content were normalized to the average content in the mutant and wild type 5S rRNA, respectively. The values for the mtu1 +/− and mtu1 −/− zebrafish are expressed as percentages of the average values for the mtu1 +/+ zebrafish. The calculations were based on three independent determinations. Graph details and symbols are explained in the legend to the Figure 3 .
    Figure Legend Snippet: Northern blot analysis of mitochondrial tRNAs. ( A, C ). Five μg of total cellular RNA samples from mutant and wild type zebrafish were electrophoresed through a denaturing polyacrylamide gel, electroblotted and hybridized with DIG-labeled oligonucleotide probes specific for mitochondrial tRNA Lys , tRNA Glu , tRNA Gln , tRNA Met , tRNA Trp , tRNA His , tRNA Leu(UUR) , cytoplasmic tRNA Glu , tRNA Ala and tRNA Gly as well as 5S rRNA probe, respectively. ( B, D ) Quantification of the levels of tRNAs. Average relative levels tRNA content were normalized to the average content in the mutant and wild type 5S rRNA, respectively. The values for the mtu1 +/− and mtu1 −/− zebrafish are expressed as percentages of the average values for the mtu1 +/+ zebrafish. The calculations were based on three independent determinations. Graph details and symbols are explained in the legend to the Figure 3 .

    Techniques Used: Northern Blot, Mutagenesis, Labeling

    APM gel electrophoresis combined with Northern blotting of mitochondrial tRNAs. ( A ) Five μg ( mtu1 +/+ ), 10 μg ( mtu1 +/− ) and 20 μg ( mtu1 −/− ) of zebrafish total RNAs were separated by polyacrylamide gel electrophoresis that contains 0.05 mg/ml APM, electroblotted onto a positively charged membrane, and hybridized with a DIG-labeled oligonucleotide probe specific for the mt-tRNA Lys . The blots were then stripped and the membrane was re-hybridized with DIG-labeled probes for mt-tRNA Glu , mt-tRNA Gln , and mt-tRNA Leu(UUR) , respectively. The retarded bands of 2-thiolated tRNAs and non-retarded bands of tRNA without thiolation are marked by arrows. ( B ) Five micrograms of total RNA from various genotype Zebrafish for APM gel to determine 2-thiolation levels of cytosolic tRNA Glu and tRNA Ala . ( C ) Proportion in vivo of the 2-thiolated tRNA levels. The proportion values for the mutant zebrafish are expressed as percentages of the average values for the wild type zebrafish. The calculations were based on three independent determinations of each tRNA in each fish. The error bars indicate standard errors; P indicates the significance, according to Student's t test, of the difference between mutant and wild type for each tRNA.
    Figure Legend Snippet: APM gel electrophoresis combined with Northern blotting of mitochondrial tRNAs. ( A ) Five μg ( mtu1 +/+ ), 10 μg ( mtu1 +/− ) and 20 μg ( mtu1 −/− ) of zebrafish total RNAs were separated by polyacrylamide gel electrophoresis that contains 0.05 mg/ml APM, electroblotted onto a positively charged membrane, and hybridized with a DIG-labeled oligonucleotide probe specific for the mt-tRNA Lys . The blots were then stripped and the membrane was re-hybridized with DIG-labeled probes for mt-tRNA Glu , mt-tRNA Gln , and mt-tRNA Leu(UUR) , respectively. The retarded bands of 2-thiolated tRNAs and non-retarded bands of tRNA without thiolation are marked by arrows. ( B ) Five micrograms of total RNA from various genotype Zebrafish for APM gel to determine 2-thiolation levels of cytosolic tRNA Glu and tRNA Ala . ( C ) Proportion in vivo of the 2-thiolated tRNA levels. The proportion values for the mutant zebrafish are expressed as percentages of the average values for the wild type zebrafish. The calculations were based on three independent determinations of each tRNA in each fish. The error bars indicate standard errors; P indicates the significance, according to Student's t test, of the difference between mutant and wild type for each tRNA.

    Techniques Used: Nucleic Acid Electrophoresis, Northern Blot, Polyacrylamide Gel Electrophoresis, Labeling, In Vivo, Mutagenesis, Fluorescence In Situ Hybridization

    Generation of mtu1 knock-out zebrafish using CRISPR/Cas9 system. ( A ) Schematic representation of CRISPR/Cas9 target site at exon 4 as used in this study. An allele, mtu1 ins32bp was produced by a 32 bp insertion in exon 4 and a truncated 145 amino acid non-functional protein. ( B – D ) Genotyping of mtu1 ins32bp by Sanger sequence, the PAGE RFLP and Western blot analyses. ( E ) The morphology of mtu1 −/− , mtu1 +/− and mtu1 +/+ zebrafish at 3 dpf. ( F ) The ratios of genotypes/phenotype of offsprings (F2) in clutches from different F1 mtu1 heterozygous crosses at 10 dpf ( n = 350).
    Figure Legend Snippet: Generation of mtu1 knock-out zebrafish using CRISPR/Cas9 system. ( A ) Schematic representation of CRISPR/Cas9 target site at exon 4 as used in this study. An allele, mtu1 ins32bp was produced by a 32 bp insertion in exon 4 and a truncated 145 amino acid non-functional protein. ( B – D ) Genotyping of mtu1 ins32bp by Sanger sequence, the PAGE RFLP and Western blot analyses. ( E ) The morphology of mtu1 −/− , mtu1 +/− and mtu1 +/+ zebrafish at 3 dpf. ( F ) The ratios of genotypes/phenotype of offsprings (F2) in clutches from different F1 mtu1 heterozygous crosses at 10 dpf ( n = 350).

    Techniques Used: Knock-Out, CRISPR, Produced, Functional Assay, Sequencing, Polyacrylamide Gel Electrophoresis, Western Blot

    Defects in hearing organs in zebrafish at 5 dpf. ( A ) Otolith morphologies of mtu1 −/− , mtu1 +/− and mtu1 +/+ zebrafish were illustrated under a Leica microscope with an objective magnification of 20 X. Lateral views of the otic vesicle were shown in the low arrows. V, ventral; P, posterior. ( B ) Quantification of sizes of posterior otolith in the mtu1 −/− ( n = 98), mtu1 +/− ( n = 95) and mtu1 +/+ ( n = 99) zebrafish. ( C ) Lateral and dorsal views of zebrafish larvae show the distribution of neuromasts along the body. A, anterior; P, posterior. ( D ) Larval of mtu1 −/− , mtu1 +/− and mtu1 +/+ zebrafish were fluorescently labeled with FM1-43FX and observed under a stereoscopic microscope with an objective magnification of 20 X. Neuromast numbers of anterior lateral line (ALL) and posterior lateral line (PLL) in mutant and wild type fishes were counted, respectively. The arrows indicated the positions where the number of neuromasts were significantly reduced in the mutant fishes. ( E ) Quantification of neuromasts numbers of posterior lateral line (PLL) from the mtu1 −/− , mtu1 +/− mutant and wild type zebrafish. The calculations were based on the numbers of mtu1 −/− ( n = 32), mtu1 +/− ( n = 20) and mtu1 +/+ ( n = 21) larvae. ( F ) The hair cells, stereocilia and nuclei of neuromas from mutant and wild type zebrafish were stained with FM1-43FX (red), phalloidin (green) and DAPI (blue), respectively. Scale bars = 10 μm. ( G ) Quantification of numbers of hair cells for the mtu1 −/− ( n = 25), mtu1 +/− ( n = 17) and mtu1 +/+ ( n = 28) zebrafish. Graph details and symbols are explained in the legend to Figure 3 .
    Figure Legend Snippet: Defects in hearing organs in zebrafish at 5 dpf. ( A ) Otolith morphologies of mtu1 −/− , mtu1 +/− and mtu1 +/+ zebrafish were illustrated under a Leica microscope with an objective magnification of 20 X. Lateral views of the otic vesicle were shown in the low arrows. V, ventral; P, posterior. ( B ) Quantification of sizes of posterior otolith in the mtu1 −/− ( n = 98), mtu1 +/− ( n = 95) and mtu1 +/+ ( n = 99) zebrafish. ( C ) Lateral and dorsal views of zebrafish larvae show the distribution of neuromasts along the body. A, anterior; P, posterior. ( D ) Larval of mtu1 −/− , mtu1 +/− and mtu1 +/+ zebrafish were fluorescently labeled with FM1-43FX and observed under a stereoscopic microscope with an objective magnification of 20 X. Neuromast numbers of anterior lateral line (ALL) and posterior lateral line (PLL) in mutant and wild type fishes were counted, respectively. The arrows indicated the positions where the number of neuromasts were significantly reduced in the mutant fishes. ( E ) Quantification of neuromasts numbers of posterior lateral line (PLL) from the mtu1 −/− , mtu1 +/− mutant and wild type zebrafish. The calculations were based on the numbers of mtu1 −/− ( n = 32), mtu1 +/− ( n = 20) and mtu1 +/+ ( n = 21) larvae. ( F ) The hair cells, stereocilia and nuclei of neuromas from mutant and wild type zebrafish were stained with FM1-43FX (red), phalloidin (green) and DAPI (blue), respectively. Scale bars = 10 μm. ( G ) Quantification of numbers of hair cells for the mtu1 −/− ( n = 25), mtu1 +/− ( n = 17) and mtu1 +/+ ( n = 28) zebrafish. Graph details and symbols are explained in the legend to Figure 3 .

    Techniques Used: Microscopy, Labeling, Mutagenesis, Staining

    Reduced density of hair cell bundles in inner ear labyrinth. ( A ) Anatomy of adult wild type zebrafish inner ear labyrinth, showing two lagenas (L), two saccule (S), and two utricle (U). ( B – D ) Hair bundle density in lagenas, saccule and utricle. The hair cells, nuclei of from mutant and wild type zebrafish were stained with phalloidin (green) and DAPI (blue), respectively. Hair cell counts were sampled at six locations of lagenas, four locations of saccule and six locations of utricle, respectively. A 1600 μm 2 box was placed at each sampling area and labeled hair cell bundles were counted within each box to determine hair cell density were selected for counting. A, anterior; D, dorsal; M, medial; P, posterior; V, ventral. ( E ). Quantification of density of hair cell bundles for the mtu1 −/− , mtu1 +/− and mtu1 +/+ zebrafish. Graph details and symbols are explained in the legend to Figure 3 .
    Figure Legend Snippet: Reduced density of hair cell bundles in inner ear labyrinth. ( A ) Anatomy of adult wild type zebrafish inner ear labyrinth, showing two lagenas (L), two saccule (S), and two utricle (U). ( B – D ) Hair bundle density in lagenas, saccule and utricle. The hair cells, nuclei of from mutant and wild type zebrafish were stained with phalloidin (green) and DAPI (blue), respectively. Hair cell counts were sampled at six locations of lagenas, four locations of saccule and six locations of utricle, respectively. A 1600 μm 2 box was placed at each sampling area and labeled hair cell bundles were counted within each box to determine hair cell density were selected for counting. A, anterior; D, dorsal; M, medial; P, posterior; V, ventral. ( E ). Quantification of density of hair cell bundles for the mtu1 −/− , mtu1 +/− and mtu1 +/+ zebrafish. Graph details and symbols are explained in the legend to Figure 3 .

    Techniques Used: Mutagenesis, Staining, Sampling, Labeling

    Enzymatic activities of respiratory chain complexes and measurement of ATP levels. ( A ) The activities of respiratory complexes were investigated by enzymatic assays on complexes I, II, III, IV and V in mitochondria isolated from mtu1 mutant and wild type zebrafish. The calculations were based on three independent determinations. ( B ) Measurement of mitochondrial and cytosolic ATP levels. Average cytosolic ATP level (presence of oligomycin for inhibition of the mitochondrial ATP synthesis) and mitochondrial ATP level (subtraction of cytosolic ATP level from total cellular ATP levels) are shown. Three independent experiments were made for each genotype of zebrafish. Graph details and symbols are explained in the legend to Figure 3 .
    Figure Legend Snippet: Enzymatic activities of respiratory chain complexes and measurement of ATP levels. ( A ) The activities of respiratory complexes were investigated by enzymatic assays on complexes I, II, III, IV and V in mitochondria isolated from mtu1 mutant and wild type zebrafish. The calculations were based on three independent determinations. ( B ) Measurement of mitochondrial and cytosolic ATP levels. Average cytosolic ATP level (presence of oligomycin for inhibition of the mitochondrial ATP synthesis) and mitochondrial ATP level (subtraction of cytosolic ATP level from total cellular ATP levels) are shown. Three independent experiments were made for each genotype of zebrafish. Graph details and symbols are explained in the legend to Figure 3 .

    Techniques Used: Isolation, Mutagenesis, Inhibition

    mtu1 −/− zebrafish at 5 dpf did not show the defects in muscle, brain and eyes. ( A ) Hematoxylin and eosin (HE) staining of skeletal muscles, brain and eye in the wild type ( mtu1 +/+ ), mtu1 +/− and mtu1 −/− zebrafish at 5 dpf. ( B ) Succinate dehydrogenase (SDH) and ( C ) cytochrome c oxidase (COX) staining of skeletal muscles, brain and eye in wild type ( mtu1 +/+ ), mtu1 +/− and mtu1 −/− zebrafish at 5 dpf. D, dorsal; P, posterior; V, ventral; L, lateral.
    Figure Legend Snippet: mtu1 −/− zebrafish at 5 dpf did not show the defects in muscle, brain and eyes. ( A ) Hematoxylin and eosin (HE) staining of skeletal muscles, brain and eye in the wild type ( mtu1 +/+ ), mtu1 +/− and mtu1 −/− zebrafish at 5 dpf. ( B ) Succinate dehydrogenase (SDH) and ( C ) cytochrome c oxidase (COX) staining of skeletal muscles, brain and eye in wild type ( mtu1 +/+ ), mtu1 +/− and mtu1 −/− zebrafish at 5 dpf. D, dorsal; P, posterior; V, ventral; L, lateral.

    Techniques Used: Staining

    8) Product Images from "Caspase Cleavage of Gelsolin Is an Inductive Cue for Pathologic Cardiac Hypertrophy"

    Article Title: Caspase Cleavage of Gelsolin Is an Inductive Cue for Pathologic Cardiac Hypertrophy

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    doi: 10.1161/JAHA.118.010404

    Gelsolin secreted by hypertrophic cardiomyocytes may be involved in the induction of hypertrophy. A, Primary cardiomyocytes were treated with conditioned hypertrophic media (Cond. Media), from phenylephrine‐ or procaspase activating compound (PAC‐1)–treated cardiomyocytes, supplemented with gelsolin antibody to block secreted gelsolin, gelsolin antibody plus gelsolin peptide, or MyoD antibody. Serum‐free medium treatment was used as a control. B, Cell size and atrial natriuretic peptide (ANP) levels were analyzed and are quantified. Phenylephrine (n=3, ** P
    Figure Legend Snippet: Gelsolin secreted by hypertrophic cardiomyocytes may be involved in the induction of hypertrophy. A, Primary cardiomyocytes were treated with conditioned hypertrophic media (Cond. Media), from phenylephrine‐ or procaspase activating compound (PAC‐1)–treated cardiomyocytes, supplemented with gelsolin antibody to block secreted gelsolin, gelsolin antibody plus gelsolin peptide, or MyoD antibody. Serum‐free medium treatment was used as a control. B, Cell size and atrial natriuretic peptide (ANP) levels were analyzed and are quantified. Phenylephrine (n=3, ** P

    Techniques Used: Blocking Assay, Aqueous Normal-phase Chromatography

    Gelsolin is an essential caspase cleavage substrate during cardiomyocyte hypertrophy. A, Cardiomyocytes infected with p35‐adenovirus or green fluorescent protein ( GFP )–adenovirus were treated with phenylephrine and subjected to immunoblot using a C‐terminal gelsolin antibody. Gelsolin cleavage was observed with GFP ‐adenovirus, whereas these fragments were not present with p35‐adenovirus. Treatment with 2 μmol/L staurosporine served as a positive (+) control. B, Gelsolin in vitro cleavage assays for caspase alone, gelsolin alone, gelsolin with caspase 3 or 7, or gelsolin with caspase and caspase inhibitor N ‐benzyloxycarbonyl‐Asp‐Glu‐Val‐Asp fluoromethyl ketone (z‐DEVD‐fmk) were probed with a C‐terminal gelsolin antibody. A smaller molecular weight gelsolin fragment (asterisk) is observed when gelsolin is incubated with caspase 3/7 and is reduced with z‐DEVD‐fmk. C, Recombinant gelsolin was subjected to an in vitro cleavage reaction, followed by SDS / PAGE and silver staining. Protein fragments were isolated and processed by liquid chromatography–tandem mass spectrometry. D, Red peptides represent those from the N‐terminal fragment (44 kDa plus 26‐kDa N‐terminal gelsolin Glutathione S‐transferase (GST) tag). Green peptides represent those from the C‐terminal fragment (≈42 kDa). The aspartic acid targeted by caspase 3/7 is highlighted in yellow. Consensus of the targeted aspartic acid residues (highlighted in yellow) in Homo sapiens (D403; NP _000168.1), Rattus norvegicus (D401; NP _001004080.1), and Mus musculus (D401; NP _666232.2). Amino acid sequences were obtained from the National Center for Biotechnology Information. E, Cardiomyocytes transfected with scrambled negative control small interfering RNA (siRNA) or gelsolin siRNA, followed by infection with GFP‐adenovirus, wild‐type gelsolin‐adenovirus, or D401A gelsolin‐adenovirus (multiplicity of infection=1) during serum‐free or phenylephrine treatment. F, Gelsolin knockdown confirmed by Western blotting, where gelsolin siRNA led to reduced gelsolin levels compared with the negative scrambled siRNA. α‐ß Tubulin was the loading control. G, During serum‐free treatment, wild‐type gelsolin‐adenovirus infection after negative siRNA transfection led to increased cell size (n=4, ** P
    Figure Legend Snippet: Gelsolin is an essential caspase cleavage substrate during cardiomyocyte hypertrophy. A, Cardiomyocytes infected with p35‐adenovirus or green fluorescent protein ( GFP )–adenovirus were treated with phenylephrine and subjected to immunoblot using a C‐terminal gelsolin antibody. Gelsolin cleavage was observed with GFP ‐adenovirus, whereas these fragments were not present with p35‐adenovirus. Treatment with 2 μmol/L staurosporine served as a positive (+) control. B, Gelsolin in vitro cleavage assays for caspase alone, gelsolin alone, gelsolin with caspase 3 or 7, or gelsolin with caspase and caspase inhibitor N ‐benzyloxycarbonyl‐Asp‐Glu‐Val‐Asp fluoromethyl ketone (z‐DEVD‐fmk) were probed with a C‐terminal gelsolin antibody. A smaller molecular weight gelsolin fragment (asterisk) is observed when gelsolin is incubated with caspase 3/7 and is reduced with z‐DEVD‐fmk. C, Recombinant gelsolin was subjected to an in vitro cleavage reaction, followed by SDS / PAGE and silver staining. Protein fragments were isolated and processed by liquid chromatography–tandem mass spectrometry. D, Red peptides represent those from the N‐terminal fragment (44 kDa plus 26‐kDa N‐terminal gelsolin Glutathione S‐transferase (GST) tag). Green peptides represent those from the C‐terminal fragment (≈42 kDa). The aspartic acid targeted by caspase 3/7 is highlighted in yellow. Consensus of the targeted aspartic acid residues (highlighted in yellow) in Homo sapiens (D403; NP _000168.1), Rattus norvegicus (D401; NP _001004080.1), and Mus musculus (D401; NP _666232.2). Amino acid sequences were obtained from the National Center for Biotechnology Information. E, Cardiomyocytes transfected with scrambled negative control small interfering RNA (siRNA) or gelsolin siRNA, followed by infection with GFP‐adenovirus, wild‐type gelsolin‐adenovirus, or D401A gelsolin‐adenovirus (multiplicity of infection=1) during serum‐free or phenylephrine treatment. F, Gelsolin knockdown confirmed by Western blotting, where gelsolin siRNA led to reduced gelsolin levels compared with the negative scrambled siRNA. α‐ß Tubulin was the loading control. G, During serum‐free treatment, wild‐type gelsolin‐adenovirus infection after negative siRNA transfection led to increased cell size (n=4, ** P

    Techniques Used: Infection, Positive Control, In Vitro, Molecular Weight, Incubation, Recombinant, SDS Page, Silver Staining, Isolation, Liquid Chromatography, Mass Spectrometry, Transfection, Negative Control, Small Interfering RNA, Western Blot

    Expression of wild‐type or N‐terminal gelsolin can induce hypertrophy in primary cardiomyocytes. Primary cardiomyocytes were infected with green fluorescent protein (GFP) control adenovirus, wild‐type gelsolin‐adenovirus, N‐terminal gelsolin‐adenovirus, C‐terminal gelsolin‐adenovirus, or D401A gelsolin‐adenovirus (multiplicity of infection [MOI]=1) during serum‐free or phenylephrine treatment (bar=40 μm). Immunofluorescence analysis was completed (A), and cell size and atrial natriuretic peptide (ANP) levels were evaluated (B). All values were normalized to the GFP‐adenovirus–infected cardiomyocytes within each treatment. A significant increase in cell size was observed after wild‐type (n=3, ** P
    Figure Legend Snippet: Expression of wild‐type or N‐terminal gelsolin can induce hypertrophy in primary cardiomyocytes. Primary cardiomyocytes were infected with green fluorescent protein (GFP) control adenovirus, wild‐type gelsolin‐adenovirus, N‐terminal gelsolin‐adenovirus, C‐terminal gelsolin‐adenovirus, or D401A gelsolin‐adenovirus (multiplicity of infection [MOI]=1) during serum‐free or phenylephrine treatment (bar=40 μm). Immunofluorescence analysis was completed (A), and cell size and atrial natriuretic peptide (ANP) levels were evaluated (B). All values were normalized to the GFP‐adenovirus–infected cardiomyocytes within each treatment. A significant increase in cell size was observed after wild‐type (n=3, ** P

    Techniques Used: Expressing, Infection, Immunofluorescence, Aqueous Normal-phase Chromatography

    Expression of wild‐type or N‐terminal gelsolin can rescue the hypertrophy response reduced by caspase inhibition. Primary cardiomyocytes were treated with 20 μmol/L of the caspase 3 inhibitor N ‐benzyloxycarbonyl‐Asp‐Glu‐Val‐Asp fluoromethyl ketone (z‐DEVD‐fmk); infected with green fluorescent protein (GFP) control adenovirus, wild‐type gelsolin‐adenovirus, N‐terminal gelsolin‐adenovirus, C‐terminal gelsolin‐adenovirus, or D401A gelsolin‐adenovirus (multiplicity of infection=1); and treated with phenylephrine for 24 hours. Immunofluorescence analysis was completed (A), and cell size and atrial natriuretic peptide (ANP) levels were evaluated (B). All values were normalized to the caspase‐inhibited and GFP‐adenovirus–infected cardiomyocytes, which were reduced in size, and ANP levels were compared with those lacking caspase inhibitor treatment (n=3, ** P
    Figure Legend Snippet: Expression of wild‐type or N‐terminal gelsolin can rescue the hypertrophy response reduced by caspase inhibition. Primary cardiomyocytes were treated with 20 μmol/L of the caspase 3 inhibitor N ‐benzyloxycarbonyl‐Asp‐Glu‐Val‐Asp fluoromethyl ketone (z‐DEVD‐fmk); infected with green fluorescent protein (GFP) control adenovirus, wild‐type gelsolin‐adenovirus, N‐terminal gelsolin‐adenovirus, C‐terminal gelsolin‐adenovirus, or D401A gelsolin‐adenovirus (multiplicity of infection=1); and treated with phenylephrine for 24 hours. Immunofluorescence analysis was completed (A), and cell size and atrial natriuretic peptide (ANP) levels were evaluated (B). All values were normalized to the caspase‐inhibited and GFP‐adenovirus–infected cardiomyocytes, which were reduced in size, and ANP levels were compared with those lacking caspase inhibitor treatment (n=3, ** P

    Techniques Used: Expressing, Inhibition, Infection, Immunofluorescence, Aqueous Normal-phase Chromatography

    9) Product Images from "Induction and decay of functional complement-fixing antibodies by the RTS,S malaria vaccine in children, and a negative impact of malaria exposure"

    Article Title: Induction and decay of functional complement-fixing antibodies by the RTS,S malaria vaccine in children, and a negative impact of malaria exposure

    Journal: BMC Medicine

    doi: 10.1186/s12916-019-1277-x

    Relationship between age and immunity. Children in RTS,S vaccine group from Manhiça (black box plots) and Ilha Josina cohorts (gray box plots) were categorized into younger (12 to 24 months; Manhiça n = 11 and Ilha Josina n = 23, respectively) and older (24 to 60 months; Manhiça n = 39 and Ilha Josina n = 26, respectively) age groups. Sera collected after vaccination (month 3, M3) were tested for C1q-fixation to CSP and NANP ( a ) and IgG-reactivity to blood-stage antigens MSP2 and AMA1 ( b ). Samples were tested in duplicate, and the mean value was used to generate box plots for samples stratified by age group. Top, center, and bottom horizontal lines represent the 75th percentile, median, and 25th percentile, respectively; upper and lower whiskers represent the highest and lowest values within 1.5× IQR, respectively; and values that exceed this range are presented as dots. Malaria-naïve negative controls from Melbourne donors were used to calculate the seropositivity cutoff values (dashed lines), and the percentages of individuals above this threshold are shown. Reactivity between unpaired samples was compared using Mann-Whitney U test
    Figure Legend Snippet: Relationship between age and immunity. Children in RTS,S vaccine group from Manhiça (black box plots) and Ilha Josina cohorts (gray box plots) were categorized into younger (12 to 24 months; Manhiça n = 11 and Ilha Josina n = 23, respectively) and older (24 to 60 months; Manhiça n = 39 and Ilha Josina n = 26, respectively) age groups. Sera collected after vaccination (month 3, M3) were tested for C1q-fixation to CSP and NANP ( a ) and IgG-reactivity to blood-stage antigens MSP2 and AMA1 ( b ). Samples were tested in duplicate, and the mean value was used to generate box plots for samples stratified by age group. Top, center, and bottom horizontal lines represent the 75th percentile, median, and 25th percentile, respectively; upper and lower whiskers represent the highest and lowest values within 1.5× IQR, respectively; and values that exceed this range are presented as dots. Malaria-naïve negative controls from Melbourne donors were used to calculate the seropositivity cutoff values (dashed lines), and the percentages of individuals above this threshold are shown. Reactivity between unpaired samples was compared using Mann-Whitney U test

    Techniques Used: MANN-WHITNEY

    RTS,S vaccine-induced immunity declines over time. A random selection of children vaccinated with RTS,S (Manhiça cohort, n = 30) was tested for C1q-fixation ( a ), IgG/IgM ( b ), and IgG subclasses ( c ) to CSP at months 3, 8.5, 21, 33, 45, and 6. Note that due to low reactivity, C1q-fixation was re-tested at a higher dilution of 1/110, in addition to 1/250, to confirm results. Samples were tested in duplicate, and the median and 95% CI of the median from each time point group are shown by the symbol and shaded area, respectively
    Figure Legend Snippet: RTS,S vaccine-induced immunity declines over time. A random selection of children vaccinated with RTS,S (Manhiça cohort, n = 30) was tested for C1q-fixation ( a ), IgG/IgM ( b ), and IgG subclasses ( c ) to CSP at months 3, 8.5, 21, 33, 45, and 6. Note that due to low reactivity, C1q-fixation was re-tested at a higher dilution of 1/110, in addition to 1/250, to confirm results. Samples were tested in duplicate, and the median and 95% CI of the median from each time point group are shown by the symbol and shaded area, respectively

    Techniques Used: Selection

    RTS,S vaccine-induced antibodies promote complement fixation to CSP. a Children in RTS,S and comparator vaccine groups from Manhiça (black box plots; N = 50 and N = 25, respectively) and Ilha Josina cohorts (gray box plots; N = 49 and N = 24, respectively) were tested for C1q-fixation to CSP. Sera collected at baseline (month 0, M0) and after vaccination (month 3, M3) were tested in duplicate, and the mean value was used to generate box plots whereby top, center, and bottom horizontal lines represent the 75th percentile, median, and 25th percentile, respectively; upper and lower whiskers represent the highest and lowest values within 1.5× IQR, respectively; and values that exceed this range are presented as dots. Malaria-naïve negative controls from Melbourne donors were used to calculate the seropositivity cutoff values (dashed lines), and the percentages of individuals above this threshold are shown. Reactivity between paired samples and unpaired samples were compared using Wilcoxon matched-pairs signed-rank test and Mann-Whitney U test, respectively. b Random selection of children in the RTS,S vaccine group from Manhiça cohort ( n = 20, M3) were tested for C5b-C9-fixation to CSP, and the mean of duplicates was graphed as scatter plots
    Figure Legend Snippet: RTS,S vaccine-induced antibodies promote complement fixation to CSP. a Children in RTS,S and comparator vaccine groups from Manhiça (black box plots; N = 50 and N = 25, respectively) and Ilha Josina cohorts (gray box plots; N = 49 and N = 24, respectively) were tested for C1q-fixation to CSP. Sera collected at baseline (month 0, M0) and after vaccination (month 3, M3) were tested in duplicate, and the mean value was used to generate box plots whereby top, center, and bottom horizontal lines represent the 75th percentile, median, and 25th percentile, respectively; upper and lower whiskers represent the highest and lowest values within 1.5× IQR, respectively; and values that exceed this range are presented as dots. Malaria-naïve negative controls from Melbourne donors were used to calculate the seropositivity cutoff values (dashed lines), and the percentages of individuals above this threshold are shown. Reactivity between paired samples and unpaired samples were compared using Wilcoxon matched-pairs signed-rank test and Mann-Whitney U test, respectively. b Random selection of children in the RTS,S vaccine group from Manhiça cohort ( n = 20, M3) were tested for C5b-C9-fixation to CSP, and the mean of duplicates was graphed as scatter plots

    Techniques Used: MANN-WHITNEY, Selection

    High variability among RTS,S vaccine-induced IgG targeting the central repeat and C-terminal regions of CSP. Children in RTS,S vaccine group from Manhiça and Ilha Josina cohorts ( N = 99, 3 M) were tested for IgG to NANP and CT and C1q-fixation to CSP, and the values were used for the following analysis. a Heat map of children arranged in descending (top to bottom) order of C1q-fixation (left), corresponding IgG to NANP and CT (middle), and for comparison IgM to NANP and CT (right). b Variability between epitope specificity was quantified by calculating the ratio of NANP-to-CT IgG. Children with low variability were considered to have equal reactivity to NANP and CT (ratio between 0.75 and 1.25 shown in white), and children exceeding this range were considered to have a NANP- or CT-skewed response (ratio > 1.25 shown in purple and ratio
    Figure Legend Snippet: High variability among RTS,S vaccine-induced IgG targeting the central repeat and C-terminal regions of CSP. Children in RTS,S vaccine group from Manhiça and Ilha Josina cohorts ( N = 99, 3 M) were tested for IgG to NANP and CT and C1q-fixation to CSP, and the values were used for the following analysis. a Heat map of children arranged in descending (top to bottom) order of C1q-fixation (left), corresponding IgG to NANP and CT (middle), and for comparison IgM to NANP and CT (right). b Variability between epitope specificity was quantified by calculating the ratio of NANP-to-CT IgG. Children with low variability were considered to have equal reactivity to NANP and CT (ratio between 0.75 and 1.25 shown in white), and children exceeding this range were considered to have a NANP- or CT-skewed response (ratio > 1.25 shown in purple and ratio

    Techniques Used:

    Functional complement-fixing antibodies target the central repeat and C-terminal regions of CSP. Children in RTS,S vaccine group from Manhiça (black box plots; N = 50) and Ilha Josina cohorts (gray box plots; N = 49) were tested for IgG ( a ) and IgM ( b ) to NANP and CT regions of CSP. Sera collected at baseline (month 0, M0) and after vaccination (month 3, M3) were tested in duplicate, and the mean value was used to generate box plots whereby top, center, and bottom horizontal lines represent the 75th percentile, median, and 25th percentile, respectively; upper and lower whiskers represent the highest and lowest values within 1.5× IQR, respectively; and values that exceed this range are presented as dots. Malaria-naïve negative controls from Melbourne donors were used to calculate the seropositivity cutoff values (dashed lines), and the percentages of individuals above this threshold are shown. Reactivity between paired samples was compared using Wilcoxon matched-pairs signed-rank test. c , d Children in RTS,S vaccine group from Manhiça and Ilha Josina cohorts ( N = 99, M3) were tested for C1q-fixation to CSP, NANP, and CT, and the values were plotted compared to IgG reactivity (c) and C1q fixation to CSP, NANP, and CT were correlated (d)
    Figure Legend Snippet: Functional complement-fixing antibodies target the central repeat and C-terminal regions of CSP. Children in RTS,S vaccine group from Manhiça (black box plots; N = 50) and Ilha Josina cohorts (gray box plots; N = 49) were tested for IgG ( a ) and IgM ( b ) to NANP and CT regions of CSP. Sera collected at baseline (month 0, M0) and after vaccination (month 3, M3) were tested in duplicate, and the mean value was used to generate box plots whereby top, center, and bottom horizontal lines represent the 75th percentile, median, and 25th percentile, respectively; upper and lower whiskers represent the highest and lowest values within 1.5× IQR, respectively; and values that exceed this range are presented as dots. Malaria-naïve negative controls from Melbourne donors were used to calculate the seropositivity cutoff values (dashed lines), and the percentages of individuals above this threshold are shown. Reactivity between paired samples was compared using Wilcoxon matched-pairs signed-rank test. c , d Children in RTS,S vaccine group from Manhiça and Ilha Josina cohorts ( N = 99, M3) were tested for C1q-fixation to CSP, NANP, and CT, and the values were plotted compared to IgG reactivity (c) and C1q fixation to CSP, NANP, and CT were correlated (d)

    Techniques Used: Functional Assay

    10) Product Images from "Intranasal Administration of Recombinant Neisseria gonorrhoeae Transferrin Binding Proteins A and B Conjugated to the Cholera Toxin B Subunit Induces Systemic and Vaginal Antibodies in Mice "

    Article Title: Intranasal Administration of Recombinant Neisseria gonorrhoeae Transferrin Binding Proteins A and B Conjugated to the Cholera Toxin B Subunit Induces Systemic and Vaginal Antibodies in Mice

    Journal: Infection and Immunity

    doi: 10.1128/IAI.73.7.3945-3953.2005

    Serum IgG levels specific for TbpA, TbpB, and Ctb. (A) Serum IgG levels specific for TbpA detected at days 17, 28, 35, and 65. (B) Serum IgG levels specific for TbpB detected at the same time points. (C) Serum IgG levels specific for Ctb detected at the same time points. Results are expressed as the geometric mean of antibody titers ×/÷ standard deviation. For all immunization groups, n = 5.
    Figure Legend Snippet: Serum IgG levels specific for TbpA, TbpB, and Ctb. (A) Serum IgG levels specific for TbpA detected at days 17, 28, 35, and 65. (B) Serum IgG levels specific for TbpB detected at the same time points. (C) Serum IgG levels specific for Ctb detected at the same time points. Results are expressed as the geometric mean of antibody titers ×/÷ standard deviation. For all immunization groups, n = 5.

    Techniques Used: CtB Assay, Standard Deviation

    IgG1 and IgG2a subtype analysis. (A) IgG1 and IgG2a antibody levels specific for TbpA detected in sera collected at day 35. (B) IgG1 and IgG2a antibody levels specific for TbpB detected in sera collected at day 35. The bars represent the geometric mean ×/÷ standard deviation. The values above the bars represent the IgG1/IgG2a ratios of the corresponding immunization groups, indicated below each graph. For all groups, n = 5.
    Figure Legend Snippet: IgG1 and IgG2a subtype analysis. (A) IgG1 and IgG2a antibody levels specific for TbpA detected in sera collected at day 35. (B) IgG1 and IgG2a antibody levels specific for TbpB detected in sera collected at day 35. The bars represent the geometric mean ×/÷ standard deviation. The values above the bars represent the IgG1/IgG2a ratios of the corresponding immunization groups, indicated below each graph. For all groups, n = 5.

    Techniques Used: Standard Deviation

    11) Product Images from "Altered Localization of Retinoid X Receptor ? Coincides with Loss of Retinoid Responsiveness in Human Breast Cancer MDA-MB-231 Cells"

    Article Title: Altered Localization of Retinoid X Receptor ? Coincides with Loss of Retinoid Responsiveness in Human Breast Cancer MDA-MB-231 Cells

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.24.9.3972-3982.2004

    Colocalization of RXRα with nuclear organelle proteins. (A) Fixed MDA-MB-231 cells were double immunostained with RXRα (e to h) and the indicated nuclear protein antibody anti-NPC (a), anti-PML (b), anti-SC-35 (c), or anti-p105 (d). Confocal overlays of double-immunostained images are shown in merged panels (i to l). (B) HMEC, MCF-7, and MDA-MB-231 cells were double immunostained with anti-RXRα and anti-SC-35. (C) RXRα antisense adenovirus was infected to MDA-MB-231 and double immunostained with anti-RXRα (green) and anti-adenovirus type 5 (red). (D) The dimerization partner (RXRγ, PPARα, PPARγ, or RARα) did not colocalize with SC-35. All antibodies were diluted at 1:200. (E) Paraffin-embedded human breast tissue sections were deparaffinized and hydrated. Antigen was retrieved in 10 mM citrate buffer in a microwave for 15 min past boiling.
    Figure Legend Snippet: Colocalization of RXRα with nuclear organelle proteins. (A) Fixed MDA-MB-231 cells were double immunostained with RXRα (e to h) and the indicated nuclear protein antibody anti-NPC (a), anti-PML (b), anti-SC-35 (c), or anti-p105 (d). Confocal overlays of double-immunostained images are shown in merged panels (i to l). (B) HMEC, MCF-7, and MDA-MB-231 cells were double immunostained with anti-RXRα and anti-SC-35. (C) RXRα antisense adenovirus was infected to MDA-MB-231 and double immunostained with anti-RXRα (green) and anti-adenovirus type 5 (red). (D) The dimerization partner (RXRγ, PPARα, PPARγ, or RARα) did not colocalize with SC-35. All antibodies were diluted at 1:200. (E) Paraffin-embedded human breast tissue sections were deparaffinized and hydrated. Antigen was retrieved in 10 mM citrate buffer in a microwave for 15 min past boiling.

    Techniques Used: Multiple Displacement Amplification, Infection

    12) Product Images from "The Brd4 Extraterminal Domain Confers Transcription Activation Independent of pTEFb by Recruiting Multiple Proteins, Including NSD3 ▿The Brd4 Extraterminal Domain Confers Transcription Activation Independent of pTEFb by Recruiting Multiple Proteins, Including NSD3 ▿ †"

    Article Title: The Brd4 Extraterminal Domain Confers Transcription Activation Independent of pTEFb by Recruiting Multiple Proteins, Including NSD3 ▿The Brd4 Extraterminal Domain Confers Transcription Activation Independent of pTEFb by Recruiting Multiple Proteins, Including NSD3 ▿ †

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.01341-10

    ). At 48 h posttransfection, anti-GFP immunoprecipitations were performed with whole-cell extract. Equal amounts of input (IN) samples along with 50% of IPs were separated by SDS-PAGE and immunoblotted with antibodies specific to GFP, NSD3, JMJD6, CHD4, and actin. The symbols *1, *2, and *3 denote the GFP band, the Brd4 band, and the Brd2/Brd3 bands, respectively.
    Figure Legend Snippet: ). At 48 h posttransfection, anti-GFP immunoprecipitations were performed with whole-cell extract. Equal amounts of input (IN) samples along with 50% of IPs were separated by SDS-PAGE and immunoblotted with antibodies specific to GFP, NSD3, JMJD6, CHD4, and actin. The symbols *1, *2, and *3 denote the GFP band, the Brd4 band, and the Brd2/Brd3 bands, respectively.

    Techniques Used: SDS Page

    The Brd4 ET domain associates with several multiprotein complexes. HA immunoprecipitations were performed with lysates from 293T cells stably expressing HA-GLTSCR1, HA-ATAD5, HA-NSD3, HA-JMJD6, or HA-CHD4, and the bound proteins were subjected to mass spectrometry and CompPASS analysis (MS or IP).
    Figure Legend Snippet: The Brd4 ET domain associates with several multiprotein complexes. HA immunoprecipitations were performed with lysates from 293T cells stably expressing HA-GLTSCR1, HA-ATAD5, HA-NSD3, HA-JMJD6, or HA-CHD4, and the bound proteins were subjected to mass spectrometry and CompPASS analysis (MS or IP).

    Techniques Used: Stable Transfection, Expressing, Mass Spectrometry

    13) Product Images from "Wavy Multistratified Amacrine Cells in the Monkey Retina Contain Immunoreactive Secretoneurin"

    Article Title: Wavy Multistratified Amacrine Cells in the Monkey Retina Contain Immunoreactive Secretoneurin

    Journal: Peptides

    doi: 10.1016/j.peptides.2017.06.005

    Interactions between dendrites of secretoneurin-IR amacrine cells (red) and melanopsin-IR (green) retinal ganglion cells. Secretoneurin-IR dendrites are closely apposed to dendrites of outer-stratifying melanopsin cells (arrowheads). A. Note that a secretoneurin-IR dendrite also contacts the soma of an outer melanopsin cell. The main figure is an orthogonal projection of 6 optical sections showing only the melanopsin signal (green), z step = 0.5 μm, scale bar = 20 μm. Insets are single optical sections displaying both melanopsin (green) and secretoneurin (red) signals, scale bars = 2 μm. B. Note the co-fasciculation of the 2 dendrites. The top figure is an orthogonal projection of 10 optical sections, z step = 0.31 μm. The others are consecutive single optical sections. Scale bar = 5 μm.
    Figure Legend Snippet: Interactions between dendrites of secretoneurin-IR amacrine cells (red) and melanopsin-IR (green) retinal ganglion cells. Secretoneurin-IR dendrites are closely apposed to dendrites of outer-stratifying melanopsin cells (arrowheads). A. Note that a secretoneurin-IR dendrite also contacts the soma of an outer melanopsin cell. The main figure is an orthogonal projection of 6 optical sections showing only the melanopsin signal (green), z step = 0.5 μm, scale bar = 20 μm. Insets are single optical sections displaying both melanopsin (green) and secretoneurin (red) signals, scale bars = 2 μm. B. Note the co-fasciculation of the 2 dendrites. The top figure is an orthogonal projection of 10 optical sections, z step = 0.31 μm. The others are consecutive single optical sections. Scale bar = 5 μm.

    Techniques Used:

    14) Product Images from "Physics of active jamming during collective cellular motion in a monolayer"

    Article Title: Physics of active jamming during collective cellular motion in a monolayer

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1510973112

    Aging of the system through maturation of adhesion proteins with time. ( A ) Tissue stained for cell−cell junctions. Cadherin concentration is observed to increase at the cell−cell contacts as the cell layer ages. ( B ) Quantification of the contrast in cadherin signal between the junction and the cytoplasm. ( C ) As the layer ages, vinculin distribution is observed to evolve from small complexes at the ends of actin filaments to a more homogeneous and uniform distribution over the whole cell−substrate contact area. (Scale bars: 20 μm.)
    Figure Legend Snippet: Aging of the system through maturation of adhesion proteins with time. ( A ) Tissue stained for cell−cell junctions. Cadherin concentration is observed to increase at the cell−cell contacts as the cell layer ages. ( B ) Quantification of the contrast in cadherin signal between the junction and the cytoplasm. ( C ) As the layer ages, vinculin distribution is observed to evolve from small complexes at the ends of actin filaments to a more homogeneous and uniform distribution over the whole cell−substrate contact area. (Scale bars: 20 μm.)

    Techniques Used: Staining, Concentration Assay

    15) Product Images from "Hepatocellular carcinoma-associated antigen 59 of Haemonchus contortus modulates the functions of PBMCs and the differentiation and maturation of monocyte-derived dendritic cells of goats in vitro"

    Article Title: Hepatocellular carcinoma-associated antigen 59 of Haemonchus contortus modulates the functions of PBMCs and the differentiation and maturation of monocyte-derived dendritic cells of goats in vitro

    Journal: Parasites & Vectors

    doi: 10.1186/s13071-019-3375-1

    Expression, purification and Western blot analysis of rHCA59 protein. Lane M: standard protein molecular weight marker. a rHCA59 protein was induced with 1 mM IPTG. Lane 1: expression of recombinant protein rHCA59 vector before purification; Lane 2: purified rHCA59 protein. b Western blot of rHCA59 protein. Lane 1: recombinant protein HCA59 was recognized by goat anti- H. contortus sera; Lane 2: membrane incubated with normal goat sera (as control). c Western blot of total HcESPs. Lane 1: HcESPs were detected by rat anti-rHCA59 protein antibodies; Lane 2: membrane incubated with normal rat sera (as control)
    Figure Legend Snippet: Expression, purification and Western blot analysis of rHCA59 protein. Lane M: standard protein molecular weight marker. a rHCA59 protein was induced with 1 mM IPTG. Lane 1: expression of recombinant protein rHCA59 vector before purification; Lane 2: purified rHCA59 protein. b Western blot of rHCA59 protein. Lane 1: recombinant protein HCA59 was recognized by goat anti- H. contortus sera; Lane 2: membrane incubated with normal goat sera (as control). c Western blot of total HcESPs. Lane 1: HcESPs were detected by rat anti-rHCA59 protein antibodies; Lane 2: membrane incubated with normal rat sera (as control)

    Techniques Used: Expressing, Purification, Western Blot, Molecular Weight, Marker, Recombinant, Plasmid Preparation, Incubation

    16) Product Images from "CTCF binding and higher order chromatin structure of the H19 locus are maintained in mitotic chromatin"

    Article Title: CTCF binding and higher order chromatin structure of the H19 locus are maintained in mitotic chromatin

    Journal: The EMBO Journal

    doi: 10.1038/sj.emboj.7600793

    The cell cycle-dependent subcellular distribution of CTCF and HP1α. ( A ) Double indirect immunofluorescence experiments were carried out in HeLa cells fixed with ethanol/acetic acid. Cells were stained with primary antibodies against CTCF and HP1α followed by FITC-conjugated (green) or Texas red dye-conjugated (red) secondary antibodies, respectively. Interphase (a–c), metaphase (d–f) and early cytokinesis (g–i) are shown. The merged panel allows the comparison of the localization of CTCF and HP1α. ( B ) Mitotic chromosome spreads were prepared with synchronized human primary amnion cells (46,XX) (a–c). Indirect immunofluorescence was carried out with the anti-CTCF antibody and an FITC-conjugated secondary antibody (green). DNA was stained with propidium iodide (red). The overlay shows colocalization of CTCF and DNA. Unfixed HeLa cell mitotic chromosome spreads were stained with Hoechst and antibodies against CTCF and HP1α (d–g). The slide was visualized for CTCF (d), HP1α (e), CTCF+HP1α (f) and CTCF+HP1α+Hoechst (g).
    Figure Legend Snippet: The cell cycle-dependent subcellular distribution of CTCF and HP1α. ( A ) Double indirect immunofluorescence experiments were carried out in HeLa cells fixed with ethanol/acetic acid. Cells were stained with primary antibodies against CTCF and HP1α followed by FITC-conjugated (green) or Texas red dye-conjugated (red) secondary antibodies, respectively. Interphase (a–c), metaphase (d–f) and early cytokinesis (g–i) are shown. The merged panel allows the comparison of the localization of CTCF and HP1α. ( B ) Mitotic chromosome spreads were prepared with synchronized human primary amnion cells (46,XX) (a–c). Indirect immunofluorescence was carried out with the anti-CTCF antibody and an FITC-conjugated secondary antibody (green). DNA was stained with propidium iodide (red). The overlay shows colocalization of CTCF and DNA. Unfixed HeLa cell mitotic chromosome spreads were stained with Hoechst and antibodies against CTCF and HP1α (d–g). The slide was visualized for CTCF (d), HP1α (e), CTCF+HP1α (f) and CTCF+HP1α+Hoechst (g).

    Techniques Used: Immunofluorescence, Staining

    17) Product Images from "A Novel Mitogen-Activated Protein Kinase Is Responsive to Raf and Mediates Growth Factor Specificity"

    Article Title: A Novel Mitogen-Activated Protein Kinase Is Responsive to Raf and Mediates Growth Factor Specificity

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.21.6.2235-2247.2001

    The Raf affinity column binds a protein of 97 kDa (p97) that is recognized by an anti-phospho ERK antibody. (A) p97 binds to active but not inactive ΔRaf-1:ER. ΔRaf-1:ER was isolated from ΔRaf-1:ER cells that were serum starved for 16 h and then either treated with ethanol (inactive Raf beads) or 1 μM E2 (active Raf beads) for 60 min as described in Materials and Methods. Active and inactive Raf beads were then loaded with cell extracts from untreated or E2-treated ΔRaf-1:ER cells, and some cells were pretreated for 10 min with 10 μM MEK inhibitor PD98059 (PD) as indicated. Binding proteins were eluted by boiling in PAGE sample buffer and analyzed by Western blotting with an anti-phospho pTEpY ERK(A) antibody. (B) p97 does not bind to the ER directly or to the antibodies used to isolate ΔRaf-1:ER. H19-7 cells were treated with bFGF, lysed in RIPA, and immunoprecipitated with antibodies against rat IgG or the ER (H222). Immunoprecipitated proteins were Western blotted with the anti-phospho ERK(A) antibody. The GST-ER fusion protein (GST-ER) was prepared and incubated with ΔRaf-1:ER cell extracts, washed, and Western blotted with the anti-phospho ERK(A) antibody. (C) p97 binds through the kinase domain of Raf-1. GST-ΔRaf was isolated on GSH beads, and 25 μg of isolated protein was incubated with increasing amounts of ΔRaf-1:ER cell extracts, washed, and Western blotted as described above. (D) Differentiating but not mitogenic signals increase binding of p97 to ΔRaf-1:ER. Activated ΔRaf-1:ER beads were incubated with 100 μg of cell lysates prepared from untreated (UT), EGF-, or FGF-treated H19-7 cells and analyzed by Western blotting as described for panel A. Cells were lysed with buffers containing either SDS and sodium deoxycholate (RIPA) or Triton X-100 (TX) as described in Materials and Methods. (E) ΔRaf-1:ER binds with high affinity to p97. Extracts from ΔRaf-1:ER cells treated with 1 μM E2 were bound to ΔRaf-1:ER beads and then eluted with NaCl, Empiger-BB, urea, or boiling (Cont) at the indicated concentrations. The eluates were then analyzed by Western blotting with anti-phospho ERK(A) antibodies as described above.
    Figure Legend Snippet: The Raf affinity column binds a protein of 97 kDa (p97) that is recognized by an anti-phospho ERK antibody. (A) p97 binds to active but not inactive ΔRaf-1:ER. ΔRaf-1:ER was isolated from ΔRaf-1:ER cells that were serum starved for 16 h and then either treated with ethanol (inactive Raf beads) or 1 μM E2 (active Raf beads) for 60 min as described in Materials and Methods. Active and inactive Raf beads were then loaded with cell extracts from untreated or E2-treated ΔRaf-1:ER cells, and some cells were pretreated for 10 min with 10 μM MEK inhibitor PD98059 (PD) as indicated. Binding proteins were eluted by boiling in PAGE sample buffer and analyzed by Western blotting with an anti-phospho pTEpY ERK(A) antibody. (B) p97 does not bind to the ER directly or to the antibodies used to isolate ΔRaf-1:ER. H19-7 cells were treated with bFGF, lysed in RIPA, and immunoprecipitated with antibodies against rat IgG or the ER (H222). Immunoprecipitated proteins were Western blotted with the anti-phospho ERK(A) antibody. The GST-ER fusion protein (GST-ER) was prepared and incubated with ΔRaf-1:ER cell extracts, washed, and Western blotted with the anti-phospho ERK(A) antibody. (C) p97 binds through the kinase domain of Raf-1. GST-ΔRaf was isolated on GSH beads, and 25 μg of isolated protein was incubated with increasing amounts of ΔRaf-1:ER cell extracts, washed, and Western blotted as described above. (D) Differentiating but not mitogenic signals increase binding of p97 to ΔRaf-1:ER. Activated ΔRaf-1:ER beads were incubated with 100 μg of cell lysates prepared from untreated (UT), EGF-, or FGF-treated H19-7 cells and analyzed by Western blotting as described for panel A. Cells were lysed with buffers containing either SDS and sodium deoxycholate (RIPA) or Triton X-100 (TX) as described in Materials and Methods. (E) ΔRaf-1:ER binds with high affinity to p97. Extracts from ΔRaf-1:ER cells treated with 1 μM E2 were bound to ΔRaf-1:ER beads and then eluted with NaCl, Empiger-BB, urea, or boiling (Cont) at the indicated concentrations. The eluates were then analyzed by Western blotting with anti-phospho ERK(A) antibodies as described above.

    Techniques Used: Affinity Column, Isolation, Binding Assay, Polyacrylamide Gel Electrophoresis, Western Blot, Immunoprecipitation, Incubation

    18) Product Images from "Role of Pex21p for Piggyback Import of Gpd1p and Pnc1p into Peroxisomes of Saccharomyces cerevisiae *"

    Article Title: Role of Pex21p for Piggyback Import of Gpd1p and Pnc1p into Peroxisomes of Saccharomyces cerevisiae *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.653451

    Pnc1p associates with Gpd1p. Gpd1p was affinity-purified from soluble fractions of a strain expressing Gpd1p-TPA under control of its own promoter. Cells were grown on high salt-containing medium and disrupted by glass bead breakage. Soluble fractions were applied to affinity chromatography at an IgG matrix, and Gpd1p-associated proteins were eluted by TEV protease cleavage ( left panel ). Wild-type cells without genomically integrated TEV-protein A served as a negative control. Gpd1p-associated proteins were separated by SDS-PAGE, and Coomassie-stained polypeptides were identified by mass spectrometry. M , molecular mass marker. Gpd1p complexes were further fractionated by calibrated size-exclusion chromatography ( right panels ). Fractions were collected, subjected to immunoblot analysis, and either stained with Amido Black dye ( lower right panel ) or immunodetected using antibodies against Gpd1p ( upper right panel ). Molecular masses of calibration proteins and calculated mass of the Gpd1p-Pnc1p complex are indicated.
    Figure Legend Snippet: Pnc1p associates with Gpd1p. Gpd1p was affinity-purified from soluble fractions of a strain expressing Gpd1p-TPA under control of its own promoter. Cells were grown on high salt-containing medium and disrupted by glass bead breakage. Soluble fractions were applied to affinity chromatography at an IgG matrix, and Gpd1p-associated proteins were eluted by TEV protease cleavage ( left panel ). Wild-type cells without genomically integrated TEV-protein A served as a negative control. Gpd1p-associated proteins were separated by SDS-PAGE, and Coomassie-stained polypeptides were identified by mass spectrometry. M , molecular mass marker. Gpd1p complexes were further fractionated by calibrated size-exclusion chromatography ( right panels ). Fractions were collected, subjected to immunoblot analysis, and either stained with Amido Black dye ( lower right panel ) or immunodetected using antibodies against Gpd1p ( upper right panel ). Molecular masses of calibration proteins and calculated mass of the Gpd1p-Pnc1p complex are indicated.

    Techniques Used: Affinity Purification, Expressing, Affinity Chromatography, Negative Control, SDS Page, Staining, Mass Spectrometry, Marker, Size-exclusion Chromatography

    19) Product Images from "Membrane-Microdomain Localization of Amyloid ?-Precursor Protein (APP) C-terminal Fragments Is Regulated by Phosphorylation of the Cytoplasmic Thr668 Residue *"

    Article Title: Membrane-Microdomain Localization of Amyloid ?-Precursor Protein (APP) C-terminal Fragments Is Regulated by Phosphorylation of the Cytoplasmic Thr668 Residue *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.334847

    In vitro kinetic analysis of phosphorylated and nonphosphorylated CTF cleavage by γ-secretase. A , in vitro γ-secretase assay with membrane preparations from wild-type mouse brain. Membrane preparations of the wild-type mouse brains were incubated at 37 °C for the indicated period of time (h). The samples were subjected to immunoblotting with anti-APP C-terminal to detect pAICD and nAICD, along with CTFs. Actin was also detected with anti-actin antibody. B and C, kinetic analysis of AICD generated by incubation of membrane preparations. The nAICD and pAICD in panel A were quantified with VersaDoc imaging analyzer and the levels of nAICD ( panel B ) and pAICD ( panel C ) at the respective incubation times were indicated as a relative ratio to the level at 4 h (1.0). D, the production ratio of pAICD to nAICD (pAICD/nAICD) at the indicated times are shown. Results are presented with mean ± S.D. ( n = 3).
    Figure Legend Snippet: In vitro kinetic analysis of phosphorylated and nonphosphorylated CTF cleavage by γ-secretase. A , in vitro γ-secretase assay with membrane preparations from wild-type mouse brain. Membrane preparations of the wild-type mouse brains were incubated at 37 °C for the indicated period of time (h). The samples were subjected to immunoblotting with anti-APP C-terminal to detect pAICD and nAICD, along with CTFs. Actin was also detected with anti-actin antibody. B and C, kinetic analysis of AICD generated by incubation of membrane preparations. The nAICD and pAICD in panel A were quantified with VersaDoc imaging analyzer and the levels of nAICD ( panel B ) and pAICD ( panel C ) at the respective incubation times were indicated as a relative ratio to the level at 4 h (1.0). D, the production ratio of pAICD to nAICD (pAICD/nAICD) at the indicated times are shown. Results are presented with mean ± S.D. ( n = 3).

    Techniques Used: In Vitro, Incubation, Generated, Imaging

    Quantification of pCTFs and nCTFs in DRM and non-DRM fractions. A , identification of APP CTFs in DRM and non-DRM fractions. Proteins in CHAPSO DRM and non-DRM fractions were analyzed by immunoblotting with anti-APP C-terminal ( upper and middle ) and anti-Thr 668 phosphorylation state-specific ( lower ) antibodies. The membrane preparation is a sample prior to fractionation. A representative result is indicated. B, CTFs levels in DRM and non-DRM fractions. Respective CTFs shown in panel A were quantified by setting the amount of pC99 in the DRM to 1.0 and measuring the amounts of other CTF species as a ratio to pC99. Results are presented with mean ± S.D. The asterisk represents the statistical significance ( p
    Figure Legend Snippet: Quantification of pCTFs and nCTFs in DRM and non-DRM fractions. A , identification of APP CTFs in DRM and non-DRM fractions. Proteins in CHAPSO DRM and non-DRM fractions were analyzed by immunoblotting with anti-APP C-terminal ( upper and middle ) and anti-Thr 668 phosphorylation state-specific ( lower ) antibodies. The membrane preparation is a sample prior to fractionation. A representative result is indicated. B, CTFs levels in DRM and non-DRM fractions. Respective CTFs shown in panel A were quantified by setting the amount of pC99 in the DRM to 1.0 and measuring the amounts of other CTF species as a ratio to pC99. Results are presented with mean ± S.D. The asterisk represents the statistical significance ( p

    Techniques Used: Fractionation

    20) Product Images from "The Bacterial Biofilm Matrix as a Platform for Protein Delivery"

    Article Title: The Bacterial Biofilm Matrix as a Platform for Protein Delivery

    Journal: mBio

    doi: 10.1128/mBio.00127-12

    A V. cholerae Δ bap1 Δ rbmA Δ rbmC triple mutant does not make a biofilm but can recruit the chitinase activity of RbmA–ChiA-2–FLAG to the cell surface. (A and B) Quantification of biofilms formed by wild-type V. cholerae (WT), an exopolysaccharide mutant (ΔvpsL), and a Δ bap1 Δ rbmA Δ rbmC mutant (triple) carrying an empty vector (pCTL) or a vector encoding RbmA (pRbmA) (A) and the pellicle formed by wild-type V. cholerae (WT) or the Δ bap1 Δ rbmA Δ rbmC triple mutant carrying either an empty vector (pCTL) or plasmids encoding RbmA-FLAG (pRbmA), RbmA-CtxB (pRbmA-CtxB), ChiA-2–FLAG (pChiA-2), or RbmA–ChiA-2–FLAG (pRbmA–ChiA-2) (B). (C and D) Chitinase activity in the cellular fraction (C) and supernatants (D) of V. cholerae Δ bap1 Δ rbmA Δ rbmC mutant carrying an empty vector or a plasmid encoding RbmA-CtxB, ChiA-2–FLAG, or RbmA–ChiA-2–FLAG. Chitinase activity in the cellular fraction of the mutant expressing RbmA–ChiA-2–FLAG was significantly different from those in strains expressing all other recombinant proteins ( P
    Figure Legend Snippet: A V. cholerae Δ bap1 Δ rbmA Δ rbmC triple mutant does not make a biofilm but can recruit the chitinase activity of RbmA–ChiA-2–FLAG to the cell surface. (A and B) Quantification of biofilms formed by wild-type V. cholerae (WT), an exopolysaccharide mutant (ΔvpsL), and a Δ bap1 Δ rbmA Δ rbmC mutant (triple) carrying an empty vector (pCTL) or a vector encoding RbmA (pRbmA) (A) and the pellicle formed by wild-type V. cholerae (WT) or the Δ bap1 Δ rbmA Δ rbmC triple mutant carrying either an empty vector (pCTL) or plasmids encoding RbmA-FLAG (pRbmA), RbmA-CtxB (pRbmA-CtxB), ChiA-2–FLAG (pChiA-2), or RbmA–ChiA-2–FLAG (pRbmA–ChiA-2) (B). (C and D) Chitinase activity in the cellular fraction (C) and supernatants (D) of V. cholerae Δ bap1 Δ rbmA Δ rbmC mutant carrying an empty vector or a plasmid encoding RbmA-CtxB, ChiA-2–FLAG, or RbmA–ChiA-2–FLAG. Chitinase activity in the cellular fraction of the mutant expressing RbmA–ChiA-2–FLAG was significantly different from those in strains expressing all other recombinant proteins ( P

    Techniques Used: Mutagenesis, Activity Assay, Plasmid Preparation, Expressing, Recombinant

    An enzymatically active RbmA–ChiA-2–FLAG fusion protein is retained in the biofilm matrix. (A) Immunofluorescent imaging of the distribution of ChiA-2–FLAG, RbmA–ChiA-2–FLAG, RbmA-FLAG, or RbmA-CtxB in a biofilm formed by wild-type V. cholerae carrying a plasmid encoding each of these proteins. The proteins were visualized with an anti-FLAG antibody or anti-CtxB antibody in the case of RbmA-CtxB. Bacterial DNA was stained with DAPI (4′,6-diamidino-2-phenylindole). As a control, a biofilm formed by wild-type V. cholerae carrying an empty vector was developed with an anti-FLAG antibody (CTL) (bar = 10 µM). (B) A magnified view of the distribution of RbmA–ChiA-2–FLAG in the biofilm (bar = 10 µM). (C and D) Chitinase activity measured in the biofilms (C) and supernatants (D) of wild-type V. cholerae carrying an empty vector (CTL) or a plasmid encoding RbmA-CtxB (RbmA-CtxB), ChiA-2–FLAG (ChiA-2), or RbmA–ChiA-2–FLAG (RbmA–ChiA-2). The chitinase activity in the biofilm of the strain expressing RbmA–ChiA-2–FLAG was significantly different from that in all other biofilms ( P ≤ 0.0003). Similarly, chitinase activity in the supernatants of strains expressing either RbmA–ChiA-2–FLAG or ChiA-2–FLAG was significantly different from that of strains carrying the control vector ( P = 0.007 and P = 0.0215, respectively) or the RbmA-CtxB fusion ( P = 0.0025 or P = 0.0149, respectively). The difference in chitinase activity between the supernatants of the strains expressing RbmA–ChiA-2–FLAG and ChiA-2–FLAG was not statistically significant ( P = 0.07).
    Figure Legend Snippet: An enzymatically active RbmA–ChiA-2–FLAG fusion protein is retained in the biofilm matrix. (A) Immunofluorescent imaging of the distribution of ChiA-2–FLAG, RbmA–ChiA-2–FLAG, RbmA-FLAG, or RbmA-CtxB in a biofilm formed by wild-type V. cholerae carrying a plasmid encoding each of these proteins. The proteins were visualized with an anti-FLAG antibody or anti-CtxB antibody in the case of RbmA-CtxB. Bacterial DNA was stained with DAPI (4′,6-diamidino-2-phenylindole). As a control, a biofilm formed by wild-type V. cholerae carrying an empty vector was developed with an anti-FLAG antibody (CTL) (bar = 10 µM). (B) A magnified view of the distribution of RbmA–ChiA-2–FLAG in the biofilm (bar = 10 µM). (C and D) Chitinase activity measured in the biofilms (C) and supernatants (D) of wild-type V. cholerae carrying an empty vector (CTL) or a plasmid encoding RbmA-CtxB (RbmA-CtxB), ChiA-2–FLAG (ChiA-2), or RbmA–ChiA-2–FLAG (RbmA–ChiA-2). The chitinase activity in the biofilm of the strain expressing RbmA–ChiA-2–FLAG was significantly different from that in all other biofilms ( P ≤ 0.0003). Similarly, chitinase activity in the supernatants of strains expressing either RbmA–ChiA-2–FLAG or ChiA-2–FLAG was significantly different from that of strains carrying the control vector ( P = 0.007 and P = 0.0215, respectively) or the RbmA-CtxB fusion ( P = 0.0025 or P = 0.0149, respectively). The difference in chitinase activity between the supernatants of the strains expressing RbmA–ChiA-2–FLAG and ChiA-2–FLAG was not statistically significant ( P = 0.07).

    Techniques Used: Imaging, Plasmid Preparation, Staining, CTL Assay, Activity Assay, Expressing

    21) Product Images from "Protective Roles of Gadd45 and MDM2 in Blueberry Anthocyanins Mediated DNA Repair of Fragmented and Non-Fragmented DNA Damage in UV-Irradiated HepG2 Cells"

    Article Title: Protective Roles of Gadd45 and MDM2 in Blueberry Anthocyanins Mediated DNA Repair of Fragmented and Non-Fragmented DNA Damage in UV-Irradiated HepG2 Cells

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms141121447

    Western blot detection of Gadd45, MDM2, p53 and p21 protein expression in UV-irradiated and BA pre-treated HepG2 cells. ( a ) Western blotting with HepG2 cells that were treated with or without BA, irradiated or non irradiated with a UV dose of 30 mJ/cm 2 and harvested at the indicated times following treatment; ( b ) The relative expression of Gadd45 value after normalizing to β-actin; ( c ) The relative expression of MDM2 value after normalizing to β-actin; ( d ) The relative expression of p21 value after normalizing to β-actin; ( e ) The relative expression of p53 value after normalizing to β-actin. * p
    Figure Legend Snippet: Western blot detection of Gadd45, MDM2, p53 and p21 protein expression in UV-irradiated and BA pre-treated HepG2 cells. ( a ) Western blotting with HepG2 cells that were treated with or without BA, irradiated or non irradiated with a UV dose of 30 mJ/cm 2 and harvested at the indicated times following treatment; ( b ) The relative expression of Gadd45 value after normalizing to β-actin; ( c ) The relative expression of MDM2 value after normalizing to β-actin; ( d ) The relative expression of p21 value after normalizing to β-actin; ( e ) The relative expression of p53 value after normalizing to β-actin. * p

    Techniques Used: Western Blot, Expressing, Irradiation

    Gene expression of Gadd45 and MDM2 by RT-PCR in UV-irradiated HepG2 cells that were pre-treated for 12 h with BA. ( a ) Lane M, marker; lane 1 control (no UV radiation); lane 2, UV radiation; lane 3, UV+ 25 μg/mL; lane 4, UV+ 50 μg/mL; lane 5, UV+ 75 μg/mL; ( b ) The relative expression of Gadd45 and MDM2 value after normalizing to β-actin. * p
    Figure Legend Snippet: Gene expression of Gadd45 and MDM2 by RT-PCR in UV-irradiated HepG2 cells that were pre-treated for 12 h with BA. ( a ) Lane M, marker; lane 1 control (no UV radiation); lane 2, UV radiation; lane 3, UV+ 25 μg/mL; lane 4, UV+ 50 μg/mL; lane 5, UV+ 75 μg/mL; ( b ) The relative expression of Gadd45 and MDM2 value after normalizing to β-actin. * p

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Irradiation, Marker

    22) Product Images from "Early Vessel Destabilization Mediated by Angiopoietin-2 and Subsequent Vessel Maturation via Angiopoietin-1 Induce Functional Neovasculature after Ischemia"

    Article Title: Early Vessel Destabilization Mediated by Angiopoietin-2 and Subsequent Vessel Maturation via Angiopoietin-1 Induce Functional Neovasculature after Ischemia

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0061831

    Apelin did not provide early neovascularization with Ang2. (A, B) LDI of blood reperfusion induced by APLN is enhanced without continuous Ang2 overexpression (rAAV.Ang2), where as the combination of APLN and Ang2 displayed no significant alteration. APLN combined with early Ang2 overexpression (rAAV.Apln+Ang2(d0-3)) induced a trend towards higher perfusion (p = 0.08). (C–E) PECAM-1 positive capillaries were slightly increased by rAAV.APLN, whereas the combination with continuous rAAV.Ang2 or rAAV.Ang2(d0-3) clearly enhaced capillary formation compared to APLN alone. Capillary maturation, indicated by pericyte coverage (E) was enhanced by rAAV.APLN with or without rAAV.Ang2(d0-3), whereas continous Ang2 reduced PC-R. (MEAN ± SEM, n = 7,* p
    Figure Legend Snippet: Apelin did not provide early neovascularization with Ang2. (A, B) LDI of blood reperfusion induced by APLN is enhanced without continuous Ang2 overexpression (rAAV.Ang2), where as the combination of APLN and Ang2 displayed no significant alteration. APLN combined with early Ang2 overexpression (rAAV.Apln+Ang2(d0-3)) induced a trend towards higher perfusion (p = 0.08). (C–E) PECAM-1 positive capillaries were slightly increased by rAAV.APLN, whereas the combination with continuous rAAV.Ang2 or rAAV.Ang2(d0-3) clearly enhaced capillary formation compared to APLN alone. Capillary maturation, indicated by pericyte coverage (E) was enhanced by rAAV.APLN with or without rAAV.Ang2(d0-3), whereas continous Ang2 reduced PC-R. (MEAN ± SEM, n = 7,* p

    Techniques Used: Over Expression

    Capillary growth and maturation profile of VEGF-A, APLN and Ang1 with or without Ang2 in vitro. (A) Capillary-like tube formation was enhanced by VEGF-A alone transfection in HMECs (1×10 4 per well of μ-slide angiogenesis plate) on 2D-matrigel. (B) 1×10 3 Pericytes labeled by DiO (green) are plated after capillary-like tube formation of HMECs with DiD labeling (red). 24 h later, co-cultures reveal a low rate of pericytes attraction by VEGF-A, which was unaffected by Ang2. (C) Apelin (APLN) promoted tube formation with or without Ang2. (D) Pericyte recruitment was enhanced by Apelin, an effect attenuated by Ang2. (E) Ang1 induced tubes formationin the absence of Ang2. (F) However, the tube maturation provided by Ang1 was abolished in the presence of Ang2. (MEAN ± SEM, n = 5, ** p
    Figure Legend Snippet: Capillary growth and maturation profile of VEGF-A, APLN and Ang1 with or without Ang2 in vitro. (A) Capillary-like tube formation was enhanced by VEGF-A alone transfection in HMECs (1×10 4 per well of μ-slide angiogenesis plate) on 2D-matrigel. (B) 1×10 3 Pericytes labeled by DiO (green) are plated after capillary-like tube formation of HMECs with DiD labeling (red). 24 h later, co-cultures reveal a low rate of pericytes attraction by VEGF-A, which was unaffected by Ang2. (C) Apelin (APLN) promoted tube formation with or without Ang2. (D) Pericyte recruitment was enhanced by Apelin, an effect attenuated by Ang2. (E) Ang1 induced tubes formationin the absence of Ang2. (F) However, the tube maturation provided by Ang1 was abolished in the presence of Ang2. (MEAN ± SEM, n = 5, ** p

    Techniques Used: In Vitro, Transfection, Labeling

    APLN provided late neovascularization, with or without continuous Ang2. (A,B) At d14, LDI indicated an increased perfusion by APLN with or without continuous Ang2 compared to control. Of note, the effect of APLN was prohibited by Ang2(d0-3). (C,D) At d14, rAAV.APLN improved capillary growth (CMF-R), when used alone or in combination with continuous Ang2 overexpression and temporary Ang2 (d0-3). (C,E) Pericyte recruitment was provided by APLN, but not if combined with continuous Ang2 overexpression. (MEAN ± SEM, n = 7,* p
    Figure Legend Snippet: APLN provided late neovascularization, with or without continuous Ang2. (A,B) At d14, LDI indicated an increased perfusion by APLN with or without continuous Ang2 compared to control. Of note, the effect of APLN was prohibited by Ang2(d0-3). (C,D) At d14, rAAV.APLN improved capillary growth (CMF-R), when used alone or in combination with continuous Ang2 overexpression and temporary Ang2 (d0-3). (C,E) Pericyte recruitment was provided by APLN, but not if combined with continuous Ang2 overexpression. (MEAN ± SEM, n = 7,* p

    Techniques Used: Over Expression

    Ang1 combined with early Ang2 (d0-3) induces enhanced neovascularization at d7. (A, B) LDI analysis revealed no alteration of perfusion at d7 by either Ang1 or Ang2 (continuous overexpression), but a significant increase in the Ang1+Ang2(d0-3) group. (C–D) Analysis of capillaries revealed an elevated CMF-R only in the Ang1 and Ang2(d0-3) group, whereas the capillarization was unaltered if Ang 1 and Ang2 were overxpressed alone. (C,E) Ang1 alone as well as Ang1+Ang2(d0-3) was capable of enhancing pericytes coverage compared to control, Ang2 alone had no effect on the pericyte/capillary ratio (PC-R). (MEAN ± SEM, n = 7,* p
    Figure Legend Snippet: Ang1 combined with early Ang2 (d0-3) induces enhanced neovascularization at d7. (A, B) LDI analysis revealed no alteration of perfusion at d7 by either Ang1 or Ang2 (continuous overexpression), but a significant increase in the Ang1+Ang2(d0-3) group. (C–D) Analysis of capillaries revealed an elevated CMF-R only in the Ang1 and Ang2(d0-3) group, whereas the capillarization was unaltered if Ang 1 and Ang2 were overxpressed alone. (C,E) Ang1 alone as well as Ang1+Ang2(d0-3) was capable of enhancing pericytes coverage compared to control, Ang2 alone had no effect on the pericyte/capillary ratio (PC-R). (MEAN ± SEM, n = 7,* p

    Techniques Used: Over Expression

    Ang1 combined with early Ang2 (d0-3) enhanced neovascularization at d14. (A,B) Ang1 improved hindlimb perfusion only, if combined with Ang2 (d0-3), a condition which also elevated (C,D) capillary/muscle fiber ratio (CMF-R) and (C,E) pericyte/capillary ratio (PC-R) to a significant higher level. (MEAN ± SEM, n = 7,* p
    Figure Legend Snippet: Ang1 combined with early Ang2 (d0-3) enhanced neovascularization at d14. (A,B) Ang1 improved hindlimb perfusion only, if combined with Ang2 (d0-3), a condition which also elevated (C,D) capillary/muscle fiber ratio (CMF-R) and (C,E) pericyte/capillary ratio (PC-R) to a significant higher level. (MEAN ± SEM, n = 7,* p

    Techniques Used:

    rAAV.VEGF-A did not provide enhanced neovascularization at d14. (A, B) Laser Doppler images and quantification of LDI of low and high VEGF-A groups displayed no difference to controls, similar to the combination of high VEGF-A with Ang2. (C) Fluorescence microscopy images of gastrocnemius muscle for capillaries (PECAM-1, red), pericytes (NG2, green) and nuclei (DAPI, blue) for the different groups. (D) Quantification of CMF-R displayed increased capillary growth for high VEGF-A with or without Ang2, whereas (E) PC-R revealed a diminished vessel maturation only for high VEGF-A combined with Ang2. (MEAN ± SEM, n = 7,* p
    Figure Legend Snippet: rAAV.VEGF-A did not provide enhanced neovascularization at d14. (A, B) Laser Doppler images and quantification of LDI of low and high VEGF-A groups displayed no difference to controls, similar to the combination of high VEGF-A with Ang2. (C) Fluorescence microscopy images of gastrocnemius muscle for capillaries (PECAM-1, red), pericytes (NG2, green) and nuclei (DAPI, blue) for the different groups. (D) Quantification of CMF-R displayed increased capillary growth for high VEGF-A with or without Ang2, whereas (E) PC-R revealed a diminished vessel maturation only for high VEGF-A combined with Ang2. (MEAN ± SEM, n = 7,* p

    Techniques Used: Fluorescence, Microscopy

    23) Product Images from "PIN2 Turnover in Arabidopsis Root Epidermal Cells Explored by the Photoconvertible Protein Dendra2"

    Article Title: PIN2 Turnover in Arabidopsis Root Epidermal Cells Explored by the Photoconvertible Protein Dendra2

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0061403

    Time-dependent photoconversion of PIN2-Dendra2 fusion protein. ( A ) The same root was analyzed for green and red fluorescence before (time 0) and after 3 s and 20 s conversion. ( B ) Efficiency of photoconversion depends on the time of illumination with blue-violet light. Green and red fluorescence signals were collected and the intensities of 150 transversal membranes within 5 roots were assessed with the ImageJ software. The interrupted line with open circles represents changes in red signal intensities, the solid line with closed circles represents decreasing in green signal intensities, bars represent SE. ( C ) Emission spectra of green and red signals of unconverted and photoconverted membranes were analyzed in three roots (represented by three lines in the graph). The HFT UV/488/543/633 was used as a beam splitter. Each point on the graph with SE bar represents the mean of 30 transversal membranes.
    Figure Legend Snippet: Time-dependent photoconversion of PIN2-Dendra2 fusion protein. ( A ) The same root was analyzed for green and red fluorescence before (time 0) and after 3 s and 20 s conversion. ( B ) Efficiency of photoconversion depends on the time of illumination with blue-violet light. Green and red fluorescence signals were collected and the intensities of 150 transversal membranes within 5 roots were assessed with the ImageJ software. The interrupted line with open circles represents changes in red signal intensities, the solid line with closed circles represents decreasing in green signal intensities, bars represent SE. ( C ) Emission spectra of green and red signals of unconverted and photoconverted membranes were analyzed in three roots (represented by three lines in the graph). The HFT UV/488/543/633 was used as a beam splitter. Each point on the graph with SE bar represents the mean of 30 transversal membranes.

    Techniques Used: Fluorescence, Software

    Localization pattern of PIN2-Dendra2 fusion protein. The roots of PIN2-Dendra2 were photoconverted before imaging. ( A ) PIN2-Dendra2 when driven by the endogenous promoter is expressed in the root tip epidermis (e) and cortex (c) identically to PIN2-EGFP that was described previously [48] . ( B ) In both PIN2-Dendra2 and PIN2-EGFP transgenic lines, the fusion proteins localized polarly in shootwards transversal membranes (arrows). ( C ) PIN2-Dendra2 similarly to PIN2-EGFP accumulates in BFA bodies (arrows). ( D ) Expression and localization pattern of PIN2-Dendra2 was confirmed by immunohistochemistry using an anti-Dendra2 antibody (green) on chemically fixed and sectioned roots. Sections were counterstained by propidium iodide (red).
    Figure Legend Snippet: Localization pattern of PIN2-Dendra2 fusion protein. The roots of PIN2-Dendra2 were photoconverted before imaging. ( A ) PIN2-Dendra2 when driven by the endogenous promoter is expressed in the root tip epidermis (e) and cortex (c) identically to PIN2-EGFP that was described previously [48] . ( B ) In both PIN2-Dendra2 and PIN2-EGFP transgenic lines, the fusion proteins localized polarly in shootwards transversal membranes (arrows). ( C ) PIN2-Dendra2 similarly to PIN2-EGFP accumulates in BFA bodies (arrows). ( D ) Expression and localization pattern of PIN2-Dendra2 was confirmed by immunohistochemistry using an anti-Dendra2 antibody (green) on chemically fixed and sectioned roots. Sections were counterstained by propidium iodide (red).

    Techniques Used: Imaging, Transgenic Assay, Expressing, Immunohistochemistry

    Western blot analysis of PIN2-Dendra2 expression. For Western blot analysis seedlings were grown and treated as described for microscopy and roots were collected after 12 h’ treatment for protein extraction. The lane marked as ‘covered’ represents extracts from seedlings whose roots grew for 12 h under anaerobic conditions under the cover slip. Control (untreated) samples, samples of untransformed seedlings and samples treated with ABA and jasmonates (MeJA and JA) were kept uncovered during the experiment. The blot was probed by a Dendra2 specific antibody (upper row), stripped and re-probed with an actin specific antibody (middle row). The lower row shows the membrane after Ponceau S staining. Bands intensities were quantified using ImageJ. The values obtained for Dendra2 were divided by the values for actin for that sample, normalized to the control and graphed. Columns in the graph represent the means of three blots, bars represent SD.
    Figure Legend Snippet: Western blot analysis of PIN2-Dendra2 expression. For Western blot analysis seedlings were grown and treated as described for microscopy and roots were collected after 12 h’ treatment for protein extraction. The lane marked as ‘covered’ represents extracts from seedlings whose roots grew for 12 h under anaerobic conditions under the cover slip. Control (untreated) samples, samples of untransformed seedlings and samples treated with ABA and jasmonates (MeJA and JA) were kept uncovered during the experiment. The blot was probed by a Dendra2 specific antibody (upper row), stripped and re-probed with an actin specific antibody (middle row). The lower row shows the membrane after Ponceau S staining. Bands intensities were quantified using ImageJ. The values obtained for Dendra2 were divided by the values for actin for that sample, normalized to the control and graphed. Columns in the graph represent the means of three blots, bars represent SD.

    Techniques Used: Western Blot, Expressing, Microscopy, Protein Extraction, Staining

    Values of green and red signal intensities in transversal plasma membranes containing PIN2-Dendra2. ( A ) Means of green and red membrane fluorescence intensities examined before and after photoconversion in arbitrary unites. The graph summarize the results obtained in the course of the whole study (with 330 roots 20 to 25 membranes per root were analyzed). ( B ) Values of green signal intensities emitted by the membranes before photoconversion were plotted against the values of red signal intensities emitted by the same membranes after 15 s’ photoconversion. The graph shows the results from a total of 2500 membranes from 100 roots used in the course of study.
    Figure Legend Snippet: Values of green and red signal intensities in transversal plasma membranes containing PIN2-Dendra2. ( A ) Means of green and red membrane fluorescence intensities examined before and after photoconversion in arbitrary unites. The graph summarize the results obtained in the course of the whole study (with 330 roots 20 to 25 membranes per root were analyzed). ( B ) Values of green signal intensities emitted by the membranes before photoconversion were plotted against the values of red signal intensities emitted by the same membranes after 15 s’ photoconversion. The graph shows the results from a total of 2500 membranes from 100 roots used in the course of study.

    Techniques Used: Fluorescence

    Representative spectra of Dendra2 before and after photoconversion. Dendra2 was expressed under the control of the 35S promoter and data for guard cells and root tips are presented. PIN2-Dendra2 driven by the endogenous promoter was analyzed in the membranes of root epidermis. Spectra were measured with a Zeiss LSM-510 Meta microscope before (labeled as unconverted) and after photoconversion (labeled as converted) for regions of interest (ROI) drawn in the coded images (left pictures in panel). Intact Dendra2 was analyzed in the nuclei (ROI1 drawn in the red color in the coded images) and in the cytoplasm (ROI2 drawn in green color in the coded images) of the guard cells and the cells of the root cap. Red lines in graphs represent the spectra emitted by nuclei, green lines represent the cytoplasm. The lower row in the panel shows the spectra emitted by the membrane-localized PIN2-Dendra2 (region drawn in red color in the coded image). The 458 nm excitation was combined with the HFT 458 beam splitter, the 488 nm excitation with the HFT 488 beam splitter and the 543 nm excitation with the HFT UV/488/543/633 beam splitter.
    Figure Legend Snippet: Representative spectra of Dendra2 before and after photoconversion. Dendra2 was expressed under the control of the 35S promoter and data for guard cells and root tips are presented. PIN2-Dendra2 driven by the endogenous promoter was analyzed in the membranes of root epidermis. Spectra were measured with a Zeiss LSM-510 Meta microscope before (labeled as unconverted) and after photoconversion (labeled as converted) for regions of interest (ROI) drawn in the coded images (left pictures in panel). Intact Dendra2 was analyzed in the nuclei (ROI1 drawn in the red color in the coded images) and in the cytoplasm (ROI2 drawn in green color in the coded images) of the guard cells and the cells of the root cap. Red lines in graphs represent the spectra emitted by nuclei, green lines represent the cytoplasm. The lower row in the panel shows the spectra emitted by the membrane-localized PIN2-Dendra2 (region drawn in red color in the coded image). The 458 nm excitation was combined with the HFT 458 beam splitter, the 488 nm excitation with the HFT 488 beam splitter and the 543 nm excitation with the HFT UV/488/543/633 beam splitter.

    Techniques Used: Microscopy, Labeling

    24) Product Images from "Intermolecular interactions of the malate synthase of Paracoccidioides spp"

    Article Title: Intermolecular interactions of the malate synthase of Paracoccidioides spp

    Journal: BMC Microbiology

    doi: 10.1186/1471-2180-13-107

    Interaction between Paracoccidioides yeast cells and pneumocytes by confocal laser scanning microscopy. Infected cell monolayers were fixed and permeabilized. Primary anti- Pb MLS and secondary antibodies Alexa Fluor 594 goat anti-rabbit IgG (red) were used. The specimens were analyzed by laser confocal microscopy using DIC ( A ) and fluorescence ( B ).
    Figure Legend Snippet: Interaction between Paracoccidioides yeast cells and pneumocytes by confocal laser scanning microscopy. Infected cell monolayers were fixed and permeabilized. Primary anti- Pb MLS and secondary antibodies Alexa Fluor 594 goat anti-rabbit IgG (red) were used. The specimens were analyzed by laser confocal microscopy using DIC ( A ) and fluorescence ( B ).

    Techniques Used: Confocal Laser Scanning Microscopy, Infection, Confocal Microscopy, Fluorescence

    25) Product Images from "Rottlerin-Mediated Inhibition of Chlamydia trachomatis Growth and Uptake of Sphingolipids Is Independent of p38-Regulated/Activated Protein Kinase (PRAK)"

    Article Title: Rottlerin-Mediated Inhibition of Chlamydia trachomatis Growth and Uptake of Sphingolipids Is Independent of p38-Regulated/Activated Protein Kinase (PRAK)

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0044733

    Replication of C. trachomatis organisms in PRAK-deficient mouse embryo fibroblast cells. Mouse embryo fibroblast cells (MEF) without (panels a–d) or with (e–h) PRAK deficiency (PRAK−/−) were infected with (b–d f–h) or without (a e) C. trachomatis (MOI = 0.5) for various periods of time as indicated on top of the figure. The cultures were processed for immunofluorescence assay with a rabbit antibody for visualizing chlamydial organisms (green), Alexa-Fluor 568 Phalloidin for host cell F-actin (red) and Hoechst dye for DNA (blue). Note that the inclusion sizes were similar in MEF with or without PRAK deficiency.
    Figure Legend Snippet: Replication of C. trachomatis organisms in PRAK-deficient mouse embryo fibroblast cells. Mouse embryo fibroblast cells (MEF) without (panels a–d) or with (e–h) PRAK deficiency (PRAK−/−) were infected with (b–d f–h) or without (a e) C. trachomatis (MOI = 0.5) for various periods of time as indicated on top of the figure. The cultures were processed for immunofluorescence assay with a rabbit antibody for visualizing chlamydial organisms (green), Alexa-Fluor 568 Phalloidin for host cell F-actin (red) and Hoechst dye for DNA (blue). Note that the inclusion sizes were similar in MEF with or without PRAK deficiency.

    Techniques Used: Infection, Immunofluorescence

    Rottlerin inhibits chlamydial growth in the absence of PRAK. MEF without (panels a–c) or with (d–f) PRAK deficiency (PRAK−/−) were infected with C. trachomatis (MOI = 0.5) and at oh (b e) or 16 h (c f) post infection, parallel cultures were treated with (b, c, e f) or without (a d) rottlerin at 1 µM. The cultures were processed 44 h post infection for immunofluorescence assay as described in Fig. 3 legend. Note that rottlerin inhibited chlamydial growth in both wild type and PRAK-deficient MEF cells.
    Figure Legend Snippet: Rottlerin inhibits chlamydial growth in the absence of PRAK. MEF without (panels a–c) or with (d–f) PRAK deficiency (PRAK−/−) were infected with C. trachomatis (MOI = 0.5) and at oh (b e) or 16 h (c f) post infection, parallel cultures were treated with (b, c, e f) or without (a d) rottlerin at 1 µM. The cultures were processed 44 h post infection for immunofluorescence assay as described in Fig. 3 legend. Note that rottlerin inhibited chlamydial growth in both wild type and PRAK-deficient MEF cells.

    Techniques Used: Infection, Immunofluorescence

    Chlamydia trachomatis acquisition of host sphingomyelin is independent of PRAK. (A) HeLa cells with (panels e–h) or without (a–d) C. trachomatis infection (MOI = 0.5) were treated without (a e) or with EGCG (1 µM, b f; 10 µM, c g) or rottlerin (1 µM, d h) 16 h post infection. Eight hours later, the cultures were subjected to BODIPY-FL-C5-ceremide labeling and visualized under a fluorescence microscope. Note that EGCG failed to block the accumulation of BODIPY-FL-sphingomyelin in the chlamydial inclusions (panel f g) while rottlerin did (h). (B) MEF without (panels a c) or with (b d) PRAK deficiency (PRAK−/−) were infected with C. trachomatis (MOI = 0.5) and 24 h post infection, the cultures were labeled with BODIPY-FL-C5-ceremide and observed as described above. Note that C. trachomatis organisms can take up BODIPY-FL-sphingomyelin from MEF cells with or without PRAK. The thick arrows point to chlamydial inclusions with while thin arrows point to the inclusions without the fluorescent sphingomyelin.
    Figure Legend Snippet: Chlamydia trachomatis acquisition of host sphingomyelin is independent of PRAK. (A) HeLa cells with (panels e–h) or without (a–d) C. trachomatis infection (MOI = 0.5) were treated without (a e) or with EGCG (1 µM, b f; 10 µM, c g) or rottlerin (1 µM, d h) 16 h post infection. Eight hours later, the cultures were subjected to BODIPY-FL-C5-ceremide labeling and visualized under a fluorescence microscope. Note that EGCG failed to block the accumulation of BODIPY-FL-sphingomyelin in the chlamydial inclusions (panel f g) while rottlerin did (h). (B) MEF without (panels a c) or with (b d) PRAK deficiency (PRAK−/−) were infected with C. trachomatis (MOI = 0.5) and 24 h post infection, the cultures were labeled with BODIPY-FL-C5-ceremide and observed as described above. Note that C. trachomatis organisms can take up BODIPY-FL-sphingomyelin from MEF cells with or without PRAK. The thick arrows point to chlamydial inclusions with while thin arrows point to the inclusions without the fluorescent sphingomyelin.

    Techniques Used: Infection, Labeling, Fluorescence, Microscopy, Blocking Assay

    26) Product Images from "The Epigenetic Bivalency of Core Pancreatic ?-Cell Transcription Factor Genes within Mouse Pluripotent Embryonic Stem Cells Is Not Affected by Knockdown of the Polycomb Repressive Complex 2, SUZ12"

    Article Title: The Epigenetic Bivalency of Core Pancreatic ?-Cell Transcription Factor Genes within Mouse Pluripotent Embryonic Stem Cells Is Not Affected by Knockdown of the Polycomb Repressive Complex 2, SUZ12

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0097820

    ChIP analysis for H3K4me3 and H3K27me3 at the key pluripotency and β-cell transcription factors in D3 and MIN6 cells. ChIP assays for the active H3K4me3 and repressive H3K27me3 histone modifications were carried out on chromatin extracts from  A ) D3 cells; and B) MIN6 cells. The presence of each histone modification at a locus within 1 kb of the transcription start site of the pluripotency ( Nanog, Oct4, Utf1  and  Sox2 ) and β-cell ( Pdx1, Nkx6.1 ,  Nkx2.2 ,  MafA  and  Pax4 ) transcription factors was then quantified by qPCR analysis. Binding values of non-immune IgG were subtracted from the binding values of antibodies for each histone modification. The data is presented as the amount of DNA specifically bound relative to the total amount of DNA, expressed as a percentage. As a benchmark to determine the level of histone binding that would indicate the significance of a histone modification at a gene of interest, the grey area represents the range two standard deviations around the mean of binding by the pluripotency genes ( Nanog ,  Oct4 ,  Utf1  and  Sox2 ) grouped together. The results are the mean and standard deviation of three independent experiments. Statistically significant differences in binding at a β-cell gene of interest in comparison to the benchmark range are denoted by ** p
    Figure Legend Snippet: ChIP analysis for H3K4me3 and H3K27me3 at the key pluripotency and β-cell transcription factors in D3 and MIN6 cells. ChIP assays for the active H3K4me3 and repressive H3K27me3 histone modifications were carried out on chromatin extracts from A ) D3 cells; and B) MIN6 cells. The presence of each histone modification at a locus within 1 kb of the transcription start site of the pluripotency ( Nanog, Oct4, Utf1 and Sox2 ) and β-cell ( Pdx1, Nkx6.1 , Nkx2.2 , MafA and Pax4 ) transcription factors was then quantified by qPCR analysis. Binding values of non-immune IgG were subtracted from the binding values of antibodies for each histone modification. The data is presented as the amount of DNA specifically bound relative to the total amount of DNA, expressed as a percentage. As a benchmark to determine the level of histone binding that would indicate the significance of a histone modification at a gene of interest, the grey area represents the range two standard deviations around the mean of binding by the pluripotency genes ( Nanog , Oct4 , Utf1 and Sox2 ) grouped together. The results are the mean and standard deviation of three independent experiments. Statistically significant differences in binding at a β-cell gene of interest in comparison to the benchmark range are denoted by ** p

    Techniques Used: Chromatin Immunoprecipitation, Modification, Real-time Polymerase Chain Reaction, Binding Assay, Standard Deviation

    Analysis of siRNA-mediated knockdown of Suz12 in D3 cells after 144 h transfection. D3 cells were transfected with 100 Gapdh , Suz12 or non-targeting ‘scrambled’ siRNA using the DharmaFECT 1 transfection reagent in antibiotic-free ES cell medium. Untreated control cells were maintained in antibiotic-free ES cell medium only. The ‘Dharmafect’ control was cultured in transfection medium without siRNA. A ) qRT-PCR analysis was performed to test the expression status of Gapdh and Suz12 at 144 h transfection. The data is expressed as the average relative gene expression ± standard deviation in comparison to the scrambled siRNA. Quantified values were normalized against two housekeeping genes, Tbp and Actb. The results are the mean and standard deviation of three independent experiments. Statistically significant differences in gene expression levels are denoted by *** p
    Figure Legend Snippet: Analysis of siRNA-mediated knockdown of Suz12 in D3 cells after 144 h transfection. D3 cells were transfected with 100 Gapdh , Suz12 or non-targeting ‘scrambled’ siRNA using the DharmaFECT 1 transfection reagent in antibiotic-free ES cell medium. Untreated control cells were maintained in antibiotic-free ES cell medium only. The ‘Dharmafect’ control was cultured in transfection medium without siRNA. A ) qRT-PCR analysis was performed to test the expression status of Gapdh and Suz12 at 144 h transfection. The data is expressed as the average relative gene expression ± standard deviation in comparison to the scrambled siRNA. Quantified values were normalized against two housekeeping genes, Tbp and Actb. The results are the mean and standard deviation of three independent experiments. Statistically significant differences in gene expression levels are denoted by *** p

    Techniques Used: Transfection, Cell Culture, Quantitative RT-PCR, Expressing, Standard Deviation

    The effect of siRNA-mediated transfection on SUZ12 and H3K27me3 binding at Gata4 and the key β-cell transcription factors in D3 cells. D3 cells were transfected with 100 uz12 or non-targeting ‘scrambled’ siRNA using the DharmaFECT 1 transfection reagent in antibio tic-free ES cell medium. Untreated control cells were maintained in antibiotic-free ES cell medium only. ChIP assays for A ) SUZ12 and B ) H3K27me3 were carried out on chromatin extracts from samples after 144 h transfection. The presence of SUZ12 and H3K27me3 at a locus within 1 kb of the transcription start site of Gata4 and the β-cell transcription factors, Pdx1 , Nkx6.1 , Nkx2.2 , MafA and Pax4 were then quantified by qPCR analysis. Binding values of non-immune IgG were subtracted from the binding values of SUZ12 or H3K27me3 antibody. The data is presented as the amount of DNA specifically bound relative to the total amount of DNA, expressed as a percentage. The results are the mean and standard deviation of three independent experiments. Statistically significant differences in SUZ12 or H3K27me3 binding at a loci are denoted by * p
    Figure Legend Snippet: The effect of siRNA-mediated transfection on SUZ12 and H3K27me3 binding at Gata4 and the key β-cell transcription factors in D3 cells. D3 cells were transfected with 100 uz12 or non-targeting ‘scrambled’ siRNA using the DharmaFECT 1 transfection reagent in antibio tic-free ES cell medium. Untreated control cells were maintained in antibiotic-free ES cell medium only. ChIP assays for A ) SUZ12 and B ) H3K27me3 were carried out on chromatin extracts from samples after 144 h transfection. The presence of SUZ12 and H3K27me3 at a locus within 1 kb of the transcription start site of Gata4 and the β-cell transcription factors, Pdx1 , Nkx6.1 , Nkx2.2 , MafA and Pax4 were then quantified by qPCR analysis. Binding values of non-immune IgG were subtracted from the binding values of SUZ12 or H3K27me3 antibody. The data is presented as the amount of DNA specifically bound relative to the total amount of DNA, expressed as a percentage. The results are the mean and standard deviation of three independent experiments. Statistically significant differences in SUZ12 or H3K27me3 binding at a loci are denoted by * p

    Techniques Used: Transfection, Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Standard Deviation

    27) Product Images from "Toll-like receptor 8 functions as a negative regulator of neurite outgrowth and inducer of neuronal apoptosis"

    Article Title: Toll-like receptor 8 functions as a negative regulator of neurite outgrowth and inducer of neuronal apoptosis

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200606016

    TLR8 stimulation in neurons does not activate the canonical TLR–NF-κB signaling pathway, but rather down-regulates IκBα and IRAK4. (A) ELISA assay for NF-κB (p65) transactivation using nuclear extracts from cortical neurons stimulated with 100 μM R-848, 500 μM loxoribine, 5 μg/ml LPS, or 10 ng/ml TNFα for the indicated times. LPS and TNFα serve as negative and positive controls, respectively. (B) Western blotting of the hallmarks of the conventional TLR-signaling pathway with lysates from neurons and Raw264.7 macrophages treated with 100 μM R-848 for the indicated times. (C) Quantification of changes in IκBα levels in R-848–stimulated neurons by band densitometry. A representative blot is shown in B. (D) Western blotting of IRAK4 in neurons stimulated with 100 μM R-848 for the indicated times. Note that TLR8 levels remain unchanged. (E) Quantification of changes in IRAK4 levels by band densitometry. A representative blot is shown in D. Data in C and E, expressed as percentage normalized to controls (100%), are the mean ± the SEM for pooled Western-blots from three independent cultures. Statistical analysis was done by t test. *, P
    Figure Legend Snippet: TLR8 stimulation in neurons does not activate the canonical TLR–NF-κB signaling pathway, but rather down-regulates IκBα and IRAK4. (A) ELISA assay for NF-κB (p65) transactivation using nuclear extracts from cortical neurons stimulated with 100 μM R-848, 500 μM loxoribine, 5 μg/ml LPS, or 10 ng/ml TNFα for the indicated times. LPS and TNFα serve as negative and positive controls, respectively. (B) Western blotting of the hallmarks of the conventional TLR-signaling pathway with lysates from neurons and Raw264.7 macrophages treated with 100 μM R-848 for the indicated times. (C) Quantification of changes in IκBα levels in R-848–stimulated neurons by band densitometry. A representative blot is shown in B. (D) Western blotting of IRAK4 in neurons stimulated with 100 μM R-848 for the indicated times. Note that TLR8 levels remain unchanged. (E) Quantification of changes in IRAK4 levels by band densitometry. A representative blot is shown in D. Data in C and E, expressed as percentage normalized to controls (100%), are the mean ± the SEM for pooled Western-blots from three independent cultures. Statistical analysis was done by t test. *, P

    Techniques Used: Enzyme-linked Immunosorbent Assay, Western Blot

    28) Product Images from "The Somatostatin 2A Receptor Is Enriched in Migrating Neurons during Rat and Human Brain Development and Stimulates Migration and Axonal Outgrowth"

    Article Title: The Somatostatin 2A Receptor Is Enriched in Migrating Neurons during Rat and Human Brain Development and Stimulates Migration and Axonal Outgrowth

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0005509

    Regional and cellular distribution of sst2A receptor immunoreactivity in the human developing cortex. A,B) Embryonic sagittal sections at GW 8 at the level of the medial cerebral cortex (A) reveals numerous receptor immunoreactive cell bodies (purple color) in the preplate (PP) (inset box) and in the subventricular zone (SVZ). By contrast the ventricular zone (VZ) is devoid of receptor immunoreactivity. The red color is due to the counterstaining of sections with neutral red. In the lateral part of the medial cerebral cortex (B), sst2A receptor immunoreactivity is detected in the marginal zone (MZ), cortical plate (CP), subplate/intermediate zone (SP/IZ) and SVZ (inset box). C–E) At GW 12 in coronal sections, albeit less intense, the pattern of receptor immunoreactivity is comparable to that observed at GW 8 with higher signals in the MZ (C,D) and SVZ (C,E). D and E are magnifications of boxed areas in C at the level of the MZ and the SVZ, respectively. Note in D that some bipolar neurons expressing the sst2A receptor are visible in the CP and in E that patches of labeling are observed in the SVZ contiguous to the VZ. F, G) In coronal sections at GW 23, the labeling is present in neurons of CP (F) as well as in presumably post-mitotic migrating neurons in the IZ (G). H–J) In coronal sections at birth, the labeling is diffusely distributed in layers II–III and V. In this latter layer some neurons positive for the sst2A receptor are also observed (I,J). Scale bars: A, B, D, E, G, 25 µm; C, F, H, 100 µm; Inset in A,B and I,J, 10 µm.
    Figure Legend Snippet: Regional and cellular distribution of sst2A receptor immunoreactivity in the human developing cortex. A,B) Embryonic sagittal sections at GW 8 at the level of the medial cerebral cortex (A) reveals numerous receptor immunoreactive cell bodies (purple color) in the preplate (PP) (inset box) and in the subventricular zone (SVZ). By contrast the ventricular zone (VZ) is devoid of receptor immunoreactivity. The red color is due to the counterstaining of sections with neutral red. In the lateral part of the medial cerebral cortex (B), sst2A receptor immunoreactivity is detected in the marginal zone (MZ), cortical plate (CP), subplate/intermediate zone (SP/IZ) and SVZ (inset box). C–E) At GW 12 in coronal sections, albeit less intense, the pattern of receptor immunoreactivity is comparable to that observed at GW 8 with higher signals in the MZ (C,D) and SVZ (C,E). D and E are magnifications of boxed areas in C at the level of the MZ and the SVZ, respectively. Note in D that some bipolar neurons expressing the sst2A receptor are visible in the CP and in E that patches of labeling are observed in the SVZ contiguous to the VZ. F, G) In coronal sections at GW 23, the labeling is present in neurons of CP (F) as well as in presumably post-mitotic migrating neurons in the IZ (G). H–J) In coronal sections at birth, the labeling is diffusely distributed in layers II–III and V. In this latter layer some neurons positive for the sst2A receptor are also observed (I,J). Scale bars: A, B, D, E, G, 25 µm; C, F, H, 100 µm; Inset in A,B and I,J, 10 µm.

    Techniques Used: Expressing, Labeling

    Regional and cellular localization of the sst2A receptor immunoreactivity in sagittal sections of the rat rhombencephalon at embryonic day 13 (E13). A) Densely packed sst2A receptor-immunoreactive cells are observed in the marginal zone contiguous to the ventricular zone of the rhombomeres (r1 to r6, arrowheads) and in the lateral reticular formation (LRtF). B–B″) Sst2A receptor-immunoreactive cells (red) are localized in the marginal zone (B) whereas proliferating cells identified by the proliferation marker Ki-67 (green) are concentrated in the ventricular zone (B′). The lack of overlap between the two signals (B″) indicates that sst2A receptor-expressing cells are predominantly post-mitotic. C) The majority of sst2A receptor-immunoreactive cells have small round perikarya and some exhibit immunolabeled processes that are oriented perpendicularly to the ventricular surface. D) A few sst2A receptor-immunoreactive cells are bipolar, displaying the morphological features of migrating neurons. E) In the LRtF, cell bodies are strongly sst2A receptor-immunoreactive. F–F″) An sst2A receptor-immunoreactive cell (red in F, F″) of the LRtF is found to be Ki-67-positive (green in F′, F″) (arrowheads). The low percentage of colocalization (F″) indicates that the majority of receptor-expressing cells are post-mitotic. G–G″) The post-mitotic feature of most sst2A receptor-immunoreactive cells (red in G, G″) of the rhombencephalon is further indicated by the colocalization (G″) with the post-mitotic neuronal marker β-tubulin (green in G′, G″) (arrowheads), as illustrated in the facial nucleus. Scale bars: A, 250 µm; B–B″, G–G″, 50 µm; C, E, 20 µm; F, 10 µm.
    Figure Legend Snippet: Regional and cellular localization of the sst2A receptor immunoreactivity in sagittal sections of the rat rhombencephalon at embryonic day 13 (E13). A) Densely packed sst2A receptor-immunoreactive cells are observed in the marginal zone contiguous to the ventricular zone of the rhombomeres (r1 to r6, arrowheads) and in the lateral reticular formation (LRtF). B–B″) Sst2A receptor-immunoreactive cells (red) are localized in the marginal zone (B) whereas proliferating cells identified by the proliferation marker Ki-67 (green) are concentrated in the ventricular zone (B′). The lack of overlap between the two signals (B″) indicates that sst2A receptor-expressing cells are predominantly post-mitotic. C) The majority of sst2A receptor-immunoreactive cells have small round perikarya and some exhibit immunolabeled processes that are oriented perpendicularly to the ventricular surface. D) A few sst2A receptor-immunoreactive cells are bipolar, displaying the morphological features of migrating neurons. E) In the LRtF, cell bodies are strongly sst2A receptor-immunoreactive. F–F″) An sst2A receptor-immunoreactive cell (red in F, F″) of the LRtF is found to be Ki-67-positive (green in F′, F″) (arrowheads). The low percentage of colocalization (F″) indicates that the majority of receptor-expressing cells are post-mitotic. G–G″) The post-mitotic feature of most sst2A receptor-immunoreactive cells (red in G, G″) of the rhombencephalon is further indicated by the colocalization (G″) with the post-mitotic neuronal marker β-tubulin (green in G′, G″) (arrowheads), as illustrated in the facial nucleus. Scale bars: A, 250 µm; B–B″, G–G″, 50 µm; C, E, 20 µm; F, 10 µm.

    Techniques Used: Marker, Expressing, Immunolabeling

    Expression of the sst2A receptor in serotoninergic neurons of the rat brainstem at E16. A) Triple-labeling with sst2A receptor (red), 5-HT (green) and DAPI (blue) in the ventral part of the brainstem illustrates that most serotoninergic neurons are sst2A receptor immunoreactive. B,B′ represent magnification of the boxed area in A. Note the extensive colocalization of sst2A receptor and 5-HT in both cell bodies and processes. C) The sst2A receptor is also expressed in serotoninergic migrating cells in the more dorsal part of the mesencephalon presumably corresponding to the dorsal raphe nucleus. D,D′ represents magnification of the boxed area in C and illustrates double-labeling in both the soma and processes of migrating neurons. Scale bars: A, C, 50 µm; B, D, 20 µm.
    Figure Legend Snippet: Expression of the sst2A receptor in serotoninergic neurons of the rat brainstem at E16. A) Triple-labeling with sst2A receptor (red), 5-HT (green) and DAPI (blue) in the ventral part of the brainstem illustrates that most serotoninergic neurons are sst2A receptor immunoreactive. B,B′ represent magnification of the boxed area in A. Note the extensive colocalization of sst2A receptor and 5-HT in both cell bodies and processes. C) The sst2A receptor is also expressed in serotoninergic migrating cells in the more dorsal part of the mesencephalon presumably corresponding to the dorsal raphe nucleus. D,D′ represents magnification of the boxed area in C and illustrates double-labeling in both the soma and processes of migrating neurons. Scale bars: A, C, 50 µm; B, D, 20 µm.

    Techniques Used: Expressing, Labeling

    Distribution of the sst2A receptor immunoreactivity in the rat hippocampus during pre- and postnatal development. A–A″) At E16, sst2A receptor immunoreactivity (red in A, A″) is localized in the intermediate zone (IZ) of the hippocampus. Note the lack of immunoreactivity in the ventricular zone (VZ). B–B″) At E21, the most intense immunolabeling is found in the intermediate zone of CA1. In addition, less intense immunolabeling is apparent in the pyramidal cell layer as well as in the strata oriens and radiatum of CA1, in the CA3 and in the developing dentate gyrus (DG). C) In the hilus of the DG, sst2A receptor immunoreactivity appears diffusely distributed. D represents magnification of the area labeled with asterisk in B and illustrates the diffuse sst2A receptor immunolabeling observed in the CA1 pyramidal cell layer. E represents magnification of boxed area in B. The sst2A receptor immunolabeling is intense in cells of the IZ whereas the subventricular zone (SVZ) is devoid of labeling. F) At P3, intense immunofluorescence is detected in the pyramidal layer, strata oriens and radiatum of CA1-3, as well as in the hilus of DG. The molecular layer of dentate gyrus is weakly immunoreactive. G represents magnification of boxed area in F and illustrates the intense sst2A receptor immunolabeling localized in CA1 pyramidal cell bodies and proximal dendrites. H represents magnification of area labeled with asterisk in F and illustrates the diffuse sst2A receptor immunolabeling observed in the hilus of the DG. Scale bars: A–A″, B–B″, F, 100 µm; C, D, E, G, H, 20 µm.
    Figure Legend Snippet: Distribution of the sst2A receptor immunoreactivity in the rat hippocampus during pre- and postnatal development. A–A″) At E16, sst2A receptor immunoreactivity (red in A, A″) is localized in the intermediate zone (IZ) of the hippocampus. Note the lack of immunoreactivity in the ventricular zone (VZ). B–B″) At E21, the most intense immunolabeling is found in the intermediate zone of CA1. In addition, less intense immunolabeling is apparent in the pyramidal cell layer as well as in the strata oriens and radiatum of CA1, in the CA3 and in the developing dentate gyrus (DG). C) In the hilus of the DG, sst2A receptor immunoreactivity appears diffusely distributed. D represents magnification of the area labeled with asterisk in B and illustrates the diffuse sst2A receptor immunolabeling observed in the CA1 pyramidal cell layer. E represents magnification of boxed area in B. The sst2A receptor immunolabeling is intense in cells of the IZ whereas the subventricular zone (SVZ) is devoid of labeling. F) At P3, intense immunofluorescence is detected in the pyramidal layer, strata oriens and radiatum of CA1-3, as well as in the hilus of DG. The molecular layer of dentate gyrus is weakly immunoreactive. G represents magnification of boxed area in F and illustrates the intense sst2A receptor immunolabeling localized in CA1 pyramidal cell bodies and proximal dendrites. H represents magnification of area labeled with asterisk in F and illustrates the diffuse sst2A receptor immunolabeling observed in the hilus of the DG. Scale bars: A–A″, B–B″, F, 100 µm; C, D, E, G, H, 20 µm.

    Techniques Used: Immunolabeling, Labeling, Immunofluorescence

    Regional, cellular and subcellular distribution of sst2A receptor immunoreactivity on coronal sections of the rat telencephalon at E16 and E18. A,B) Intense sst2A receptor immunoreactivity is detected at E16 in the post-mitotic areas of the lateral ganglionic eminence (LGE) and the caudal ganglionic eminence (CGE) (B). Note the presence of sst2A receptor immunoreactivity in the cortex (cx) and hippocampus (hi). C represents magnification of boxed area in B and illustrates that the sst2A receptor immunoreactivity is found in cell bodies and short processes in the CGE. D,E) Pre-embedding immunogold immunohistochemistry of the sst2A receptor in the CGE illustrates that high density of immunoparticles are localized intracellularly. However, sst2A receptor-immunoreactive particles are also found in association with the plasma membrane (arrowheads in D). Note that in a neuronal process the majority of the immunoparticles are membrane-associated (arrowheads in E). F represents magnification of the boxed area in A. Numerous cells are immunoreactive for sst2A in the LGE. G represents high magnification of the area labeled with an arrow on A and illustrates that fibers are also sst2A receptor-immunolabeled. H,I) High magnification confocal microscopic analysis in the CGE demonstrates redistribution of receptors upon agonist stimulation. In control conditions, sst2A receptor immunoreactivity outlines the periphery of cells (H). Forty minutes after agonist administration, receptor immunoreactivity is confined to small puncta in the cytoplasm (I). J,K) At E18, intense sst2A receptor immunoreactivity is observed in the dorso-medial part of the caudate-putamen in rostral (J) and caudal (K) sections close to the ventricular surface. Scattered sst2A receptor immunoreactivity is also evident in the medial part of the developing caudate-putamen (asterisk). L represents magnification of boxed area on J. The sst2A receptor immunoreactivity is observed in large number of cells and their short processes in the dorsal caudate-putamen. Note the lack of sst2A receptor immunoreactivity in the subventricular zone (SVZ). M,N are high magnifications from the area labeled with asterisk on J. The sst2A receptor is expressed in neuronal perikarya and processes in the medial part of the caudate-putamen. Scale bars: A, B, 200 µm; C, F, G, L, 20 µm; D, 500 nm; E, 250 nm; H, I, M, N, 10 µm; J, K, 500 µm.
    Figure Legend Snippet: Regional, cellular and subcellular distribution of sst2A receptor immunoreactivity on coronal sections of the rat telencephalon at E16 and E18. A,B) Intense sst2A receptor immunoreactivity is detected at E16 in the post-mitotic areas of the lateral ganglionic eminence (LGE) and the caudal ganglionic eminence (CGE) (B). Note the presence of sst2A receptor immunoreactivity in the cortex (cx) and hippocampus (hi). C represents magnification of boxed area in B and illustrates that the sst2A receptor immunoreactivity is found in cell bodies and short processes in the CGE. D,E) Pre-embedding immunogold immunohistochemistry of the sst2A receptor in the CGE illustrates that high density of immunoparticles are localized intracellularly. However, sst2A receptor-immunoreactive particles are also found in association with the plasma membrane (arrowheads in D). Note that in a neuronal process the majority of the immunoparticles are membrane-associated (arrowheads in E). F represents magnification of the boxed area in A. Numerous cells are immunoreactive for sst2A in the LGE. G represents high magnification of the area labeled with an arrow on A and illustrates that fibers are also sst2A receptor-immunolabeled. H,I) High magnification confocal microscopic analysis in the CGE demonstrates redistribution of receptors upon agonist stimulation. In control conditions, sst2A receptor immunoreactivity outlines the periphery of cells (H). Forty minutes after agonist administration, receptor immunoreactivity is confined to small puncta in the cytoplasm (I). J,K) At E18, intense sst2A receptor immunoreactivity is observed in the dorso-medial part of the caudate-putamen in rostral (J) and caudal (K) sections close to the ventricular surface. Scattered sst2A receptor immunoreactivity is also evident in the medial part of the developing caudate-putamen (asterisk). L represents magnification of boxed area on J. The sst2A receptor immunoreactivity is observed in large number of cells and their short processes in the dorsal caudate-putamen. Note the lack of sst2A receptor immunoreactivity in the subventricular zone (SVZ). M,N are high magnifications from the area labeled with asterisk on J. The sst2A receptor is expressed in neuronal perikarya and processes in the medial part of the caudate-putamen. Scale bars: A, B, 200 µm; C, F, G, L, 20 µm; D, 500 nm; E, 250 nm; H, I, M, N, 10 µm; J, K, 500 µm.

    Techniques Used: Hi-C, Immunohistochemistry, Labeling, Immunolabeling

    Regional and cellular distribution of the sst2A receptor immunofluorescence in the human cerebral cortex at GW 19. A–A″) Intensely labeled sst2A receptor-immunoreactive neurons (red in A′–A″) form chain-like clusters in the middle part of the cortical plate (CP). Note that long sst2A receptor-immunoreactive radial processes reach the pial surface (PS). B–B″) Receptor-immunolabeled cells and processes (red in B, B″) are closely apposed by vimentin-positive processes (green in B′B″), suggesting migration of sst2A-labeled cells on radial glia. C–C″) Sst2A receptor-immunoreactive processes (red in C, C″) are contacted by fibers that are immunoreactive for SRIF (green in D, D″) (arrowheads), the endogen ligand of the receptor. Scale bars: 20 µm.
    Figure Legend Snippet: Regional and cellular distribution of the sst2A receptor immunofluorescence in the human cerebral cortex at GW 19. A–A″) Intensely labeled sst2A receptor-immunoreactive neurons (red in A′–A″) form chain-like clusters in the middle part of the cortical plate (CP). Note that long sst2A receptor-immunoreactive radial processes reach the pial surface (PS). B–B″) Receptor-immunolabeled cells and processes (red in B, B″) are closely apposed by vimentin-positive processes (green in B′B″), suggesting migration of sst2A-labeled cells on radial glia. C–C″) Sst2A receptor-immunoreactive processes (red in C, C″) are contacted by fibers that are immunoreactive for SRIF (green in D, D″) (arrowheads), the endogen ligand of the receptor. Scale bars: 20 µm.

    Techniques Used: Immunofluorescence, Labeling, Immunolabeling, Migration

    Immunofluorescence of the sst2A receptor in coronal sections through the rat neocortical wall between E14 and P5. A–A″′) At E14, the sst2A receptor immunoreactivity is detected in the preplate (PP). Receptor immunoreactivity is observed in cell bodies and basal processes perpendicular to the pial surface (A″′). B–B″′) At E16, intense receptor immunoreactivity is confined to neuronal cells located in the subplate/intermediate zone (SP/IZ; defined by arrowheads). Immunolabeling is located in cell bodies and small processes of closely packed and presumably migrating neurons (B″′). C–C″′) At E18, the sst2A receptor immunoreactivity is confined to cells in the intermediate zone but absent from the adjacent subplate (defined by arrowheads). D–D″′) At E21, sst2A receptor immunoreactivity is concentrated in the subventricular zone (SVZ) and the adjacent deep part of IZ. Immunoreactivity is apparent in cell bodies and radially oriented processes (D, D″′). E) At P5, the sst2A receptor immunoreactivity is diffusely distributed over the neuropil. The labeling intensity decreases towards the deep layers. At high magnification, receptor immunoreactivity appears diffusely distributed within the neuropil (E″′). A″′, B″′, C″′, D″′ and E″′ represent magnifications of boxed areas on A″, B″, C″, D″ and E″, respectively. CP/MZ, cortical plate/marginal zone; CP, cortical plate; MZ, marginal zone; VZ, ventricular zone; I–VI, cortical layers I to VI; WM, white matter. Scale bars: A–A″, B–B″, 50 µm; C–C″, D–D″, E–E″, 100 µm; A″′–E″′,10 µm.
    Figure Legend Snippet: Immunofluorescence of the sst2A receptor in coronal sections through the rat neocortical wall between E14 and P5. A–A″′) At E14, the sst2A receptor immunoreactivity is detected in the preplate (PP). Receptor immunoreactivity is observed in cell bodies and basal processes perpendicular to the pial surface (A″′). B–B″′) At E16, intense receptor immunoreactivity is confined to neuronal cells located in the subplate/intermediate zone (SP/IZ; defined by arrowheads). Immunolabeling is located in cell bodies and small processes of closely packed and presumably migrating neurons (B″′). C–C″′) At E18, the sst2A receptor immunoreactivity is confined to cells in the intermediate zone but absent from the adjacent subplate (defined by arrowheads). D–D″′) At E21, sst2A receptor immunoreactivity is concentrated in the subventricular zone (SVZ) and the adjacent deep part of IZ. Immunoreactivity is apparent in cell bodies and radially oriented processes (D, D″′). E) At P5, the sst2A receptor immunoreactivity is diffusely distributed over the neuropil. The labeling intensity decreases towards the deep layers. At high magnification, receptor immunoreactivity appears diffusely distributed within the neuropil (E″′). A″′, B″′, C″′, D″′ and E″′ represent magnifications of boxed areas on A″, B″, C″, D″ and E″, respectively. CP/MZ, cortical plate/marginal zone; CP, cortical plate; MZ, marginal zone; VZ, ventricular zone; I–VI, cortical layers I to VI; WM, white matter. Scale bars: A–A″, B–B″, 50 µm; C–C″, D–D″, E–E″, 100 µm; A″′–E″′,10 µm.

    Techniques Used: Immunofluorescence, Immunolabeling, Labeling

    Effect of sst2A receptor activation on in vitro granule cell migration. A) Representative image of an external granular layer (EGL) microexplant after 3 days in vitro in culture. The core of the explant and surrounding scattered migrating granules cells are labeled with DAPI (blue). Neuronal processes are labeled by neuronal class III β-tubulin immunoreactivity (green). B) In individual granule cells, sst2A receptor immunoreactivity is visible in both neuronal perikarya and processes (red). C) Illustration of sst2A receptor immunolabeling (red) in a β-tubulin-immunoreactive (green) axon. Note the sst2A-immunoreactive puncta in a growth cone structure (arrow). D, E) In comparison to control (D) the number of migrating granule cells is significantly increased in 100 nm octreotide-treated EGL (E) microexplants. The octreotide-induced granule cell migration increase is dose-dependent as revealed by quantitative analysis (F). *p
    Figure Legend Snippet: Effect of sst2A receptor activation on in vitro granule cell migration. A) Representative image of an external granular layer (EGL) microexplant after 3 days in vitro in culture. The core of the explant and surrounding scattered migrating granules cells are labeled with DAPI (blue). Neuronal processes are labeled by neuronal class III β-tubulin immunoreactivity (green). B) In individual granule cells, sst2A receptor immunoreactivity is visible in both neuronal perikarya and processes (red). C) Illustration of sst2A receptor immunolabeling (red) in a β-tubulin-immunoreactive (green) axon. Note the sst2A-immunoreactive puncta in a growth cone structure (arrow). D, E) In comparison to control (D) the number of migrating granule cells is significantly increased in 100 nm octreotide-treated EGL (E) microexplants. The octreotide-induced granule cell migration increase is dose-dependent as revealed by quantitative analysis (F). *p

    Techniques Used: Activation Assay, In Vitro, Migration, Labeling, Immunolabeling

    Subcellular localization of sst2A receptor immunoreactivity in neocortical cells at E16. A–C) Pre-embedding immunogold immunohistochemistry of the sst2A receptor in the developing cortex at E16 demonstrates localization of immunoparticles at the internal surface of the plasma membrane (arrowheads). D,E) High magnification confocal microscopic analysis reveals agonist-induced redistribution of surface receptors to intracellular compartments. In control conditions, sst2A receptor immunoreactivity outlines the periphery of cells (D). Forty minutes after agonist administration, accumulation of immunoreactive puncta in the cytoplasm become evident (E). Scale bars, A, C, 500 nm; B, 1 µm; D, E, 10 µm.
    Figure Legend Snippet: Subcellular localization of sst2A receptor immunoreactivity in neocortical cells at E16. A–C) Pre-embedding immunogold immunohistochemistry of the sst2A receptor in the developing cortex at E16 demonstrates localization of immunoparticles at the internal surface of the plasma membrane (arrowheads). D,E) High magnification confocal microscopic analysis reveals agonist-induced redistribution of surface receptors to intracellular compartments. In control conditions, sst2A receptor immunoreactivity outlines the periphery of cells (D). Forty minutes after agonist administration, accumulation of immunoreactive puncta in the cytoplasm become evident (E). Scale bars, A, C, 500 nm; B, 1 µm; D, E, 10 µm.

    Techniques Used: Immunohistochemistry

    Regional, cellular and subcellular localization of sst2A receptor immunoreactivity on sagittal (A–H) and coronal (I–K) sections of the rat mesencephalon and diencephalon between E14 and E18. A) At E16, intense sst2A receptor immunoreactivity is observed in the substantia nigra (SN; boxed area) and along the medial forebrain bundle (mfb; arrowhead). B–B″) At E16, the sst2A receptor (red in B, B″) and tyrosine hydroxylase (TH) (green in B′, B″) immunoreactivities extensively overlap both in the SN and in emerging processes of the mfb. C–C″) At E18, sst2A receptor immunoreactivity is dramatically decreased in both the SN and the mfb. D–D″) High magnification microscopic images illustrate numerous sst2A receptor-immunoreactive fibers (red in D,D′) in the mfb at E16. Some of them are TH-positive (green in D′,D″) (arrowheads). E–E″) Some sst2A receptor-immunolabeled axons (red in E, E″) of the mfb express 5-HT (green in E′, E″) (arrowheads). F,G) Pre-embedding immunogold immunohistochemistry of the sst2A receptor in the mfb at E16 illustrates very high density of immunoparticles in axons (F) and growth cone-like structures (G). Note that although the majority of immunoparticles are intracellular, some are found associated to the plasma membrane. H) At E14, intense sst2A receptor immunolabeling is observed on sagittal sections in the developing hypothalamus (boxed area). I) Illustration of receptor immunoreactivity on coronal section at the level of hypothalamic area at E16. Note the receptor immunoreactivity in the caudal ganglionic eminence (CGE) (arrow). J,K) J represents magnification of boxed area in I. At high magnification, sst2A receptor immunoreactivity is found at the periphery of numerous hypothalamic neurons. III, third ventricle; HA, hypothalamic area. Scale bars: A, 500 µm; B–B″, C–C″, 50 µm; D–D″, E–E″, J, 20 µm; F, G, 1 µm; H, 250 µm; I, 200 µm; K, 10 µm.
    Figure Legend Snippet: Regional, cellular and subcellular localization of sst2A receptor immunoreactivity on sagittal (A–H) and coronal (I–K) sections of the rat mesencephalon and diencephalon between E14 and E18. A) At E16, intense sst2A receptor immunoreactivity is observed in the substantia nigra (SN; boxed area) and along the medial forebrain bundle (mfb; arrowhead). B–B″) At E16, the sst2A receptor (red in B, B″) and tyrosine hydroxylase (TH) (green in B′, B″) immunoreactivities extensively overlap both in the SN and in emerging processes of the mfb. C–C″) At E18, sst2A receptor immunoreactivity is dramatically decreased in both the SN and the mfb. D–D″) High magnification microscopic images illustrate numerous sst2A receptor-immunoreactive fibers (red in D,D′) in the mfb at E16. Some of them are TH-positive (green in D′,D″) (arrowheads). E–E″) Some sst2A receptor-immunolabeled axons (red in E, E″) of the mfb express 5-HT (green in E′, E″) (arrowheads). F,G) Pre-embedding immunogold immunohistochemistry of the sst2A receptor in the mfb at E16 illustrates very high density of immunoparticles in axons (F) and growth cone-like structures (G). Note that although the majority of immunoparticles are intracellular, some are found associated to the plasma membrane. H) At E14, intense sst2A receptor immunolabeling is observed on sagittal sections in the developing hypothalamus (boxed area). I) Illustration of receptor immunoreactivity on coronal section at the level of hypothalamic area at E16. Note the receptor immunoreactivity in the caudal ganglionic eminence (CGE) (arrow). J,K) J represents magnification of boxed area in I. At high magnification, sst2A receptor immunoreactivity is found at the periphery of numerous hypothalamic neurons. III, third ventricle; HA, hypothalamic area. Scale bars: A, 500 µm; B–B″, C–C″, 50 µm; D–D″, E–E″, J, 20 µm; F, G, 1 µm; H, 250 µm; I, 200 µm; K, 10 µm.

    Techniques Used: Immunolabeling, Immunohistochemistry

    Immunofluorescence of sst2A receptor in the rat perinatal rostral migratory stream. A–A″) In sagittal sections at P0, an intense band of sst2A receptor immunoreactivity is observed from the anterior subventricular zone (SVZa), through the rostral migratory stream (RMS) and ending in the olfactory bulb (OB). From the SVZa, shown in detail in the high magnification insets, chains of immunoreactive neurons perpendicular to the SVZa long axis extend into the white matter of the overlying cerebral cortex. B–B″ represents high magnification of the area labeled with asterisk in A. The sst2A receptor immunoreactive cells are principally localized along the ventral and dorsal surface of RMS. C) High magnification of sst2A receptor immunoreactivity at the entrance of RMS into the olfactory bulb at P5 illustrates immunoreactive cells at the surface of the stream as well as embedded in central position. D) In the dorsal part of the RMS, sst2A receptor-immunoreactive cells (red) contain NeuN labeling in their nuclei (green; arrowhead). E) Sst2A receptor- (red) and NeuN- (blue) double-labeled cells (arrowhead) of the RMS do not contain BrdU immunoreactivity (green), demonstrating that receptor expression is restricted to post-mitotic neurons. Scale bars: A–A″, 500 µm; B–B″, C, 100 µm; D, 20 µm; E, 10 µm.
    Figure Legend Snippet: Immunofluorescence of sst2A receptor in the rat perinatal rostral migratory stream. A–A″) In sagittal sections at P0, an intense band of sst2A receptor immunoreactivity is observed from the anterior subventricular zone (SVZa), through the rostral migratory stream (RMS) and ending in the olfactory bulb (OB). From the SVZa, shown in detail in the high magnification insets, chains of immunoreactive neurons perpendicular to the SVZa long axis extend into the white matter of the overlying cerebral cortex. B–B″ represents high magnification of the area labeled with asterisk in A. The sst2A receptor immunoreactive cells are principally localized along the ventral and dorsal surface of RMS. C) High magnification of sst2A receptor immunoreactivity at the entrance of RMS into the olfactory bulb at P5 illustrates immunoreactive cells at the surface of the stream as well as embedded in central position. D) In the dorsal part of the RMS, sst2A receptor-immunoreactive cells (red) contain NeuN labeling in their nuclei (green; arrowhead). E) Sst2A receptor- (red) and NeuN- (blue) double-labeled cells (arrowhead) of the RMS do not contain BrdU immunoreactivity (green), demonstrating that receptor expression is restricted to post-mitotic neurons. Scale bars: A–A″, 500 µm; B–B″, C, 100 µm; D, 20 µm; E, 10 µm.

    Techniques Used: Immunofluorescence, Labeling, Expressing

    Regional and cellular distribution of sst2A receptor immunoreactivity in the rat developing locus coeruleus. (A) At E18, strong sst2A receptor immunoreactivity is found not only in the rhombic lip (rl) and external granular layer (EGL) but also in the rostro-ventral part of the cerebellum between the cerebellar ventricular area (IV) and the ventral hindbrain (boxed area). B–B″) The large, elongated sst2A receptor-immunoreactive cells (red in B, B″) lie parallel with the ventricular surface. These neurons also express tyrosine hydroxylase (TH; green in B′, B″), a marker of catecholaminergic neurons. C) At E21, intense sst2A receptor immunoreactivity (red in C, C″) is observed in the developing locus coeruleus (LC) and overlap with TH immunolabeling (green in D′,D″). D represents high magnification of boxed area in C. Note that intense sst2A receptor immunoreactivity (red) outlines the periphery of TH-positive (green) neurons (arrowheads). E) At P3, the locus coeruleus exhibits also strong sst2A receptor immunoreactivity (red). The blue labeling represents DAPI staining. CB, cerebellum; IV, fourth ventricle. Scale bars: A, 200 µm; B–B″, C–C″, E, 100 µm; D–D″, 20 µm.
    Figure Legend Snippet: Regional and cellular distribution of sst2A receptor immunoreactivity in the rat developing locus coeruleus. (A) At E18, strong sst2A receptor immunoreactivity is found not only in the rhombic lip (rl) and external granular layer (EGL) but also in the rostro-ventral part of the cerebellum between the cerebellar ventricular area (IV) and the ventral hindbrain (boxed area). B–B″) The large, elongated sst2A receptor-immunoreactive cells (red in B, B″) lie parallel with the ventricular surface. These neurons also express tyrosine hydroxylase (TH; green in B′, B″), a marker of catecholaminergic neurons. C) At E21, intense sst2A receptor immunoreactivity (red in C, C″) is observed in the developing locus coeruleus (LC) and overlap with TH immunolabeling (green in D′,D″). D represents high magnification of boxed area in C. Note that intense sst2A receptor immunoreactivity (red) outlines the periphery of TH-positive (green) neurons (arrowheads). E) At P3, the locus coeruleus exhibits also strong sst2A receptor immunoreactivity (red). The blue labeling represents DAPI staining. CB, cerebellum; IV, fourth ventricle. Scale bars: A, 200 µm; B–B″, C–C″, E, 100 µm; D–D″, 20 µm.

    Techniques Used: Marker, Immunolabeling, Labeling, Staining

    Regional and cellular distribution of sst2A receptor immunoreactivity on coronal sections of the prenatal human cerebellum. A) At GW 19, intense receptor immunoreactivity (red in A, A″) is observed in the deep part of the external granular layer (EGL). Note the large number of DAPI-positive cell nuclei (blue in A′, A″) in the superficial EGL. B–B″′ represent high magnification of boxed area in A. The sst2A receptor immunoreactivity (red in B, B″′) is mainly distributed in the deep part of the EGL whereas SRIF-immunoreactive cells (green in B′, B″′) are mainly located in the deep part of the molecular layer (ML). C–C″′) At GW 20, the high density sst2A receptor immunoreactivity (red in C, C″′) in the deep EGL is still present. In addition, intense receptor immunolabeling is detected in the internal granular layer (IGL; asterisk) and overlap with NeuN-immunoreactive cells (green in C′–C″′). D represents magnification of boxed area in C. The sst2A receptor is expressed in cells bodies located in the deep part of the EGL. E–E″′) In the IGL, the vast majority of NeuN- (green in E′, E″′) and DAPI- (blue in E″, E″′) positive cell nuclei are outlined by sst2A receptor immunoreactivity (red in E, E″′) (arrowheads), suggesting that the receptor is expressed by migrating granule cells. Scale bars: A–A″, C–C″′, 100 µm; B–B″′, D, E–E″′, 20 µm.
    Figure Legend Snippet: Regional and cellular distribution of sst2A receptor immunoreactivity on coronal sections of the prenatal human cerebellum. A) At GW 19, intense receptor immunoreactivity (red in A, A″) is observed in the deep part of the external granular layer (EGL). Note the large number of DAPI-positive cell nuclei (blue in A′, A″) in the superficial EGL. B–B″′ represent high magnification of boxed area in A. The sst2A receptor immunoreactivity (red in B, B″′) is mainly distributed in the deep part of the EGL whereas SRIF-immunoreactive cells (green in B′, B″′) are mainly located in the deep part of the molecular layer (ML). C–C″′) At GW 20, the high density sst2A receptor immunoreactivity (red in C, C″′) in the deep EGL is still present. In addition, intense receptor immunolabeling is detected in the internal granular layer (IGL; asterisk) and overlap with NeuN-immunoreactive cells (green in C′–C″′). D represents magnification of boxed area in C. The sst2A receptor is expressed in cells bodies located in the deep part of the EGL. E–E″′) In the IGL, the vast majority of NeuN- (green in E′, E″′) and DAPI- (blue in E″, E″′) positive cell nuclei are outlined by sst2A receptor immunoreactivity (red in E, E″′) (arrowheads), suggesting that the receptor is expressed by migrating granule cells. Scale bars: A–A″, C–C″′, 100 µm; B–B″′, D, E–E″′, 20 µm.

    Techniques Used: Immunolabeling

    Regional, cellular and subcellular distribution of sst2A receptor immunoreactivity in sagittal sections of the rat cerebellum during pre- and postnatal development. A) At E14, sst2A receptor immunoreactivity is detected in the developing cerebellum (boxed area). Note the strong expression of the receptor in the developing hypothalamus (arrowhead) and rhombencephalon (asterisk). B) The sst2A receptor immunoreactivity is intense at the outer border of the cerebellar neuroepithelium (asterisk) and the adjacent upper component of the rhombic lip (rl). C) At E16, strong cellular sst2A receptor labeling is evident in the dorsal part of the cerebellum, where the progenitors of the external granular layer (EGL) migrate. D,E) Pre-embedding immunogold immunohistochemistry of the sst2A receptor in the developing external germinal layer at E16 illustrates that immunoparticles are predominantly localized at the internal surface of the plasma membrane (arrowheads). F,G) High magnification confocal microscopic analysis of the developing EGL reveals redistribution of surface receptors to intracellular compartments upon agonist stimulation. In control conditions, sst2A receptor immunoreactivity outlines the periphery of neurons (F). Forty minutes after agonist administration, accumulation of immunoreactive puncta in the cytoplasm become evident (G). H) At P5, intense sst2A receptor immunofluorescence is observed in the EGL. I–I″ represent magnification of boxed area in H. The sst2A receptor-immunoreactive neurons (red) are predominantly located in the deep part of EGL (I). The Ki-67-immuonreactive proliferative neurons (green) are distributed predominantly in the superficial EGL (I′). Accordingly only few sst2A receptor-immunoreactive neurons are Ki-67-positive (I″; arrowheads). J–J″) In the EGL, most sst2A receptor-immunolabeled neurons (red in J, J″) are positive for the neuronal-specific nuclear protein NeuN (green in J′, J″) (arrowheads) and demonstrate the post-mitotic nature of sst2A-positive EGL neurons. K, K″) At P5, the large unipolar calretinin-immunoreactive brush cells (green in K′, K″) are sst2A receptor immunoreactive (red in K, K″). Note the colocalization of sst2A receptor and calretinin in a long brush cell process (arrowhead). cb, cerebellar neuroepithelium; CB, cerebellum. Scale bars: A, 250 µm; B, H, K–K″, 50 µm; C, 20 µm, D, 200 nm; E, 400 nm. F, G, I–I″, J–J″, 10 µm.
    Figure Legend Snippet: Regional, cellular and subcellular distribution of sst2A receptor immunoreactivity in sagittal sections of the rat cerebellum during pre- and postnatal development. A) At E14, sst2A receptor immunoreactivity is detected in the developing cerebellum (boxed area). Note the strong expression of the receptor in the developing hypothalamus (arrowhead) and rhombencephalon (asterisk). B) The sst2A receptor immunoreactivity is intense at the outer border of the cerebellar neuroepithelium (asterisk) and the adjacent upper component of the rhombic lip (rl). C) At E16, strong cellular sst2A receptor labeling is evident in the dorsal part of the cerebellum, where the progenitors of the external granular layer (EGL) migrate. D,E) Pre-embedding immunogold immunohistochemistry of the sst2A receptor in the developing external germinal layer at E16 illustrates that immunoparticles are predominantly localized at the internal surface of the plasma membrane (arrowheads). F,G) High magnification confocal microscopic analysis of the developing EGL reveals redistribution of surface receptors to intracellular compartments upon agonist stimulation. In control conditions, sst2A receptor immunoreactivity outlines the periphery of neurons (F). Forty minutes after agonist administration, accumulation of immunoreactive puncta in the cytoplasm become evident (G). H) At P5, intense sst2A receptor immunofluorescence is observed in the EGL. I–I″ represent magnification of boxed area in H. The sst2A receptor-immunoreactive neurons (red) are predominantly located in the deep part of EGL (I). The Ki-67-immuonreactive proliferative neurons (green) are distributed predominantly in the superficial EGL (I′). Accordingly only few sst2A receptor-immunoreactive neurons are Ki-67-positive (I″; arrowheads). J–J″) In the EGL, most sst2A receptor-immunolabeled neurons (red in J, J″) are positive for the neuronal-specific nuclear protein NeuN (green in J′, J″) (arrowheads) and demonstrate the post-mitotic nature of sst2A-positive EGL neurons. K, K″) At P5, the large unipolar calretinin-immunoreactive brush cells (green in K′, K″) are sst2A receptor immunoreactive (red in K, K″). Note the colocalization of sst2A receptor and calretinin in a long brush cell process (arrowhead). cb, cerebellar neuroepithelium; CB, cerebellum. Scale bars: A, 250 µm; B, H, K–K″, 50 µm; C, 20 µm, D, 200 nm; E, 400 nm. F, G, I–I″, J–J″, 10 µm.

    Techniques Used: Expressing, Labeling, Immunohistochemistry, Immunofluorescence, Immunolabeling

    Effect of sst2A receptor agonist on axonal and dendritic patterning. A–A″) Representative image of sst2A receptor localization in a primary hippocampal cell after 24 h in vitro in culture. Receptor immunoreactivity (green in A, A″) is present in the cell body and processes. Cell morphology is revealed by actin-binding protein phalloidin (red in A′, A″). Note that sst2A receptor immunoreactivity is also present in growth cones (insets in A–A″). B–D) Representative images of neurons from control (ctrl; B), 10 nM octreotide-treated (10 nM oct.; C) and 50 nM octreotide-treated (50 nM oct.; D) cultures. Arrows depict the axonal process which appears longer when cells are treated with 50 nM octreotide. E) Quantitative analysis reveals that the axon length (right panel) is significantly increased in the 50 nM oct. group when compared to the control group. The mean cell body surface (left panel) and the mean dendritic length (middle panel) are not modified by sst2A receptor agonist treatments. Values (mean±SEM) are expressed in relation to an arbitrary unit (100%) of the control values. *p
    Figure Legend Snippet: Effect of sst2A receptor agonist on axonal and dendritic patterning. A–A″) Representative image of sst2A receptor localization in a primary hippocampal cell after 24 h in vitro in culture. Receptor immunoreactivity (green in A, A″) is present in the cell body and processes. Cell morphology is revealed by actin-binding protein phalloidin (red in A′, A″). Note that sst2A receptor immunoreactivity is also present in growth cones (insets in A–A″). B–D) Representative images of neurons from control (ctrl; B), 10 nM octreotide-treated (10 nM oct.; C) and 50 nM octreotide-treated (50 nM oct.; D) cultures. Arrows depict the axonal process which appears longer when cells are treated with 50 nM octreotide. E) Quantitative analysis reveals that the axon length (right panel) is significantly increased in the 50 nM oct. group when compared to the control group. The mean cell body surface (left panel) and the mean dendritic length (middle panel) are not modified by sst2A receptor agonist treatments. Values (mean±SEM) are expressed in relation to an arbitrary unit (100%) of the control values. *p

    Techniques Used: In Vitro, Binding Assay, Modification

    29) Product Images from "Bacteria-Induced Uroplakin Signaling Mediates Bladder Response to Infection"

    Article Title: Bacteria-Induced Uroplakin Signaling Mediates Bladder Response to Infection

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1000415

    Co-localization of uroplakins and FimH binding sites on PD07i cell surface. The surface-expressed uroplakins on PD07i cells were detected using antisera against individual uroplakins Ia (B), Ib (E), II (H), and IIIa (K), followed by Alexa Fluor 594-conjugated donkey anti-rabbit IgG; while FimH was localized using biotinylated FimH/C complex, followed with FITC-conjugated streptavidin (A, D, G, J). Arrows mark the co-localization of surface-expressed uroplakins and FimH binding sties (A–L).
    Figure Legend Snippet: Co-localization of uroplakins and FimH binding sites on PD07i cell surface. The surface-expressed uroplakins on PD07i cells were detected using antisera against individual uroplakins Ia (B), Ib (E), II (H), and IIIa (K), followed by Alexa Fluor 594-conjugated donkey anti-rabbit IgG; while FimH was localized using biotinylated FimH/C complex, followed with FITC-conjugated streptavidin (A, D, G, J). Arrows mark the co-localization of surface-expressed uroplakins and FimH binding sties (A–L).

    Techniques Used: Binding Assay, IA

    30) Product Images from "C1q Governs Deposition of Circulating Immune Complexes and Leukocyte Fc? Receptors Mediate Subsequent Neutrophil Recruitment"

    Article Title: C1q Governs Deposition of Circulating Immune Complexes and Leukocyte Fc? Receptors Mediate Subsequent Neutrophil Recruitment

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20040501

    ICs deposit in cremaster muscle venules after induction of vascular permeability and are accessible to circulating leukocytes. (A–F) Deposits of colloidal carbon and ICs (BSA/anti-BSA) were visualized using phase contrast and fluorescent microscopy, respectively, in cremaster whole mounts of mice given prelabeled (Cy-3 anti–rabbit IgG) ICs followed by colloidal carbon i.v. ( n = 3 per experiment). (A and B) Mice were killed after colloidal carbon and IC injection and their cremasters were harvested. No deposits of colloidal carbon (A) or ICs (B) were seen (bar, 100 μm). (C and D) The cremaster was exteriorized after colloidal carbon and IC injection, and then harvested. Granular deposits of colloidal carbon (C) and ICs (D) delineated venules (bar, 50 μm). (E and F) Histamine or VEGF (not depicted) was given i.p. between colloidal carbon and IC injections. Mice were then killed and their cremasters were harvested. Colloidal carbon (E) and ICs (F) deposited, mimicking that after cremaster exteriorization (bar, 50 μm). (G–I) Mice were given FITC-prelabeled ICs (G and H) or BSA and FITC anti–rabbit IgG (I). Their cremasters were then exteriorized under anesthesia and visualized with IVM. (G) IC deposits occurred in postcapillary venules (arrows), but not arterioles (arrowhead; bar, 35 μm). (H) Granular IC deposits delineated venules, but did not extend into capillaries (arrows; bar, 35 μm). (I) Diffuse vascular staining was seen in the muscles of mice given BSA and FITC secondary Ab (bar, 70 μm). (J) FITC-dextran, given i.v., diffused into the extravascular space around venules (arrows), but not arterioles (arrowhead) of exteriorized cremasters of mice given ICs (H), PBS, or BSA (not depicted), indicating surgical-induced permeability (bar, 240 μm). (K and L) Unlabeled ICs (K) or BSA (L) were injected i.v. after cremaster exteriorization in anesthetized mice. 1 μm secondary Ab-coupled microspheres were then given, followed by Cy3-labeled secondary Ab. More microspheres (yellow) accumulated in venules after IC than BSA injection. Cy3-Ab (red) identified IC deposits only in vessels of IC-injected mice (bar, 50 μm). See Video 1.
    Figure Legend Snippet: ICs deposit in cremaster muscle venules after induction of vascular permeability and are accessible to circulating leukocytes. (A–F) Deposits of colloidal carbon and ICs (BSA/anti-BSA) were visualized using phase contrast and fluorescent microscopy, respectively, in cremaster whole mounts of mice given prelabeled (Cy-3 anti–rabbit IgG) ICs followed by colloidal carbon i.v. ( n = 3 per experiment). (A and B) Mice were killed after colloidal carbon and IC injection and their cremasters were harvested. No deposits of colloidal carbon (A) or ICs (B) were seen (bar, 100 μm). (C and D) The cremaster was exteriorized after colloidal carbon and IC injection, and then harvested. Granular deposits of colloidal carbon (C) and ICs (D) delineated venules (bar, 50 μm). (E and F) Histamine or VEGF (not depicted) was given i.p. between colloidal carbon and IC injections. Mice were then killed and their cremasters were harvested. Colloidal carbon (E) and ICs (F) deposited, mimicking that after cremaster exteriorization (bar, 50 μm). (G–I) Mice were given FITC-prelabeled ICs (G and H) or BSA and FITC anti–rabbit IgG (I). Their cremasters were then exteriorized under anesthesia and visualized with IVM. (G) IC deposits occurred in postcapillary venules (arrows), but not arterioles (arrowhead; bar, 35 μm). (H) Granular IC deposits delineated venules, but did not extend into capillaries (arrows; bar, 35 μm). (I) Diffuse vascular staining was seen in the muscles of mice given BSA and FITC secondary Ab (bar, 70 μm). (J) FITC-dextran, given i.v., diffused into the extravascular space around venules (arrows), but not arterioles (arrowhead) of exteriorized cremasters of mice given ICs (H), PBS, or BSA (not depicted), indicating surgical-induced permeability (bar, 240 μm). (K and L) Unlabeled ICs (K) or BSA (L) were injected i.v. after cremaster exteriorization in anesthetized mice. 1 μm secondary Ab-coupled microspheres were then given, followed by Cy3-labeled secondary Ab. More microspheres (yellow) accumulated in venules after IC than BSA injection. Cy3-Ab (red) identified IC deposits only in vessels of IC-injected mice (bar, 50 μm). See Video 1.

    Techniques Used: Permeability, Microscopy, Mouse Assay, Injection, Staining, Labeling

    31) Product Images from "Healing Potential of Picrorhiza kurroa (Scrofulariaceae) rhizomes against indomethacin-induced gastric ulceration: a mechanistic exploration."

    Article Title: Healing Potential of Picrorhiza kurroa (Scrofulariaceae) rhizomes against indomethacin-induced gastric ulceration: a mechanistic exploration.

    Journal: BMC Complementary and Alternative Medicine

    doi: 10.1186/1472-6882-8-3

    The changes in the tissue EGF expression due to acute gastric ulceration of mice and its regulation by PK and Omez on the 3 rd day of ulceration. The EGF immunostaining was carried out using the peroxidase conjugate. Original magnification × 400. a – normal mice, b – ulcerated untreated mice, c – ulcerated PK-treated mice, d – ulcerated Omez-treated mice.
    Figure Legend Snippet: The changes in the tissue EGF expression due to acute gastric ulceration of mice and its regulation by PK and Omez on the 3 rd day of ulceration. The EGF immunostaining was carried out using the peroxidase conjugate. Original magnification × 400. a – normal mice, b – ulcerated untreated mice, c – ulcerated PK-treated mice, d – ulcerated Omez-treated mice.

    Techniques Used: Expressing, Mouse Assay, Immunostaining

    Comparative time dependent activity of PK and Omez in regulating the expression of tissue VEGF in acute gastric ulcerated mice. The EGF expression was quantified using Biovis MV500 software. Data are expressed as means ± SEM for fifteen mice. a P
    Figure Legend Snippet: Comparative time dependent activity of PK and Omez in regulating the expression of tissue VEGF in acute gastric ulcerated mice. The EGF expression was quantified using Biovis MV500 software. Data are expressed as means ± SEM for fifteen mice. a P

    Techniques Used: Activity Assay, Expressing, Mouse Assay, Software

    Comparative time dependent activity of PK and Omez in regulating the expression of tissue EGF in acute gastric ulcerated mice. The EGF expression was quantified using Biovis MV500 software. Data are expressed as means ± SEM for fifteen mice. a P
    Figure Legend Snippet: Comparative time dependent activity of PK and Omez in regulating the expression of tissue EGF in acute gastric ulcerated mice. The EGF expression was quantified using Biovis MV500 software. Data are expressed as means ± SEM for fifteen mice. a P

    Techniques Used: Activity Assay, Expressing, Mouse Assay, Software

    32) Product Images from "Neurofilament-dependent Radial Growth of Motor Axons and Axonal Organization of Neurofilaments Does Not Require the Neurofilament Heavy Subunit (NF-H) or Its Phosphorylation "

    Article Title: Neurofilament-dependent Radial Growth of Motor Axons and Axonal Organization of Neurofilaments Does Not Require the Neurofilament Heavy Subunit (NF-H) or Its Phosphorylation

    Journal: The Journal of Cell Biology

    doi:

    Levels of neurofilament subunits NF-L, NF-M, and NF-H in mice with zero, one, or two copies of a disrupted NF-H gene. ( A ) Total tissue extracts from 5-wk-old brain, spinal cord, and sciatic nerves were fractionated on 7% SDS–polyacrylamide gels and stained with ( A ) Coomassie blue or ( B–I ) electroblotted to nitrocellulose. ( B ) NF-H detected with a peptide antibody recognizing the extreme COOH terminus of NF-H ( Xu et al., 1993 ); ( C ) phosphorylated NF-H and NF-M detected with monoclonal antibody SMI-31; ( D ) nonphosphorylated NF-H detected with monoclonal antibody SMI-32; ( E ) NF-M detected with monoclonal antibody RM 044 ( Tu et al., 1995 ); ( F ) NF-L detected with a polyclonal peptide antibody recognizing the COOH terminus of NF-L ( Xu et al., 1993 ); ( G ) α-tubulin detected with monoclonal antibody DM1A; ( H ) the neuron-specific class III, β-tubulin isotype with mAb TuJ1 ( Lee et al., 1990 ); and ( I ) plectin detected with polyclonal antiserum P21 ( Wiche and Baker, 1982 ). (Plectin migrates with a mobility of ∼500 kD in brain and spinal cord but at ∼160 kD in nerve samples using both this antibody and monoclonal antibody 10F6 [ Foisner et al., 1991 ]; not shown.) Lanes 10–14 , quantitation standards for the neurofilament subunits provided by a twofold dilution series of a neurofilament preparation. Molecular masses (kD) are indicated at left. (Lanes 1–3 of D–F represent four times longer exposures than lanes 4–14 .)
    Figure Legend Snippet: Levels of neurofilament subunits NF-L, NF-M, and NF-H in mice with zero, one, or two copies of a disrupted NF-H gene. ( A ) Total tissue extracts from 5-wk-old brain, spinal cord, and sciatic nerves were fractionated on 7% SDS–polyacrylamide gels and stained with ( A ) Coomassie blue or ( B–I ) electroblotted to nitrocellulose. ( B ) NF-H detected with a peptide antibody recognizing the extreme COOH terminus of NF-H ( Xu et al., 1993 ); ( C ) phosphorylated NF-H and NF-M detected with monoclonal antibody SMI-31; ( D ) nonphosphorylated NF-H detected with monoclonal antibody SMI-32; ( E ) NF-M detected with monoclonal antibody RM 044 ( Tu et al., 1995 ); ( F ) NF-L detected with a polyclonal peptide antibody recognizing the COOH terminus of NF-L ( Xu et al., 1993 ); ( G ) α-tubulin detected with monoclonal antibody DM1A; ( H ) the neuron-specific class III, β-tubulin isotype with mAb TuJ1 ( Lee et al., 1990 ); and ( I ) plectin detected with polyclonal antiserum P21 ( Wiche and Baker, 1982 ). (Plectin migrates with a mobility of ∼500 kD in brain and spinal cord but at ∼160 kD in nerve samples using both this antibody and monoclonal antibody 10F6 [ Foisner et al., 1991 ]; not shown.) Lanes 10–14 , quantitation standards for the neurofilament subunits provided by a twofold dilution series of a neurofilament preparation. Molecular masses (kD) are indicated at left. (Lanes 1–3 of D–F represent four times longer exposures than lanes 4–14 .)

    Techniques Used: Mouse Assay, Staining, Quantitation Assay

    Levels of neurofilament subunits NF-L, NF-M, and NF-H in mice with zero, one, or two copies of a disrupted NF-H gene. ( A ) Total tissue extracts from 5-wk-old brain, spinal cord, and sciatic nerves were fractionated on 7% SDS–polyacrylamide gels and stained with ( A ) Coomassie blue or ( B–I ) electroblotted to nitrocellulose. ( B ) NF-H detected with a peptide antibody recognizing the extreme COOH terminus of NF-H ( Xu et al., 1993 ); ( C ) phosphorylated NF-H and NF-M detected with monoclonal antibody SMI-31; ( D ) nonphosphorylated NF-H detected with monoclonal antibody SMI-32; ( E ) NF-M detected with monoclonal antibody RM 044 ( Tu et al., 1995 ); ( F ) NF-L detected with a polyclonal peptide antibody recognizing the COOH terminus of NF-L ( Xu et al., 1993 ); ( G ) α-tubulin detected with monoclonal antibody DM1A; ( H ) the neuron-specific class III, β-tubulin isotype with mAb TuJ1 ( Lee et al., 1990 ); and ( I ) plectin detected with polyclonal antiserum P21 ( Wiche and Baker, 1982 ). (Plectin migrates with a mobility of ∼500 kD in brain and spinal cord but at ∼160 kD in nerve samples using both this antibody and monoclonal antibody 10F6 [ Foisner et al., 1991 ]; not shown.) Lanes 10–14 , quantitation standards for the neurofilament subunits provided by a twofold dilution series of a neurofilament preparation. Molecular masses (kD) are indicated at left. (Lanes 1–3 of D–F represent four times longer exposures than lanes 4–14 .)
    Figure Legend Snippet: Levels of neurofilament subunits NF-L, NF-M, and NF-H in mice with zero, one, or two copies of a disrupted NF-H gene. ( A ) Total tissue extracts from 5-wk-old brain, spinal cord, and sciatic nerves were fractionated on 7% SDS–polyacrylamide gels and stained with ( A ) Coomassie blue or ( B–I ) electroblotted to nitrocellulose. ( B ) NF-H detected with a peptide antibody recognizing the extreme COOH terminus of NF-H ( Xu et al., 1993 ); ( C ) phosphorylated NF-H and NF-M detected with monoclonal antibody SMI-31; ( D ) nonphosphorylated NF-H detected with monoclonal antibody SMI-32; ( E ) NF-M detected with monoclonal antibody RM 044 ( Tu et al., 1995 ); ( F ) NF-L detected with a polyclonal peptide antibody recognizing the COOH terminus of NF-L ( Xu et al., 1993 ); ( G ) α-tubulin detected with monoclonal antibody DM1A; ( H ) the neuron-specific class III, β-tubulin isotype with mAb TuJ1 ( Lee et al., 1990 ); and ( I ) plectin detected with polyclonal antiserum P21 ( Wiche and Baker, 1982 ). (Plectin migrates with a mobility of ∼500 kD in brain and spinal cord but at ∼160 kD in nerve samples using both this antibody and monoclonal antibody 10F6 [ Foisner et al., 1991 ]; not shown.) Lanes 10–14 , quantitation standards for the neurofilament subunits provided by a twofold dilution series of a neurofilament preparation. Molecular masses (kD) are indicated at left. (Lanes 1–3 of D–F represent four times longer exposures than lanes 4–14 .)

    Techniques Used: Mouse Assay, Staining, Quantitation Assay

    Disruption of the mouse NF-H gene by homologous recombination. ( A ) Strategy for disruption of the mouse NF-H gene. A targeting construct for disruption of the NF-H gene was constructed by inserting a 1.7-kb gene encoding resistance to neomycin in place of 1.6 kb NF-H putative promoter and the first 34 codons of the gene. The four NF-H exons are indicated by filled boxes interrupted by three introns. ATG denotes the NF-H translation initiation codon. Unique HindIII ( H3 ) and EcoRV ( RV ) sites were introduced into the disrupted gene allele after homologous recombination. RI , EcoRI; WT , wild type; MT , mutant; PGK , phosphoglycerate kinase promoter; NEO , neomycin phosphotransferase gene; TK , thymidine kinase gene. ( B–D ) Screening of ( B and C ) ES and ( D ) mouse tail DNAs for targeted inactivation of the NF-H gene. ( B ) Genomic DNA blot of ES cell DNA after digestion with HindIII was probed with a segment 3′ to the targeted domain (the highlighted EcoRV-AatII fragment in A ). The normal NF-H allele produces an 18-kb fragment; the targeted allele produces a 10-kb fragment. ( C ) Genomic DNA blot of ES cell DNA after digestion with EcoRV was probed with a 5′ probe (the EcoRI-NdeI fragment denoted in A ). The normal allele produces a 15-kb fragment; the targeted allele produces a 7-kb fragment. ( B and C ) Lane 1 , wild-type ES cell DNA; lanes 2 and 3 , DNA from two targeted ES cells. ( D ) EcoRV-digested mouse tail DNA probed with the 5′ probe. DNAs are from a mouse with (lane 1 ) two wild-type alleles or (lane 2 ) heterozygous or (lane 3 ) homozygous for disruption of the NF-H gene. ( E ) NF-L, NF-M, NF-H, and βIII-tubulin mRNA levels in mice with zero, one, or two copies of a disrupted NF-H gene. 20 μg of total RNA isolated from 5-wk-old brains and spinal cords of control mice and mice heterozygous or homozygous for disruption of the NF-H gene were fractionated on 1% formaldehyde agarose gels, blotted on to nylon membranes, and probed with radiolabeled cDNA sequences for each subunit (see Materials and Methods). Lanes 1 , 3 , and 5 , brain RNAs from wild-type, heterozygous, and homozygous mice. Lanes 2 , 4 , and 6 , spinal cord RNAs from wild-type, heterozygous, and homozygous mice.
    Figure Legend Snippet: Disruption of the mouse NF-H gene by homologous recombination. ( A ) Strategy for disruption of the mouse NF-H gene. A targeting construct for disruption of the NF-H gene was constructed by inserting a 1.7-kb gene encoding resistance to neomycin in place of 1.6 kb NF-H putative promoter and the first 34 codons of the gene. The four NF-H exons are indicated by filled boxes interrupted by three introns. ATG denotes the NF-H translation initiation codon. Unique HindIII ( H3 ) and EcoRV ( RV ) sites were introduced into the disrupted gene allele after homologous recombination. RI , EcoRI; WT , wild type; MT , mutant; PGK , phosphoglycerate kinase promoter; NEO , neomycin phosphotransferase gene; TK , thymidine kinase gene. ( B–D ) Screening of ( B and C ) ES and ( D ) mouse tail DNAs for targeted inactivation of the NF-H gene. ( B ) Genomic DNA blot of ES cell DNA after digestion with HindIII was probed with a segment 3′ to the targeted domain (the highlighted EcoRV-AatII fragment in A ). The normal NF-H allele produces an 18-kb fragment; the targeted allele produces a 10-kb fragment. ( C ) Genomic DNA blot of ES cell DNA after digestion with EcoRV was probed with a 5′ probe (the EcoRI-NdeI fragment denoted in A ). The normal allele produces a 15-kb fragment; the targeted allele produces a 7-kb fragment. ( B and C ) Lane 1 , wild-type ES cell DNA; lanes 2 and 3 , DNA from two targeted ES cells. ( D ) EcoRV-digested mouse tail DNA probed with the 5′ probe. DNAs are from a mouse with (lane 1 ) two wild-type alleles or (lane 2 ) heterozygous or (lane 3 ) homozygous for disruption of the NF-H gene. ( E ) NF-L, NF-M, NF-H, and βIII-tubulin mRNA levels in mice with zero, one, or two copies of a disrupted NF-H gene. 20 μg of total RNA isolated from 5-wk-old brains and spinal cords of control mice and mice heterozygous or homozygous for disruption of the NF-H gene were fractionated on 1% formaldehyde agarose gels, blotted on to nylon membranes, and probed with radiolabeled cDNA sequences for each subunit (see Materials and Methods). Lanes 1 , 3 , and 5 , brain RNAs from wild-type, heterozygous, and homozygous mice. Lanes 2 , 4 , and 6 , spinal cord RNAs from wild-type, heterozygous, and homozygous mice.

    Techniques Used: Homologous Recombination, Construct, Mutagenesis, Mouse Assay, Isolation

    33) Product Images from "Albumin fibrillization induces apoptosis via integrin/FAK/Akt pathway"

    Article Title: Albumin fibrillization induces apoptosis via integrin/FAK/Akt pathway

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-9-2

    Interaction between fibrillar BSA and integrin α5β1 . (A) T47D cell lines were pre-treated with or without 0.67 μM goat IgG or 0.67 μM goat anti-integrin α5β1 antibody for 30 min as indicated, then incubated with 2 μM F-BSA (BSA-S200) in serum-free medium for 8 h. Cell viability was determined by the MTT assay. Data are means ± S.D. (n = 3). (B) Integrin α5β1 protein was linked to protein A/G beads by use of anti-integrin α5β1 antibody, then incubated with F-BSA (BSA-S200) or G-BSA (BSA) overnight. The immunocomplexes were separated by SDS-PAGE and immunoblotted with anti-integrin α5 and anti-BSA antibodies.
    Figure Legend Snippet: Interaction between fibrillar BSA and integrin α5β1 . (A) T47D cell lines were pre-treated with or without 0.67 μM goat IgG or 0.67 μM goat anti-integrin α5β1 antibody for 30 min as indicated, then incubated with 2 μM F-BSA (BSA-S200) in serum-free medium for 8 h. Cell viability was determined by the MTT assay. Data are means ± S.D. (n = 3). (B) Integrin α5β1 protein was linked to protein A/G beads by use of anti-integrin α5β1 antibody, then incubated with F-BSA (BSA-S200) or G-BSA (BSA) overnight. The immunocomplexes were separated by SDS-PAGE and immunoblotted with anti-integrin α5 and anti-BSA antibodies.

    Techniques Used: Incubation, MTT Assay, SDS Page

    Fibrillar BSA induced cytotoxicity via the integrin/FAK/Akt pathway . (A) BHK-21 cells were treated with 3 μM F-BSA (BSA-S200) in serum-free medium for the indicated time, and cell lysates were analyzed by western blotting with anti-phospho-FAK(Tyr576/577), anti-phospho-FAK(Tyr397), and anti-phospho-Akt (p-Akt) antibodies. (B) BHK-21 cells were pre-treated for 30 min with or without 1 μM goat IgG or 1 μM goat anti-integrin α5β1 antibody as indicated, then treated with 3 μM F-BSA (BSA-S200) in serum-free medium for 15 min. Cell lysates were analyzed by western blotting with anti-phospho-Akt (p-Akt) and anti-phospho-GSK-3β (p-GSK-3β) antibodies. (C) BHK-21 cells were treated with increasing concentrations of G-BSA (BSA) in serum-free medium as indicated, and cell lysates were analyzed by western blotting with anti-phospho-Akt (p-Akt) antibody. (D) BHK-21 cells were treated with or without 1 μM anti-integrin α5β1 antibody in serum-free medium for 30 min, and cell lysates were analyzed by western blotting with anti-phospho-Akt (p-Akt) antibody.
    Figure Legend Snippet: Fibrillar BSA induced cytotoxicity via the integrin/FAK/Akt pathway . (A) BHK-21 cells were treated with 3 μM F-BSA (BSA-S200) in serum-free medium for the indicated time, and cell lysates were analyzed by western blotting with anti-phospho-FAK(Tyr576/577), anti-phospho-FAK(Tyr397), and anti-phospho-Akt (p-Akt) antibodies. (B) BHK-21 cells were pre-treated for 30 min with or without 1 μM goat IgG or 1 μM goat anti-integrin α5β1 antibody as indicated, then treated with 3 μM F-BSA (BSA-S200) in serum-free medium for 15 min. Cell lysates were analyzed by western blotting with anti-phospho-Akt (p-Akt) and anti-phospho-GSK-3β (p-GSK-3β) antibodies. (C) BHK-21 cells were treated with increasing concentrations of G-BSA (BSA) in serum-free medium as indicated, and cell lysates were analyzed by western blotting with anti-phospho-Akt (p-Akt) antibody. (D) BHK-21 cells were treated with or without 1 μM anti-integrin α5β1 antibody in serum-free medium for 30 min, and cell lysates were analyzed by western blotting with anti-phospho-Akt (p-Akt) antibody.

    Techniques Used: Western Blot

    34) Product Images from "Albumin fibrillization induces apoptosis via integrin/FAK/Akt pathway"

    Article Title: Albumin fibrillization induces apoptosis via integrin/FAK/Akt pathway

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-9-2

    Interaction between fibrillar BSA and integrin α5β1 . (A) T47D cell lines were pre-treated with or without 0.67 μM goat IgG or 0.67 μM goat anti-integrin α5β1 antibody for 30 min as indicated, then incubated with 2 μM F-BSA (BSA-S200) in serum-free medium for 8 h. Cell viability was determined by the MTT assay. Data are means ± S.D. (n = 3). (B) Integrin α5β1 protein was linked to protein A/G beads by use of anti-integrin α5β1 antibody, then incubated with F-BSA (BSA-S200) or G-BSA (BSA) overnight. The immunocomplexes were separated by SDS-PAGE and immunoblotted with anti-integrin α5 and anti-BSA antibodies.
    Figure Legend Snippet: Interaction between fibrillar BSA and integrin α5β1 . (A) T47D cell lines were pre-treated with or without 0.67 μM goat IgG or 0.67 μM goat anti-integrin α5β1 antibody for 30 min as indicated, then incubated with 2 μM F-BSA (BSA-S200) in serum-free medium for 8 h. Cell viability was determined by the MTT assay. Data are means ± S.D. (n = 3). (B) Integrin α5β1 protein was linked to protein A/G beads by use of anti-integrin α5β1 antibody, then incubated with F-BSA (BSA-S200) or G-BSA (BSA) overnight. The immunocomplexes were separated by SDS-PAGE and immunoblotted with anti-integrin α5 and anti-BSA antibodies.

    Techniques Used: Incubation, MTT Assay, SDS Page

    Fibrillar BSA induced cytotoxicity via the integrin/FAK/Akt pathway . (A) BHK-21 cells were treated with 3 μM F-BSA (BSA-S200) in serum-free medium for the indicated time, and cell lysates were analyzed by western blotting with anti-phospho-FAK(Tyr576/577), anti-phospho-FAK(Tyr397), and anti-phospho-Akt (p-Akt) antibodies. (B) BHK-21 cells were pre-treated for 30 min with or without 1 μM goat IgG or 1 μM goat anti-integrin α5β1 antibody as indicated, then treated with 3 μM F-BSA (BSA-S200) in serum-free medium for 15 min. Cell lysates were analyzed by western blotting with anti-phospho-Akt (p-Akt) and anti-phospho-GSK-3β (p-GSK-3β) antibodies. (C) BHK-21 cells were treated with increasing concentrations of G-BSA (BSA) in serum-free medium as indicated, and cell lysates were analyzed by western blotting with anti-phospho-Akt (p-Akt) antibody. (D) BHK-21 cells were treated with or without 1 μM anti-integrin α5β1 antibody in serum-free medium for 30 min, and cell lysates were analyzed by western blotting with anti-phospho-Akt (p-Akt) antibody.
    Figure Legend Snippet: Fibrillar BSA induced cytotoxicity via the integrin/FAK/Akt pathway . (A) BHK-21 cells were treated with 3 μM F-BSA (BSA-S200) in serum-free medium for the indicated time, and cell lysates were analyzed by western blotting with anti-phospho-FAK(Tyr576/577), anti-phospho-FAK(Tyr397), and anti-phospho-Akt (p-Akt) antibodies. (B) BHK-21 cells were pre-treated for 30 min with or without 1 μM goat IgG or 1 μM goat anti-integrin α5β1 antibody as indicated, then treated with 3 μM F-BSA (BSA-S200) in serum-free medium for 15 min. Cell lysates were analyzed by western blotting with anti-phospho-Akt (p-Akt) and anti-phospho-GSK-3β (p-GSK-3β) antibodies. (C) BHK-21 cells were treated with increasing concentrations of G-BSA (BSA) in serum-free medium as indicated, and cell lysates were analyzed by western blotting with anti-phospho-Akt (p-Akt) antibody. (D) BHK-21 cells were treated with or without 1 μM anti-integrin α5β1 antibody in serum-free medium for 30 min, and cell lysates were analyzed by western blotting with anti-phospho-Akt (p-Akt) antibody.

    Techniques Used: Western Blot

    35) Product Images from "Enalapril stimulates collagen biosynthesis through prolidase-dependent mechanism in cultured fibroblasts"

    Article Title: Enalapril stimulates collagen biosynthesis through prolidase-dependent mechanism in cultured fibroblasts

    Journal: Naunyn-Schmiedeberg's Archives of Pharmacology

    doi: 10.1007/s00210-015-1114-5

    Western blot analysis for prolidase ( a ), α 2 integrin receptor ( b ), β 1 integrin receptor ( c ), IGF receptor ( d ), TGF-β1 ( e ), and NF-κB p65 ( f ) in control human skin fibroblasts ( lane 1 ) and cultured in the medium containing 0.5 mM of enalapril ( lane 2 ) or 0.5 mM of enalaprilat ( lane 3 ). The mean values of six pooled cell homogenate extracts from six separate experiments are presented. The intensity of the bands was quantified by densitometric analysis. Densitometry was done with BioSpectrum Imaging System and presented as an arbitrary units. The same amount of supernatant protein (20 μg) was run in each lane. The expression of β -actin served as a control for protein loading ( g )
    Figure Legend Snippet: Western blot analysis for prolidase ( a ), α 2 integrin receptor ( b ), β 1 integrin receptor ( c ), IGF receptor ( d ), TGF-β1 ( e ), and NF-κB p65 ( f ) in control human skin fibroblasts ( lane 1 ) and cultured in the medium containing 0.5 mM of enalapril ( lane 2 ) or 0.5 mM of enalaprilat ( lane 3 ). The mean values of six pooled cell homogenate extracts from six separate experiments are presented. The intensity of the bands was quantified by densitometric analysis. Densitometry was done with BioSpectrum Imaging System and presented as an arbitrary units. The same amount of supernatant protein (20 μg) was run in each lane. The expression of β -actin served as a control for protein loading ( g )

    Techniques Used: Western Blot, Cell Culture, Imaging, Expressing

    36) Product Images from "The Effect of Growth-Mimicking Continuous Strain on the Early Stages of Skeletal Development in Micromass Culture"

    Article Title: The Effect of Growth-Mimicking Continuous Strain on the Early Stages of Skeletal Development in Micromass Culture

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0124948

    Chondrogenic differentiation. (A) Glycosaminoglycan (GAG) deposition is quantified in whole samples after 60 hours in culture, and normalized to DNA content. Data are normalized to the non-strained condition and represent means ± standard deviations, n≥5. (B) Samples cultured for 60hrs are stained with Alcian Blue to visualize glycosaminoglycan deposition. Color images are converted to gray scale, and an example of this conversion is shown for the non-strained condition. Scale bar is 200μm. (C) The mean fluorescent intensity (MFI) of cells stained for Sox9 was quantified using flow cytometry as a measure of the relative expression of Sox9 after 60 hrs. Data are normalized to the non-strained condition and represent means ± standard deviations, n≥4.
    Figure Legend Snippet: Chondrogenic differentiation. (A) Glycosaminoglycan (GAG) deposition is quantified in whole samples after 60 hours in culture, and normalized to DNA content. Data are normalized to the non-strained condition and represent means ± standard deviations, n≥5. (B) Samples cultured for 60hrs are stained with Alcian Blue to visualize glycosaminoglycan deposition. Color images are converted to gray scale, and an example of this conversion is shown for the non-strained condition. Scale bar is 200μm. (C) The mean fluorescent intensity (MFI) of cells stained for Sox9 was quantified using flow cytometry as a measure of the relative expression of Sox9 after 60 hrs. Data are normalized to the non-strained condition and represent means ± standard deviations, n≥4.

    Techniques Used: Cell Culture, Staining, Flow Cytometry, Cytometry, Expressing

    37) Product Images from "Th1-Biased Immunomodulation and Therapeutic Potential of Artemisia annua in Murine Visceral Leishmaniasis"

    Article Title: Th1-Biased Immunomodulation and Therapeutic Potential of Artemisia annua in Murine Visceral Leishmaniasis

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0003321

    FT-specific antibody response in sera of infected mice upon treatment with AAL (n-hexane fraction of Artemisia annua leaves), AAS (n-hexane fraction of Artemisia annua seeds), ART (Artemisinin), AMB (Amphotericin B) compared with infected control (INF). Sera from treated and control animals were analyzed for FT specific anti-IgG1 and anti-IgG2a levels by ELISA. Data represent mean ± SE for five animals per group. Data were tested by ANOVA. Differences between means were assessed for statistical significance by Tukey's test (**, P ≤ 0.01; ***, P ≤ 0.001). Results are from one of three representative experiments.
    Figure Legend Snippet: FT-specific antibody response in sera of infected mice upon treatment with AAL (n-hexane fraction of Artemisia annua leaves), AAS (n-hexane fraction of Artemisia annua seeds), ART (Artemisinin), AMB (Amphotericin B) compared with infected control (INF). Sera from treated and control animals were analyzed for FT specific anti-IgG1 and anti-IgG2a levels by ELISA. Data represent mean ± SE for five animals per group. Data were tested by ANOVA. Differences between means were assessed for statistical significance by Tukey's test (**, P ≤ 0.01; ***, P ≤ 0.001). Results are from one of three representative experiments.

    Techniques Used: Infection, Mouse Assay, Atomic Absorption Spectroscopy, Enzyme-linked Immunosorbent Assay

    38) Product Images from "CD146 acts as a novel receptor for netrin-1 in promoting angiogenesis and vascular development"

    Article Title: CD146 acts as a novel receptor for netrin-1 in promoting angiogenesis and vascular development

    Journal: Cell Research

    doi: 10.1038/cr.2015.15

    CD146 is required for netrin-1-induced angiogenesis in mouse models. (A) Aortic rings were prepared from WT or CD146 EC-KO mice. Control or netrin-1 (50 ng/ml) was directly added to the culture medium. (B) The effect of anti-CD146 antibody AA98 was tested in the aortic-ring assay. Control mIgG or AA98 (100 μg/ml) was added to the culture medium in the presence of control or netrin-1 (50 ng/ml). After culturing for 5-6 days, the number of sprouts from each ring was quantified. n = 10 in each group and results are presented as average number of sprouts per ring (means ± SEM). (C) The Matrigel-plug assay for angiogenesis was carried out using WT or CD146 EC-KO mice. The plugs were mixed with control or netrin-1 (200 ng/ml) and then injected subcutaneously into mice in the corresponding groups. (D) The effect of anti-CD146 antibody AA98 on netrin-1-induced angiogenesis was tested in the Matrigel-plug assay. The plugs were pre-mixed with AA98 or control mIgG (100 μg/ml) and injected into the WT mice. 10 days post injection, the Matrigel plugs were sectioned and immunostained with anti-CD31 antibody. The number of blood vessels in each section was scored. n = 5 in each group and results are presented as average number of blood vessels/mm 2 (means ± SEM). Scale bar, 200 μm. * P
    Figure Legend Snippet: CD146 is required for netrin-1-induced angiogenesis in mouse models. (A) Aortic rings were prepared from WT or CD146 EC-KO mice. Control or netrin-1 (50 ng/ml) was directly added to the culture medium. (B) The effect of anti-CD146 antibody AA98 was tested in the aortic-ring assay. Control mIgG or AA98 (100 μg/ml) was added to the culture medium in the presence of control or netrin-1 (50 ng/ml). After culturing for 5-6 days, the number of sprouts from each ring was quantified. n = 10 in each group and results are presented as average number of sprouts per ring (means ± SEM). (C) The Matrigel-plug assay for angiogenesis was carried out using WT or CD146 EC-KO mice. The plugs were mixed with control or netrin-1 (200 ng/ml) and then injected subcutaneously into mice in the corresponding groups. (D) The effect of anti-CD146 antibody AA98 on netrin-1-induced angiogenesis was tested in the Matrigel-plug assay. The plugs were pre-mixed with AA98 or control mIgG (100 μg/ml) and injected into the WT mice. 10 days post injection, the Matrigel plugs were sectioned and immunostained with anti-CD31 antibody. The number of blood vessels in each section was scored. n = 5 in each group and results are presented as average number of blood vessels/mm 2 (means ± SEM). Scale bar, 200 μm. * P

    Techniques Used: Mouse Assay, Aortic Ring Assay, Matrigel Assay, Injection

    39) Product Images from "Changes in the expression of DNA-binding/differentiation protein inhibitors in neurons and glial cells of the gerbil hippocampus following transient global cerebral ischemia"

    Article Title: Changes in the expression of DNA-binding/differentiation protein inhibitors in neurons and glial cells of the gerbil hippocampus following transient global cerebral ischemia

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2014.3084

    Double immunofluorescence staining for (A) ID1 (green), (B) GAD67 (red), (C) ID1+GAD67 (merged image), (D) ID4 (green), (E) Iba-1 (red) and (F) ID4+Iba-1 (merged image) in the Cornu Ammonis region CA1 5 days after ischemia-reperfusion. ID1 + cells colocalize with GAD67 + GABAergic interneurons (C, arrows); ID4 + cells colocalize with GFAP + astrocytes (F, arrows). ID, inhibitors of DNA binding/differentiation proteins; GAD67, glutamic acid decarboxylase 67; GABA, γ-aminobutyric-acid; Iba-1, ionized calcium-binding adapter molecule 1; GFAP, glial fibrillary acidic protein; SO, stratum oriens; SP, stratum pyramidale; and SR, stratum radiatum. Scale bar=20 μm.
    Figure Legend Snippet: Double immunofluorescence staining for (A) ID1 (green), (B) GAD67 (red), (C) ID1+GAD67 (merged image), (D) ID4 (green), (E) Iba-1 (red) and (F) ID4+Iba-1 (merged image) in the Cornu Ammonis region CA1 5 days after ischemia-reperfusion. ID1 + cells colocalize with GAD67 + GABAergic interneurons (C, arrows); ID4 + cells colocalize with GFAP + astrocytes (F, arrows). ID, inhibitors of DNA binding/differentiation proteins; GAD67, glutamic acid decarboxylase 67; GABA, γ-aminobutyric-acid; Iba-1, ionized calcium-binding adapter molecule 1; GFAP, glial fibrillary acidic protein; SO, stratum oriens; SP, stratum pyramidale; and SR, stratum radiatum. Scale bar=20 μm.

    Techniques Used: Double Immunofluorescence Staining, Binding Assay

    Immunohistochemical detection of ID4 in the Cornu Ammonis region CA3 (A) of the sham-operated and (B-F) ischemia groups. In the ischemia groups, ID4 immunoreactivity is not significantly changed compared to the sham-operated group. SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum; h, hours; and d, days. Scale bar=50 μm.
    Figure Legend Snippet: Immunohistochemical detection of ID4 in the Cornu Ammonis region CA3 (A) of the sham-operated and (B-F) ischemia groups. In the ischemia groups, ID4 immunoreactivity is not significantly changed compared to the sham-operated group. SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum; h, hours; and d, days. Scale bar=50 μm.

    Techniques Used: Immunohistochemistry

    Immunohistochemical detection of ID4 in the Cornu Ammonis region CA1 (A) of the sham-operated and (B-F) ischemia groups. ID4 immunoreactivity is weakly detected in the processes (arrow head) of the stratum oriens (SO) and stratum radiatum (SR) in the sham-operated group 2 days after ischemia-reperfusion and is markedly increased from 5 days (arrows) after ischemia-reperfusion. ID, inhibitors of DNA binding/differentiation proteins; SP, stratum pyramidale; h, hours; and d, days. Scale bar=50 μm.
    Figure Legend Snippet: Immunohistochemical detection of ID4 in the Cornu Ammonis region CA1 (A) of the sham-operated and (B-F) ischemia groups. ID4 immunoreactivity is weakly detected in the processes (arrow head) of the stratum oriens (SO) and stratum radiatum (SR) in the sham-operated group 2 days after ischemia-reperfusion and is markedly increased from 5 days (arrows) after ischemia-reperfusion. ID, inhibitors of DNA binding/differentiation proteins; SP, stratum pyramidale; h, hours; and d, days. Scale bar=50 μm.

    Techniques Used: Immunohistochemistry, Binding Assay

    (A) Western blot analysis of ID1 and ID4 in the Cornu Ammonis region CA1 of the sham-operated and the ischemia groups, using β-actin as the loading control. The intensity of the immunoblot bands corresponding to (B) ID1 and (C) ID4 was quantified and expressed as relative optical density (ROD)% values relative to the sham-operated group. * P
    Figure Legend Snippet: (A) Western blot analysis of ID1 and ID4 in the Cornu Ammonis region CA1 of the sham-operated and the ischemia groups, using β-actin as the loading control. The intensity of the immunoblot bands corresponding to (B) ID1 and (C) ID4 was quantified and expressed as relative optical density (ROD)% values relative to the sham-operated group. * P

    Techniques Used: Western Blot

    40) Product Images from "Anti‐Remodeling and Anti‐Fibrotic Effects of the Neuregulin‐1β Glial Growth Factor 2 in a Large Animal Model of Heart Failure"

    Article Title: Anti‐Remodeling and Anti‐Fibrotic Effects of the Neuregulin‐1β Glial Growth Factor 2 in a Large Animal Model of Heart Failure

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    doi: 10.1161/JAHA.113.000773

    Representative immunohistochemistry of swine LV tissues from untreated (A) and GGF2‐treated (B) post‐MI pigs. Tissues were stained with DAPI (blue), phalloidin (red), and anti‐αSMA (green). C, Representative cytofluorographic dot plots showing the percentage of αSMA+ fibroblasts incubated in the absence ( vehicle, upper panel ) or presence of 30 ng/mL NRG‐1β ( lower panel ) for 48 hours; (D) Graphic representation of data from flow cytometric analysis of αSMA expression in cardiac fibroblasts incubated in the absence ( vehicle, open bar ) or presence of 30 ng/mL NRG‐1β ( closed bar ) for 48 hours. Number of αSMA+ cells was calculated from percentage of αSMA‐expressing and total number of cells; E, Mean fluorescence intensity of αSMA expression in cardiac fibroblasts as assessed by flow cytometry. Data represent mean±SEM from 3 independent experiments. P‐ values indicate significance level calculated by t test. GGF2 indicates glial growth factor 2; LV, left ventricular; MI, myocardial infarction; NRG‐1β, neuregulin‐1β; αSMA, α smooth muscle.
    Figure Legend Snippet: Representative immunohistochemistry of swine LV tissues from untreated (A) and GGF2‐treated (B) post‐MI pigs. Tissues were stained with DAPI (blue), phalloidin (red), and anti‐αSMA (green). C, Representative cytofluorographic dot plots showing the percentage of αSMA+ fibroblasts incubated in the absence ( vehicle, upper panel ) or presence of 30 ng/mL NRG‐1β ( lower panel ) for 48 hours; (D) Graphic representation of data from flow cytometric analysis of αSMA expression in cardiac fibroblasts incubated in the absence ( vehicle, open bar ) or presence of 30 ng/mL NRG‐1β ( closed bar ) for 48 hours. Number of αSMA+ cells was calculated from percentage of αSMA‐expressing and total number of cells; E, Mean fluorescence intensity of αSMA expression in cardiac fibroblasts as assessed by flow cytometry. Data represent mean±SEM from 3 independent experiments. P‐ values indicate significance level calculated by t test. GGF2 indicates glial growth factor 2; LV, left ventricular; MI, myocardial infarction; NRG‐1β, neuregulin‐1β; αSMA, α smooth muscle.

    Techniques Used: Immunohistochemistry, Staining, Incubation, Flow Cytometry, Expressing, Fluorescence, Cytometry

    Immunohistochemistry of rat cardiac fibroblasts stimulated for 48 hours with TGFβ or NRG‐1β (1 or 50 ng/mL, respectively) or pre‐treated with 50 ng/mL NRG1‐β for 24 hours before treatment with TGFβ (1 ng/mL for an additional 24 hours). After fixation, cells were stained with anti‐αSMA to label myofibroblasts, anti‐collagen I, phalloidin to stain actin filaments, and DAPI to visualize nuclei. Representative images of 3 independent experiments are shown. NRG‐1β indicates neuregulin‐1β; αSMA, α‐smooth muscle actin.
    Figure Legend Snippet: Immunohistochemistry of rat cardiac fibroblasts stimulated for 48 hours with TGFβ or NRG‐1β (1 or 50 ng/mL, respectively) or pre‐treated with 50 ng/mL NRG1‐β for 24 hours before treatment with TGFβ (1 ng/mL for an additional 24 hours). After fixation, cells were stained with anti‐αSMA to label myofibroblasts, anti‐collagen I, phalloidin to stain actin filaments, and DAPI to visualize nuclei. Representative images of 3 independent experiments are shown. NRG‐1β indicates neuregulin‐1β; αSMA, α‐smooth muscle actin.

    Techniques Used: Immunohistochemistry, Staining

    A. Representative Western blot analysis of NRG‐1β‐treated rat cardiac fibroblasts treated with 50 ng/mL of recombinant NRG‐1β at various doses for 48 hours (lanes 1 to 6), 1 ng/mL TGFβ for 48 hours (lane 7) or with NRG‐1β for 24 hours followed by 1 ng/mL TGFβ or 24 hours (lanes 8 to 13) and probed with anti‐α‐smooth muscle actin (αSMA) or phospho‐SMAD3 (pSMAD3). B and C, Graph of Western blot analyses for αSMA and pSMAD3, respectively. NRG‐1β inhibited basal and TGFβ induced αSMA at all concentrations ( P
    Figure Legend Snippet: A. Representative Western blot analysis of NRG‐1β‐treated rat cardiac fibroblasts treated with 50 ng/mL of recombinant NRG‐1β at various doses for 48 hours (lanes 1 to 6), 1 ng/mL TGFβ for 48 hours (lane 7) or with NRG‐1β for 24 hours followed by 1 ng/mL TGFβ or 24 hours (lanes 8 to 13) and probed with anti‐α‐smooth muscle actin (αSMA) or phospho‐SMAD3 (pSMAD3). B and C, Graph of Western blot analyses for αSMA and pSMAD3, respectively. NRG‐1β inhibited basal and TGFβ induced αSMA at all concentrations ( P

    Techniques Used: Western Blot, Recombinant

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    Produced:

    Article Title: Cryo-EM structure of the bacteria killing type IV secretion system core complex from Xanthomonas citri
    Article Snippet: .. Primary antibodies produced in rabbit against VirB7 (AbVirB7; 1:20,000 dilution), VirB8 (AbVirB8; 1:10,000), VirB9 (AbVirB9; 1:4,000), VirB10 (AbVirB10; 1:5,000) and GFP (AbGFP; Sigma-Aldrich G1544; 1:6,000) were used. .. Secondary goat anti-rabbit IgG-AP conjugate (Bio Rad 1706518; 1:5,000) was used for AbVirB7 and AbVirB8 with BCIP (VWR 0885) and NBT (Sigma-Aldrich N6876) for detection, and secondary goat anti-rabbit IgG-IRDye® 800CW (Li-Cor 32211; 1:8,000) was used for AbVirB9, AbVirB10 and AbGFP prior to visualization using an Odyssey imaging system (Li-Cor).

    ALP Assay:

    Article Title: Human ex vivo 3D bone model recapitulates osteocyte response to metastatic prostate cancer
    Article Snippet: .. Samples were stained with rabbit anti-human sclerostin (1:10, ab75914), rabbit anti-human ALP (1:10, ab75699), rabbit anti-human active caspase-3 (1:100, ab2302), rabbit anti-human FGF23 (1:50, ab192497), or rabbit anti-human Dkk-1 (1:50, ab61034) overnight at 4 °C, followed by incubation with a secondary stain (1:100 TRITC-conjugated goat anti-rabbit IgG, ab50598) for 1 h at room temperature and counterstained with DAPI containing mounting medium (Fluoroshield with DAPI, Sigma). .. To identify PCa cells, samples were stained with mouse anti-human pan cytokeratin (1:100, ab86734) followed by secondary staining with Alexa-Fluor 488-conjugated goat anti-mouse IgG (1:100, ab150113).

    Incubation:

    Article Title: Filamin A Phosphorylation at Serine 2152 by the Serine/Threonine Kinase Ndr2 Controls TCR-Induced LFA-1 Activation in T Cells
    Article Snippet: .. Cells were permeabilized with 0.1% Triton X-100 in PBS, blocked with 5% horse serum in PBS, and incubated with Ndr2 rabbit Abs and Cy3-labeled CD3 mAb clone 145-2C11) or Ndr2 rabbit Abs in combination with TRITC-conjugated phalloidin (Sigma Aldrich). .. Bound Ndr2 antibodies were detected with FITC-conjugated goat anti-rabbit IgG (Dianova).

    Article Title: Human ex vivo 3D bone model recapitulates osteocyte response to metastatic prostate cancer
    Article Snippet: .. Samples were stained with rabbit anti-human sclerostin (1:10, ab75914), rabbit anti-human ALP (1:10, ab75699), rabbit anti-human active caspase-3 (1:100, ab2302), rabbit anti-human FGF23 (1:50, ab192497), or rabbit anti-human Dkk-1 (1:50, ab61034) overnight at 4 °C, followed by incubation with a secondary stain (1:100 TRITC-conjugated goat anti-rabbit IgG, ab50598) for 1 h at room temperature and counterstained with DAPI containing mounting medium (Fluoroshield with DAPI, Sigma). .. To identify PCa cells, samples were stained with mouse anti-human pan cytokeratin (1:100, ab86734) followed by secondary staining with Alexa-Fluor 488-conjugated goat anti-mouse IgG (1:100, ab150113).

    Staining:

    Article Title: Human ex vivo 3D bone model recapitulates osteocyte response to metastatic prostate cancer
    Article Snippet: .. Samples were stained with rabbit anti-human sclerostin (1:10, ab75914), rabbit anti-human ALP (1:10, ab75699), rabbit anti-human active caspase-3 (1:100, ab2302), rabbit anti-human FGF23 (1:50, ab192497), or rabbit anti-human Dkk-1 (1:50, ab61034) overnight at 4 °C, followed by incubation with a secondary stain (1:100 TRITC-conjugated goat anti-rabbit IgG, ab50598) for 1 h at room temperature and counterstained with DAPI containing mounting medium (Fluoroshield with DAPI, Sigma). .. To identify PCa cells, samples were stained with mouse anti-human pan cytokeratin (1:100, ab86734) followed by secondary staining with Alexa-Fluor 488-conjugated goat anti-mouse IgG (1:100, ab150113).

    Marker:

    Article Title: Staufen1 links RNA stress granules and autophagy in a model of neurodegeneration
    Article Snippet: .. Antibodies The antibodies used for western blotting and their dilutions were as follows: mouse anti-Ataxin-2 antibody (Clone 22/Ataxin-2) [(1:4000), BD Biosciences, Cat# 611378], rabbit anti-Staufen antibody [(1:5000), Novus biologicals, NBP1-33202], DDX6 antibody [(1:5000), Novus biologicals, NB200-191], RGS8 antibody [(1:5000), Novus Biologicals, NBP2-20153], LC3B Antibody [(1:7000), Novus biologicals, NB100-2220], TDP-43 antibody [(1:7000), Proteintech, Cat# 10782-2-AP], monoclonal anti-FLAG M2 antibody [(1:10,000), Sigma-Aldrich, F3165], monoclonal Anti-Calbindin-D-28K antibody [(1:5000), Sigma-Aldrich, C9848], monoclonal anti-β-Actin−peroxidase antibody (clone AC-15) [(1:20,000), Sigma-Aldrich, A3854], PCP-2 antibody (F-3) [(1:3000), Santa Cruz, sc-137064], Homer-3 antibody (E-6) [(1:2000), Santa Cruz, sc-376155], Anti-PCP4 antibody [(1:5000), Abcam, ab197377], Anti-FAM107B antibody [(1:5000), Abcam, ab175148], rabbit anti-PABP antibody [(1:4000), Abcam, ab153930], p21 Waf1/Cip1 (12D1) rabbit mAb [(1:7000), Cell Signaling, Cat# 2947], SQSTM1/p62 antibody [(1:4000), Cell Signaling, Cat# 5114], Cyclin B1 (V152) mouse mAb [(1:5000), Cell Signaling, Cat# 4135], anti-Polyglutamine-Expansion diseases marker antibody, clone 5TF1-1C2 [(1:3000), EMD Millipore, MAB1574], rabbit anti-neomycin phosphotransferase II (NPTII) antibody [(1:5000), EMD Millipore, AC113], anti-Myc-HRP antibody [(1:5000), Invitrogen, P/N 46-0709], 6 × -His Tag Monoclonal Antibody (HIS.H8), HRP [(1:10,000), ThermoFisher Scientific, MA1-21315-HRP] and sheep-anti-Digoxigenin-POD, Fab fragments [(1:10,000), Roche Life Science, Cat# 11207733910]. .. The secondary antibodies were: Peroxidase-conjugated horse anti-mouse IgG (H + L) antibody [(1:5000), Vector laboratories, PI-2000] and Peroxidase-conjugated AffiniPure goat anti-rabbit IgG (H + L) antibody [(1:5000), Jackson ImmunoResearch Laboratories, Cat# 111-035-144].

    Western Blot:

    Article Title: Staufen1 links RNA stress granules and autophagy in a model of neurodegeneration
    Article Snippet: .. Antibodies The antibodies used for western blotting and their dilutions were as follows: mouse anti-Ataxin-2 antibody (Clone 22/Ataxin-2) [(1:4000), BD Biosciences, Cat# 611378], rabbit anti-Staufen antibody [(1:5000), Novus biologicals, NBP1-33202], DDX6 antibody [(1:5000), Novus biologicals, NB200-191], RGS8 antibody [(1:5000), Novus Biologicals, NBP2-20153], LC3B Antibody [(1:7000), Novus biologicals, NB100-2220], TDP-43 antibody [(1:7000), Proteintech, Cat# 10782-2-AP], monoclonal anti-FLAG M2 antibody [(1:10,000), Sigma-Aldrich, F3165], monoclonal Anti-Calbindin-D-28K antibody [(1:5000), Sigma-Aldrich, C9848], monoclonal anti-β-Actin−peroxidase antibody (clone AC-15) [(1:20,000), Sigma-Aldrich, A3854], PCP-2 antibody (F-3) [(1:3000), Santa Cruz, sc-137064], Homer-3 antibody (E-6) [(1:2000), Santa Cruz, sc-376155], Anti-PCP4 antibody [(1:5000), Abcam, ab197377], Anti-FAM107B antibody [(1:5000), Abcam, ab175148], rabbit anti-PABP antibody [(1:4000), Abcam, ab153930], p21 Waf1/Cip1 (12D1) rabbit mAb [(1:7000), Cell Signaling, Cat# 2947], SQSTM1/p62 antibody [(1:4000), Cell Signaling, Cat# 5114], Cyclin B1 (V152) mouse mAb [(1:5000), Cell Signaling, Cat# 4135], anti-Polyglutamine-Expansion diseases marker antibody, clone 5TF1-1C2 [(1:3000), EMD Millipore, MAB1574], rabbit anti-neomycin phosphotransferase II (NPTII) antibody [(1:5000), EMD Millipore, AC113], anti-Myc-HRP antibody [(1:5000), Invitrogen, P/N 46-0709], 6 × -His Tag Monoclonal Antibody (HIS.H8), HRP [(1:10,000), ThermoFisher Scientific, MA1-21315-HRP] and sheep-anti-Digoxigenin-POD, Fab fragments [(1:10,000), Roche Life Science, Cat# 11207733910]. .. The secondary antibodies were: Peroxidase-conjugated horse anti-mouse IgG (H + L) antibody [(1:5000), Vector laboratories, PI-2000] and Peroxidase-conjugated AffiniPure goat anti-rabbit IgG (H + L) antibody [(1:5000), Jackson ImmunoResearch Laboratories, Cat# 111-035-144].

    Article Title: The mechanism of miR-142-3p in coronary microembolization-induced myocardiac injury via regulating target gene IRAK-1
    Article Snippet: .. Western blot analysis Total proteins obtained from the cardiac tissues and cardiomyocytes were separated by 10–15% SDS–PAGE and then electrotransferred onto PVDF membranes (Millipore, Atlanta, GA, US). .. The membranes were blocked with 5% bovine serum albumin or non-fat milk for 1.5 h at room temperature, followed by incubation at 4 °C overnight with primary antibodies against IRAK-1, NF-κB p65, TNF-α, IL-1β, IL-6, or GAPDH.

    Recombinant:

    Article Title: ClC-2 knockdown prevents cerebrovascular remodeling via inhibition of the Wnt/β-catenin signaling pathway
    Article Snippet: .. Angiotensin II (AngII), bromodeoxyuridine (BrdU) antibody, rabbit anti-mouse-cy3 antibody, recombinant Wnt3a, and hematoxylin and eosin solutions were obtained from Sigma-Aldrich. .. Cell culture Human brain vascular smooth muscle cells (HBVSMCs) were purchased from Creative Bioarray (CSC-7824 W, NY, USA) and cultured in SuperCult Smooth Muscle Cell Medium (Creative Bioarray) containing 10% FBS, 100 μg/ml streptomycin and 100 U/ml penicillin in a humidified incubator with 5% CO2 and 95% O2 at 37 °C.

    SDS Page:

    Article Title: The mechanism of miR-142-3p in coronary microembolization-induced myocardiac injury via regulating target gene IRAK-1
    Article Snippet: .. Western blot analysis Total proteins obtained from the cardiac tissues and cardiomyocytes were separated by 10–15% SDS–PAGE and then electrotransferred onto PVDF membranes (Millipore, Atlanta, GA, US). .. The membranes were blocked with 5% bovine serum albumin or non-fat milk for 1.5 h at room temperature, followed by incubation at 4 °C overnight with primary antibodies against IRAK-1, NF-κB p65, TNF-α, IL-1β, IL-6, or GAPDH.

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  • 99
    Millipore duolink in situ pla probe anti rabbit plus
    TrkA and APP interaction in septal primary neurons measured by proximity ligation assay. (A) Confocal microscopy analysis of double-staining of APP (rabbit APP-CT A8717, red) and Trk (mouse Trk B3, green) in primary septal neurons showing co-localization of APP and TrkA. (B) The TrkA/APP complex was visualized by <t>PLA,</t> which generates red dots when the two proteins are in close proximity. Mouse and rabbit anti TrkA-CT (mouse Trk B3 and rabbit TrkA ab7261) and anti APP-CT (rabbit APP-CT A8717 and mouse APP clone C1-6.1) were used as primary antibodies and anti-mouse MINUS and anti-rabbit <t>PLUS</t> were used as secondary antibodies. Red fluorescent dot represents single interaction between TrkA and APP. (C) In addition to PLA (red dots), rat primary septal neurons were immunostained with goat anti ChAt (green) and with DAPI for nuclei (blue). (D) PLA assay using mouse anti APP (C1-6.1) and rabbit anti TrkB (sc-119) or TrkC (sc-117).
    Duolink In Situ Pla Probe Anti Rabbit Plus, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/duolink in situ pla probe anti rabbit plus/product/Millipore
    Average 99 stars, based on 33 article reviews
    Price from $9.99 to $1999.99
    duolink in situ pla probe anti rabbit plus - by Bioz Stars, 2020-09
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    88
    Millipore goat anti rabbit igg fitc conjugated
    Alcohol Induces HDAC2 Protein and this Effect is Inhibited by TSA After reaching confluency, SK-N-MC were pre-incubated with TSA for 2 hours, then treated with EtOH (0.1%) for 48 hours. In figure 3a, 10 µg of protein were analyzed using western blot with primary anti-HDAC2 and secondary <t>anti-IgG-HRP</t> antibodies. GAPDH was used as a loading control. Data presented show a representative blot indicating modulation of HDAC2 protein expression and a bar graph representing the mean ± SE of % densitometry values of HDAC2 protein levels (% control) of three independent experiments. # represents significance compared to control. * represents significance compared to EtOH treatment. For the flow cytometry experiments, 1 × 10 6 cells were fixed and permeabilized prior to intracellular staining with primary anti-HDAC2 and secondary <t>anti-IgG-FITC</t> antibody. Data presented in figure 3b show a representative histogram overlay of the gated cells. The bar graph represents the mean ± SE of % of gated cells expressing HDAC2. 10000 events were analyzed per sample. The gray and black histograms represent the unlabeled and secondary antibody controls respectively; the green histogram is the untreated control (~52%), blue represents EtOH (~69 %), purple represents TSA (~45%), and orange represents TSA + EtOH (~49%) treated cells. Data are representative of three independent experiments.
    Goat Anti Rabbit Igg Fitc Conjugated, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/goat anti rabbit igg fitc conjugated/product/Millipore
    Average 88 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    goat anti rabbit igg fitc conjugated - by Bioz Stars, 2020-09
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    Image Search Results


    TrkA and APP interaction in septal primary neurons measured by proximity ligation assay. (A) Confocal microscopy analysis of double-staining of APP (rabbit APP-CT A8717, red) and Trk (mouse Trk B3, green) in primary septal neurons showing co-localization of APP and TrkA. (B) The TrkA/APP complex was visualized by PLA, which generates red dots when the two proteins are in close proximity. Mouse and rabbit anti TrkA-CT (mouse Trk B3 and rabbit TrkA ab7261) and anti APP-CT (rabbit APP-CT A8717 and mouse APP clone C1-6.1) were used as primary antibodies and anti-mouse MINUS and anti-rabbit PLUS were used as secondary antibodies. Red fluorescent dot represents single interaction between TrkA and APP. (C) In addition to PLA (red dots), rat primary septal neurons were immunostained with goat anti ChAt (green) and with DAPI for nuclei (blue). (D) PLA assay using mouse anti APP (C1-6.1) and rabbit anti TrkB (sc-119) or TrkC (sc-117).

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Association of TrkA and APP Is Promoted by NGF and Reduced by Cell Death-Promoting Agents

    doi: 10.3389/fnmol.2017.00015

    Figure Lengend Snippet: TrkA and APP interaction in septal primary neurons measured by proximity ligation assay. (A) Confocal microscopy analysis of double-staining of APP (rabbit APP-CT A8717, red) and Trk (mouse Trk B3, green) in primary septal neurons showing co-localization of APP and TrkA. (B) The TrkA/APP complex was visualized by PLA, which generates red dots when the two proteins are in close proximity. Mouse and rabbit anti TrkA-CT (mouse Trk B3 and rabbit TrkA ab7261) and anti APP-CT (rabbit APP-CT A8717 and mouse APP clone C1-6.1) were used as primary antibodies and anti-mouse MINUS and anti-rabbit PLUS were used as secondary antibodies. Red fluorescent dot represents single interaction between TrkA and APP. (C) In addition to PLA (red dots), rat primary septal neurons were immunostained with goat anti ChAt (green) and with DAPI for nuclei (blue). (D) PLA assay using mouse anti APP (C1-6.1) and rabbit anti TrkB (sc-119) or TrkC (sc-117).

    Article Snippet: Coverslips were washed three times for 5 min PBS 1X and then incubated with PLA Probe Anti-mouse MINUS (DUO92004) and Anti-rabbit PLUS (DUO92002) for 1 h at 37°C.

    Techniques: Proximity Ligation Assay, Confocal Microscopy, Double Staining

    Alcohol Induces HDAC2 Protein and this Effect is Inhibited by TSA After reaching confluency, SK-N-MC were pre-incubated with TSA for 2 hours, then treated with EtOH (0.1%) for 48 hours. In figure 3a, 10 µg of protein were analyzed using western blot with primary anti-HDAC2 and secondary anti-IgG-HRP antibodies. GAPDH was used as a loading control. Data presented show a representative blot indicating modulation of HDAC2 protein expression and a bar graph representing the mean ± SE of % densitometry values of HDAC2 protein levels (% control) of three independent experiments. # represents significance compared to control. * represents significance compared to EtOH treatment. For the flow cytometry experiments, 1 × 10 6 cells were fixed and permeabilized prior to intracellular staining with primary anti-HDAC2 and secondary anti-IgG-FITC antibody. Data presented in figure 3b show a representative histogram overlay of the gated cells. The bar graph represents the mean ± SE of % of gated cells expressing HDAC2. 10000 events were analyzed per sample. The gray and black histograms represent the unlabeled and secondary antibody controls respectively; the green histogram is the untreated control (~52%), blue represents EtOH (~69 %), purple represents TSA (~45%), and orange represents TSA + EtOH (~49%) treated cells. Data are representative of three independent experiments.

    Journal: Alcoholism, clinical and experimental research

    Article Title: Effects of Alcohol on Histone Deacetylase 2 (HDAC2) and the Neuroprotective Role of Trichostatin A (TSA)

    doi: 10.1111/j.1530-0277.2011.01492.x

    Figure Lengend Snippet: Alcohol Induces HDAC2 Protein and this Effect is Inhibited by TSA After reaching confluency, SK-N-MC were pre-incubated with TSA for 2 hours, then treated with EtOH (0.1%) for 48 hours. In figure 3a, 10 µg of protein were analyzed using western blot with primary anti-HDAC2 and secondary anti-IgG-HRP antibodies. GAPDH was used as a loading control. Data presented show a representative blot indicating modulation of HDAC2 protein expression and a bar graph representing the mean ± SE of % densitometry values of HDAC2 protein levels (% control) of three independent experiments. # represents significance compared to control. * represents significance compared to EtOH treatment. For the flow cytometry experiments, 1 × 10 6 cells were fixed and permeabilized prior to intracellular staining with primary anti-HDAC2 and secondary anti-IgG-FITC antibody. Data presented in figure 3b show a representative histogram overlay of the gated cells. The bar graph represents the mean ± SE of % of gated cells expressing HDAC2. 10000 events were analyzed per sample. The gray and black histograms represent the unlabeled and secondary antibody controls respectively; the green histogram is the untreated control (~52%), blue represents EtOH (~69 %), purple represents TSA (~45%), and orange represents TSA + EtOH (~49%) treated cells. Data are representative of three independent experiments.

    Article Snippet: The HDAC2 protein was detected with the primary monoclonal antibody, rabbit anti-histone deacetylase 2 (Millipore) and secondary antibody, goat anti-rabbit IgG FITC-conjugated (Millipore).

    Techniques: Incubation, Western Blot, Expressing, Flow Cytometry, Cytometry, Staining