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 2713 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 "Novel mouse monoclonal antibodies specifically recognize Aspergillus fumigatus galactomannan"

    Article Title: Novel mouse monoclonal antibodies specifically recognize Aspergillus fumigatus galactomannan

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0193938

    Immunofluorescence labeling of the Aspergillus fumigatus WT strain and ΔUgm1 mutant with mAbs 7B8 and 8G4. (A, B) Wild-type parental strain of A . fumigatus in fluorescence microscopy and light microscopy studies, respectively. (C, D) Ugm1 mutant of A . fumigatus without galactomannan in fluorescence microscopy and light microscopy studies, respectively. Binding of mAbs with Aspergillus cells was detected with goat TRITC-conjugated anti-mouse IgG antibody. Scale bar = 100 μm.
    Figure Legend Snippet: Immunofluorescence labeling of the Aspergillus fumigatus WT strain and ΔUgm1 mutant with mAbs 7B8 and 8G4. (A, B) Wild-type parental strain of A . fumigatus in fluorescence microscopy and light microscopy studies, respectively. (C, D) Ugm1 mutant of A . fumigatus without galactomannan in fluorescence microscopy and light microscopy studies, respectively. Binding of mAbs with Aspergillus cells was detected with goat TRITC-conjugated anti-mouse IgG antibody. Scale bar = 100 μm.

    Techniques Used: Immunofluorescence, Labeling, Mutagenesis, Fluorescence, Microscopy, Light Microscopy, Binding Assay

    Binding of selected mAbs with biotinylated pentasaccharide GM-1. Serial three-fold dilutions of (A) mAb 7B8 starting from 180 nM and (B) mAb 8G4 starting from 60 nM were used as analytes. Fitted traces are depicted as smooth black lines. A global analysis of the interaction demonstrated a good quality fit and experimentally determined dissociation and association rate constants, k d and k a , were (3.0 ± 0.1) × 10 −4 s −1 and (5.6 ± 0.1) × 10 4 M −1 s −1 for 7B8 antibody, and (1.5 ± 0.1) × 10 −3 s −1 and (2.3 ± 0.1) × 10 5 M −1 s −1 for 8G4 antibody, respectively. Equilibrium constants, calculated as K D = k d /k a , were 5.3 × 10 −9 M and 6.4 × 10 −9 M, respectively.
    Figure Legend Snippet: Binding of selected mAbs with biotinylated pentasaccharide GM-1. Serial three-fold dilutions of (A) mAb 7B8 starting from 180 nM and (B) mAb 8G4 starting from 60 nM were used as analytes. Fitted traces are depicted as smooth black lines. A global analysis of the interaction demonstrated a good quality fit and experimentally determined dissociation and association rate constants, k d and k a , were (3.0 ± 0.1) × 10 −4 s −1 and (5.6 ± 0.1) × 10 4 M −1 s −1 for 7B8 antibody, and (1.5 ± 0.1) × 10 −3 s −1 and (2.3 ± 0.1) × 10 5 M −1 s −1 for 8G4 antibody, respectively. Equilibrium constants, calculated as K D = k d /k a , were 5.3 × 10 −9 M and 6.4 × 10 −9 M, respectively.

    Techniques Used: Binding Assay

    Specific binding of mAbs 7B8 and 8G4 with A . fumigatus , A . flavus , and C . albicans . Cells were grown in Sabouraud broth, fixed, and incubated with mAbs 7B8 and 8G4. Binding of mAbs with fungal cells was detected with Alexa Fluor 488-conjugated anti-mouse IgG antibody staining in (A) confocal microscopy, and (B) DIC microscopy. Scale bar = 10 μm.
    Figure Legend Snippet: Specific binding of mAbs 7B8 and 8G4 with A . fumigatus , A . flavus , and C . albicans . Cells were grown in Sabouraud broth, fixed, and incubated with mAbs 7B8 and 8G4. Binding of mAbs with fungal cells was detected with Alexa Fluor 488-conjugated anti-mouse IgG antibody staining in (A) confocal microscopy, and (B) DIC microscopy. Scale bar = 10 μm.

    Techniques Used: Binding Assay, Incubation, Staining, Confocal Microscopy, Microscopy

    Investigation of oligosaccharide specificity of mAbs 7B8 and 8G4 using ELISA. (A) Composition of thematic glycoarray built using oligosaccharide ligands representing key structural elements of A . fumigatus galactomannan chain, and (B) assay for carbohydrate specificity of 7B8 and 8G4 mAbs.
    Figure Legend Snippet: Investigation of oligosaccharide specificity of mAbs 7B8 and 8G4 using ELISA. (A) Composition of thematic glycoarray built using oligosaccharide ligands representing key structural elements of A . fumigatus galactomannan chain, and (B) assay for carbohydrate specificity of 7B8 and 8G4 mAbs.

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Binding of fungal and bacterial cultures with mAbs 7B8 and 8G4. (A) Sandwich enzyme-linked immunosorbent assay (ELISA) with 7B8 mAb: the wells of microtiter plates were coated with 7B8 mAb and incubated with serial dilutions of microbial supernatants; ELISA was performed with horseradish peroxidase-conjugated 7B8 mAb. (B) Sandwich ELISA with 8G4 mAb: the wells of microtiter plates were coated with 8G4 mAb and incubated with serial dilutions of microbial supernatants; ELISA was performed with horseradish peroxidase-conjugated 8G4 mAb.
    Figure Legend Snippet: Binding of fungal and bacterial cultures with mAbs 7B8 and 8G4. (A) Sandwich enzyme-linked immunosorbent assay (ELISA) with 7B8 mAb: the wells of microtiter plates were coated with 7B8 mAb and incubated with serial dilutions of microbial supernatants; ELISA was performed with horseradish peroxidase-conjugated 7B8 mAb. (B) Sandwich ELISA with 8G4 mAb: the wells of microtiter plates were coated with 8G4 mAb and incubated with serial dilutions of microbial supernatants; ELISA was performed with horseradish peroxidase-conjugated 8G4 mAb.

    Techniques Used: Binding Assay, Sandwich ELISA, Enzyme-linked Immunosorbent Assay, Incubation

    3) 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

    4) Product Images from "Immunogenicity of a Virus-Like-Particle Vaccine Containing Multiple Antigenic Epitopes of Toxoplasma gondii Against Acute and Chronic Toxoplasmosis in Mice"

    Article Title: Immunogenicity of a Virus-Like-Particle Vaccine Containing Multiple Antigenic Epitopes of Toxoplasma gondii Against Acute and Chronic Toxoplasmosis in Mice

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2019.00592

    Plasmid identification and protein expression analysis. (A) Five recombinant plasmids, i.e., pET-30a (+) /HBc Δ , HBc ΔH82 , HBc ΔH301 , HBc ΔR82 , and HBc ΔR301 , were analyzed by agarose gel electrophoresis. They were digested into large fragments (4,193 bp) and small fragments (1,503, 2,097, 2,094, 2,094p, and 2,091bp, respectively) by two endonucleases (5'- Apa I and 3'- Xho I, respectively). Lane M: protein marker; Lanes 1–5: recombinant plasmids, i.e., pET-30a (+) /HBc Δ , HBc ΔH82 , HBc ΔH301 , HBc ΔR82 , and HBc ΔR301 . (B) Expression of the chimeric HBc VLPs in Escherichia.coli tested by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) and Coomassie Blue staining. Lane M: protein marker; Lane 1: lysates of the pET-30a (+) transformant; Lanes 2-6: lysates of the pET-30a (+)/HBc Δ , HBc ΔH82 , HBc ΔH301 , HBc ΔR82 , and HBc ΔR301 transformants. HBc Δ (18.3 KDa), HBc ΔH82 (23.2 KDa), HBc ΔH301 (23.0 KDa), HBc ΔR82 (23.0 KDa),and HBc ΔR301 (22.8 KDa) proteins have been indicated by arrows. (C) Purification of the chimeric proteins analyzed by SDS-PAGE and Coomassie Blue staining. The efficiency of the protein purification and molecular mass of the chimeric HBc VLPs were ensured by using the unpurified proteins as templates. Lane M: protein marker; Lanes 1,3,5,7,and 9: unpurified HBc Δ , HBc ΔH82 , HBc ΔH301 , HBc ΔR82 , and HBc ΔR301 protein, respectively; Lanes 2, 4, 6, 8, and 10: purified HBc Δ , HBc ΔH82 , HBc ΔH301 , HBc ΔR82 , and HBc ΔR301 proteins, respectively (indicated by arrows). (D) Western blot analysis of the expressed proteins (using an anti-His tag mAb). Lane M: protein marker; Lanes 1–5: purified HBc Δ , HBc ΔH82 , HBc ΔH301 , HBc ΔR82 , and HBc ΔR301 proteins.
    Figure Legend Snippet: Plasmid identification and protein expression analysis. (A) Five recombinant plasmids, i.e., pET-30a (+) /HBc Δ , HBc ΔH82 , HBc ΔH301 , HBc ΔR82 , and HBc ΔR301 , were analyzed by agarose gel electrophoresis. They were digested into large fragments (4,193 bp) and small fragments (1,503, 2,097, 2,094, 2,094p, and 2,091bp, respectively) by two endonucleases (5'- Apa I and 3'- Xho I, respectively). Lane M: protein marker; Lanes 1–5: recombinant plasmids, i.e., pET-30a (+) /HBc Δ , HBc ΔH82 , HBc ΔH301 , HBc ΔR82 , and HBc ΔR301 . (B) Expression of the chimeric HBc VLPs in Escherichia.coli tested by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) and Coomassie Blue staining. Lane M: protein marker; Lane 1: lysates of the pET-30a (+) transformant; Lanes 2-6: lysates of the pET-30a (+)/HBc Δ , HBc ΔH82 , HBc ΔH301 , HBc ΔR82 , and HBc ΔR301 transformants. HBc Δ (18.3 KDa), HBc ΔH82 (23.2 KDa), HBc ΔH301 (23.0 KDa), HBc ΔR82 (23.0 KDa),and HBc ΔR301 (22.8 KDa) proteins have been indicated by arrows. (C) Purification of the chimeric proteins analyzed by SDS-PAGE and Coomassie Blue staining. The efficiency of the protein purification and molecular mass of the chimeric HBc VLPs were ensured by using the unpurified proteins as templates. Lane M: protein marker; Lanes 1,3,5,7,and 9: unpurified HBc Δ , HBc ΔH82 , HBc ΔH301 , HBc ΔR82 , and HBc ΔR301 protein, respectively; Lanes 2, 4, 6, 8, and 10: purified HBc Δ , HBc ΔH82 , HBc ΔH301 , HBc ΔR82 , and HBc ΔR301 proteins, respectively (indicated by arrows). (D) Western blot analysis of the expressed proteins (using an anti-His tag mAb). Lane M: protein marker; Lanes 1–5: purified HBc Δ , HBc ΔH82 , HBc ΔH301 , HBc ΔR82 , and HBc ΔR301 proteins.

    Techniques Used: Plasmid Preparation, Expressing, Recombinant, Positron Emission Tomography, Agarose Gel Electrophoresis, Marker, Polyacrylamide Gel Electrophoresis, Staining, Purification, SDS Page, Protein Purification, Western Blot

    Electron microscopic characterization of VLPs. Transmission electron microphotographs of the chimeric HBc particles [HBc Δ (A) , HBc ΔH82 (B) , HBc ΔH301 (C) , HBc ΔR82 (D) , and HBc ΔR301 (E) ] showing regular morphology of the VLPs in each preparation. Images were generated using 1% phosphotungstic acid as a negative stain. Magnification 120, 000×. Scale bar, 100 nm.
    Figure Legend Snippet: Electron microscopic characterization of VLPs. Transmission electron microphotographs of the chimeric HBc particles [HBc Δ (A) , HBc ΔH82 (B) , HBc ΔH301 (C) , HBc ΔR82 (D) , and HBc ΔR301 (E) ] showing regular morphology of the VLPs in each preparation. Images were generated using 1% phosphotungstic acid as a negative stain. Magnification 120, 000×. Scale bar, 100 nm.

    Techniques Used: Transmission Assay, Generated, Staining

    Structure of the chimeric HBc virus-like particles (VLPs). (A) Gene structure of the chimeric HBc VLPs. HBc Δ represents the truncated HBc particles (aa 1–149). A CD8 + T cell epitope (HF10 or ROP7; green) and a B cell epitope (SAG1 82−102 or SAG1 301−320 ; red) of Toxoplasma gondii were inserted between aa78 and 79 of HBc Δ particles (MIR), with a Gln(Q) and Asp(D) linker at both ends. Futhermore, a CD4 + cell epitope (AS15) (blue) of T. gondii was fused into the C terminal of HBc Δ particles. (B) The monomer (a) and polymer (b) structures of the chimeric HBc VLPs. The inserted CD8 + T cell, CD4 + T cell, and B cell epitopes are shown in green, blue, and red, respectively.
    Figure Legend Snippet: Structure of the chimeric HBc virus-like particles (VLPs). (A) Gene structure of the chimeric HBc VLPs. HBc Δ represents the truncated HBc particles (aa 1–149). A CD8 + T cell epitope (HF10 or ROP7; green) and a B cell epitope (SAG1 82−102 or SAG1 301−320 ; red) of Toxoplasma gondii were inserted between aa78 and 79 of HBc Δ particles (MIR), with a Gln(Q) and Asp(D) linker at both ends. Futhermore, a CD4 + cell epitope (AS15) (blue) of T. gondii was fused into the C terminal of HBc Δ particles. (B) The monomer (a) and polymer (b) structures of the chimeric HBc VLPs. The inserted CD8 + T cell, CD4 + T cell, and B cell epitopes are shown in green, blue, and red, respectively.

    Techniques Used:

    5) 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

    6) 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

    7) Product Images from "Comparison of microbiota and allergen profile in house dust from homes of allergic and non-allergic subjects- results from the GUSTO study"

    Article Title: Comparison of microbiota and allergen profile in house dust from homes of allergic and non-allergic subjects- results from the GUSTO study

    Journal: The World Allergy Organization Journal

    doi: 10.1186/s40413-018-0212-5

    Concentration of indoor allergens, ( a ) Der p 1 ( b ) Tropomyosin ( c ) Mite Group 2 ( d ) Blo t 5 ( e ) Der f 1 ( f ) Can f 1( g ) Fel d 1 of dust samples from the bed (○), play Area (□), and sofa (∆) of allergic (open) and non-allergic (close) subjects. Red line represents the median of each group. Dotted lines represent the lower limit of detection of each allergen. Asterisk (*) represents significant difference of  P -value
    Figure Legend Snippet: Concentration of indoor allergens, ( a ) Der p 1 ( b ) Tropomyosin ( c ) Mite Group 2 ( d ) Blo t 5 ( e ) Der f 1 ( f ) Can f 1( g ) Fel d 1 of dust samples from the bed (○), play Area (□), and sofa (∆) of allergic (open) and non-allergic (close) subjects. Red line represents the median of each group. Dotted lines represent the lower limit of detection of each allergen. Asterisk (*) represents significant difference of P -value

    Techniques Used: Concentration Assay

    8) 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

    9) 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

    10) 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

    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 "Major histocompatibility complex (MHC) class II but not MHC class I molecules are required for efficient control of Strongyloides venezuelensis infection in mice"

    Article Title: Major histocompatibility complex (MHC) class II but not MHC class I molecules are required for efficient control of Strongyloides venezuelensis infection in mice

    Journal: Immunology

    doi: 10.1111/j.1365-2567.2008.02995.x

    Levels of specific immunoglobulin M (IgM) (a), IgA (b), IgE (c), total IgG (d), IgG1 (e) and IgG2a (f) in serum samples of wild-type (WT), major histocompatibility complex (MHC) I −/− and MHC II −/− mice, after subcutaneous (s.c.) infection with 3000 Strongyloides venezuelensis L3 larvae. The results are expressed as enzyme-linked immunosorbent assay (ELISA) index (EI) values (values of EI > 1·0 are considered positive; horizontal dashed line) and are given as mean ± standard error of the mean (SEM) ( n = 6). Results are representative of two or three independent experiments. * P
    Figure Legend Snippet: Levels of specific immunoglobulin M (IgM) (a), IgA (b), IgE (c), total IgG (d), IgG1 (e) and IgG2a (f) in serum samples of wild-type (WT), major histocompatibility complex (MHC) I −/− and MHC II −/− mice, after subcutaneous (s.c.) infection with 3000 Strongyloides venezuelensis L3 larvae. The results are expressed as enzyme-linked immunosorbent assay (ELISA) index (EI) values (values of EI > 1·0 are considered positive; horizontal dashed line) and are given as mean ± standard error of the mean (SEM) ( n = 6). Results are representative of two or three independent experiments. * P

    Techniques Used: Mouse Assay, Infection, Enzyme-linked Immunosorbent Assay

    Levels of specific immunoglobulin M (IgM) (a), IgA (b), IgE (c), total IgG (d), IgG1 (e) and IgG2a (f) in serum samples of wild-type (WT), major histocompatibility complex (MHC) I −/− and MHC II −/− mice, after subcutaneous (s.c.) infection with 3000 Strongyloides venezuelensis L3 larvae. The results are expressed as enzyme-linked immunosorbent assay (ELISA) index (EI) values (values of EI > 1·0 are considered positive; horizontal dashed line) and are given as mean ± standard error of the mean (SEM) ( n = 6). Results are representative of two or three independent experiments. * P
    Figure Legend Snippet: Levels of specific immunoglobulin M (IgM) (a), IgA (b), IgE (c), total IgG (d), IgG1 (e) and IgG2a (f) in serum samples of wild-type (WT), major histocompatibility complex (MHC) I −/− and MHC II −/− mice, after subcutaneous (s.c.) infection with 3000 Strongyloides venezuelensis L3 larvae. The results are expressed as enzyme-linked immunosorbent assay (ELISA) index (EI) values (values of EI > 1·0 are considered positive; horizontal dashed line) and are given as mean ± standard error of the mean (SEM) ( n = 6). Results are representative of two or three independent experiments. * P

    Techniques Used: Mouse Assay, Infection, Enzyme-linked Immunosorbent Assay

    15) Product Images from "Major histocompatibility complex (MHC) class II but not MHC class I molecules are required for efficient control of Strongyloides venezuelensis infection in mice"

    Article Title: Major histocompatibility complex (MHC) class II but not MHC class I molecules are required for efficient control of Strongyloides venezuelensis infection in mice

    Journal: Immunology

    doi: 10.1111/j.1365-2567.2008.02995.x

    Levels of specific immunoglobulin M (IgM) (a), IgA (b), IgE (c), total IgG (d), IgG1 (e) and IgG2a (f) in serum samples of wild-type (WT), major histocompatibility complex (MHC) I −/− and MHC II −/− mice, after subcutaneous (s.c.) infection with 3000 Strongyloides venezuelensis L3 larvae. The results are expressed as enzyme-linked immunosorbent assay (ELISA) index (EI) values (values of EI > 1·0 are considered positive; horizontal dashed line) and are given as mean ± standard error of the mean (SEM) ( n = 6). Results are representative of two or three independent experiments. * P
    Figure Legend Snippet: Levels of specific immunoglobulin M (IgM) (a), IgA (b), IgE (c), total IgG (d), IgG1 (e) and IgG2a (f) in serum samples of wild-type (WT), major histocompatibility complex (MHC) I −/− and MHC II −/− mice, after subcutaneous (s.c.) infection with 3000 Strongyloides venezuelensis L3 larvae. The results are expressed as enzyme-linked immunosorbent assay (ELISA) index (EI) values (values of EI > 1·0 are considered positive; horizontal dashed line) and are given as mean ± standard error of the mean (SEM) ( n = 6). Results are representative of two or three independent experiments. * P

    Techniques Used: Mouse Assay, Infection, Enzyme-linked Immunosorbent Assay

    Levels of specific immunoglobulin M (IgM) (a), IgA (b), IgE (c), total IgG (d), IgG1 (e) and IgG2a (f) in serum samples of wild-type (WT), major histocompatibility complex (MHC) I −/− and MHC II −/− mice, after subcutaneous (s.c.) infection with 3000 Strongyloides venezuelensis L3 larvae. The results are expressed as enzyme-linked immunosorbent assay (ELISA) index (EI) values (values of EI > 1·0 are considered positive; horizontal dashed line) and are given as mean ± standard error of the mean (SEM) ( n = 6). Results are representative of two or three independent experiments. * P
    Figure Legend Snippet: Levels of specific immunoglobulin M (IgM) (a), IgA (b), IgE (c), total IgG (d), IgG1 (e) and IgG2a (f) in serum samples of wild-type (WT), major histocompatibility complex (MHC) I −/− and MHC II −/− mice, after subcutaneous (s.c.) infection with 3000 Strongyloides venezuelensis L3 larvae. The results are expressed as enzyme-linked immunosorbent assay (ELISA) index (EI) values (values of EI > 1·0 are considered positive; horizontal dashed line) and are given as mean ± standard error of the mean (SEM) ( n = 6). Results are representative of two or three independent experiments. * P

    Techniques Used: Mouse Assay, Infection, Enzyme-linked Immunosorbent Assay

    16) 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

    17) Product Images from "Intracellular Localization Map of Human Herpesvirus 8 Proteins ▿"

    Article Title: Intracellular Localization Map of Human Herpesvirus 8 Proteins ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.01716-07

    Influence of Myc tag and cell permeabilization on subcellular localization and immunocytochemical accessibility of HHV-8-encoded proteins. (A) Myc-tagged and untagged HHV-8-encoded proteins were expressed in HeLa cells and detected with either specific antibodies against the different HHV-8 proteins (nontagged proteins) or against the Myc tag. As a negative control, cells were transfected with the vector control (pcDNA4-Myc/His) and stained with antibodies against the viral proteins. Secondary antibodies were conjugated with Alexa Fluor 488. Cells permeabilized by Triton X-100 (B) and saponin (C) were subjected to immunocytochemical analysis of Myc-tagged K8, ORF20, and ORF54. For staining an antibody against the Myc tag was used. Secondary antibodies were conjugated with Alexa Fluor 488. No differences in localization and staining sensitivity were observed under the conditions used. Pictures were obtained using an epifluorescence microscope. The bar in K2 represents 10 μm. The same magnification was used in all panels.
    Figure Legend Snippet: Influence of Myc tag and cell permeabilization on subcellular localization and immunocytochemical accessibility of HHV-8-encoded proteins. (A) Myc-tagged and untagged HHV-8-encoded proteins were expressed in HeLa cells and detected with either specific antibodies against the different HHV-8 proteins (nontagged proteins) or against the Myc tag. As a negative control, cells were transfected with the vector control (pcDNA4-Myc/His) and stained with antibodies against the viral proteins. Secondary antibodies were conjugated with Alexa Fluor 488. Cells permeabilized by Triton X-100 (B) and saponin (C) were subjected to immunocytochemical analysis of Myc-tagged K8, ORF20, and ORF54. For staining an antibody against the Myc tag was used. Secondary antibodies were conjugated with Alexa Fluor 488. No differences in localization and staining sensitivity were observed under the conditions used. Pictures were obtained using an epifluorescence microscope. The bar in K2 represents 10 μm. The same magnification was used in all panels.

    Techniques Used: Negative Control, Transfection, Plasmid Preparation, Staining, Microscopy

    18) 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

    19) 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

    20) 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

    21) Product Images from "Immature truncated O-glycophenotype of cancer directly induces oncogenic features"

    Article Title: Immature truncated O-glycophenotype of cancer directly induces oncogenic features

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

    doi: 10.1073/pnas.1406619111

    Truncated O-glycans affect the biology of human pancreatic cancer. ( A ) Depiction of common biosynthetic pathways of mucin-type O-glycans illustrating truncated O-glycan structures associated with cancer (boxed) and the key regulatory role the core 1 synthase (C1GalT1) and its private chaperone COSMC play in O-glycan extension. Deleterious mutations in COSMC result in proteasomal degradation of the core 1 synthase and expression of Tn and STn truncated O-glycans. ( B ) Heat-map summary of hypermethylation of COSMC promoter in pancreatic tumor tissues from nine cases with, and nine cases without, expression of T-synthase/core 1 and truncated Tn, STn O-glycans [immunohistochemistry (IHC)], respectively. ( C , Upper ) Example of IHC expression of core 1 synthase and truncated O-glycans Tn, STn, and ST/T in hypermethylated COSMC promoter in a primary (upper row) and metastatic (lower row) pancreas tumor. ( Lower ) Examples of hypomethylated COSMC promoter in a primary (upper row) and metastatic (lower row) pancreas tumor. ( D ) Migratory ( Left ) and invasive ( Right ) properties of T3M4 wild-type, COSMC KO, and COSMC reexpression (CR) cells quantified following transwell migration ( n = 3) and invasion through matrigel ( n = 3). Significantly increased migration and invasion were observed in KO cells compared with wild-type and COSMC reexpresseion (CR) cells. ( E ) Significantly increased pancreatic tumor volume (mm 3 ) ( Left ) and tumor weight ( Right ) in KO cells ( n = 15) compared with wild-type ( n = 10) and COSMC reexpression (CR) ( n = 15) cells with orthotopic T3M4 tumor cell implantation. ( F ) IHC demonstrating high expression of STn in T3M4 COSMC KO implanted mouse pancreas tumor tissues and reduced expression in implanted T3M4 wild-type and CR cells.
    Figure Legend Snippet: Truncated O-glycans affect the biology of human pancreatic cancer. ( A ) Depiction of common biosynthetic pathways of mucin-type O-glycans illustrating truncated O-glycan structures associated with cancer (boxed) and the key regulatory role the core 1 synthase (C1GalT1) and its private chaperone COSMC play in O-glycan extension. Deleterious mutations in COSMC result in proteasomal degradation of the core 1 synthase and expression of Tn and STn truncated O-glycans. ( B ) Heat-map summary of hypermethylation of COSMC promoter in pancreatic tumor tissues from nine cases with, and nine cases without, expression of T-synthase/core 1 and truncated Tn, STn O-glycans [immunohistochemistry (IHC)], respectively. ( C , Upper ) Example of IHC expression of core 1 synthase and truncated O-glycans Tn, STn, and ST/T in hypermethylated COSMC promoter in a primary (upper row) and metastatic (lower row) pancreas tumor. ( Lower ) Examples of hypomethylated COSMC promoter in a primary (upper row) and metastatic (lower row) pancreas tumor. ( D ) Migratory ( Left ) and invasive ( Right ) properties of T3M4 wild-type, COSMC KO, and COSMC reexpression (CR) cells quantified following transwell migration ( n = 3) and invasion through matrigel ( n = 3). Significantly increased migration and invasion were observed in KO cells compared with wild-type and COSMC reexpresseion (CR) cells. ( E ) Significantly increased pancreatic tumor volume (mm 3 ) ( Left ) and tumor weight ( Right ) in KO cells ( n = 15) compared with wild-type ( n = 10) and COSMC reexpression (CR) ( n = 15) cells with orthotopic T3M4 tumor cell implantation. ( F ) IHC demonstrating high expression of STn in T3M4 COSMC KO implanted mouse pancreas tumor tissues and reduced expression in implanted T3M4 wild-type and CR cells.

    Techniques Used: Expressing, Immunohistochemistry, Migration

    22) Product Images from "ESCRT-dependent targeting of plasma membrane localized KCa3.1 to the lysosomes"

    Article Title: ESCRT-dependent targeting of plasma membrane localized KCa3.1 to the lysosomes

    Journal: American Journal of Physiology - Cell Physiology

    doi: 10.1152/ajpcell.00120.2010

    Membrane Ca 2+ -activated K + channel KCa3.1 is targeted to the lysosomes for degradation. A : cells were transfected with biotin ligase acceptor peptide (BLAP)-KCa3.1, and the channel at the cell surface was labeled with streptavidin-Alexa 488 in human embryonic kidney (HEK) cells ( top ) and streptavidin-Alexa 555 in human microvascular endothelial cells (HMEC-1) ( bottom ). The fate of the channel inside the cells was addressed by immunofluorescence (IF) upon incubation at 37°C for the indicated periods of time. At time 0 , the channel is localized at the plasma membrane. After 1 h at 37°C, nearly all the channel is endocytosed in both HEK and HMEC-1 cells. Following 5 h at 37°C, the endocytosed KCa3.1 is extensively degraded in both cell types, as evidenced by the reduced fluorescence signal. Nuclei were labeled with DAPI (blue). HEK cells are shown as single confocal sections, while HMEC-1 cells are shown as projection images from multiple z-sections. B : the endocytosed BLAP-KCa3.1 (labeled in red) colocalizes with the lysosomal marker lysosome-associated membrane protein 2 (Lamp2; labeled in green) in both HEK and HMEC-1 cells. A confocal optical section imaged through a representative cell is shown. C : ultrastructural analysis using transmission electron microscopy confirms that membrane KCa3.1 is delivered to the lysosomes, following endocytosis. D : cells were transfected with BLAP-KCa3.1 and the channel at the cell surface was labeled as in A . Cells were incubated for 5 h at 37°C either in the presence or absence of lysosomal protease inhibitors leupeptin (100 μM)/pepstatin (1 μg/ml; L/P). At time 0 , the channel is localized at the plasma membrane. At the end of the incubation period, the endocytosed KCa3.1 is extensively degraded in control cells, whereas in cells treated with L/P, this process is inhibited, as shown by the strong fluorescent signal still present. E : degradation rate for plasma membrane localized BLAP-KCa3.1 was evaluated in HEK cells by specifically biotinylating the channel using recombinant biotin ligase (BirA). Subsequently, the channel was labeled with streptavidin and the cells were incubated at 37°C for 0, 1, 3, 5, 8, or 12 h in the presence or absence of L/P. Representative blots are shown at left ; 20 μg protein was loaded per lane. The data were quantified by densitometry and are plotted as shown ( n = 3; * P
    Figure Legend Snippet: Membrane Ca 2+ -activated K + channel KCa3.1 is targeted to the lysosomes for degradation. A : cells were transfected with biotin ligase acceptor peptide (BLAP)-KCa3.1, and the channel at the cell surface was labeled with streptavidin-Alexa 488 in human embryonic kidney (HEK) cells ( top ) and streptavidin-Alexa 555 in human microvascular endothelial cells (HMEC-1) ( bottom ). The fate of the channel inside the cells was addressed by immunofluorescence (IF) upon incubation at 37°C for the indicated periods of time. At time 0 , the channel is localized at the plasma membrane. After 1 h at 37°C, nearly all the channel is endocytosed in both HEK and HMEC-1 cells. Following 5 h at 37°C, the endocytosed KCa3.1 is extensively degraded in both cell types, as evidenced by the reduced fluorescence signal. Nuclei were labeled with DAPI (blue). HEK cells are shown as single confocal sections, while HMEC-1 cells are shown as projection images from multiple z-sections. B : the endocytosed BLAP-KCa3.1 (labeled in red) colocalizes with the lysosomal marker lysosome-associated membrane protein 2 (Lamp2; labeled in green) in both HEK and HMEC-1 cells. A confocal optical section imaged through a representative cell is shown. C : ultrastructural analysis using transmission electron microscopy confirms that membrane KCa3.1 is delivered to the lysosomes, following endocytosis. D : cells were transfected with BLAP-KCa3.1 and the channel at the cell surface was labeled as in A . Cells were incubated for 5 h at 37°C either in the presence or absence of lysosomal protease inhibitors leupeptin (100 μM)/pepstatin (1 μg/ml; L/P). At time 0 , the channel is localized at the plasma membrane. At the end of the incubation period, the endocytosed KCa3.1 is extensively degraded in control cells, whereas in cells treated with L/P, this process is inhibited, as shown by the strong fluorescent signal still present. E : degradation rate for plasma membrane localized BLAP-KCa3.1 was evaluated in HEK cells by specifically biotinylating the channel using recombinant biotin ligase (BirA). Subsequently, the channel was labeled with streptavidin and the cells were incubated at 37°C for 0, 1, 3, 5, 8, or 12 h in the presence or absence of L/P. Representative blots are shown at left ; 20 μg protein was loaded per lane. The data were quantified by densitometry and are plotted as shown ( n = 3; * P

    Techniques Used: Transfection, Labeling, Immunofluorescence, Incubation, Fluorescence, Marker, Transmission Assay, Electron Microscopy, Recombinant

    23) Product Images from "Construction of human liver cancer vascular endothelium cDNA expression library and screening of the endothelium-associated antigen genes"

    Article Title: Construction of human liver cancer vascular endothelium cDNA expression library and screening of the endothelium-associated antigen genes

    Journal: World Journal of Gastroenterology

    doi: 10.3748/wjg.v10.i10.1402

    Immunofluorescence analysis of endothelial cells stained with the sera diluted 1:5000 isolated from the mice immunized with HLCVECs. A: HLCVECs; B: Activated HUVECs; C: HUVECs.
    Figure Legend Snippet: Immunofluorescence analysis of endothelial cells stained with the sera diluted 1:5000 isolated from the mice immunized with HLCVECs. A: HLCVECs; B: Activated HUVECs; C: HUVECs.

    Techniques Used: Immunofluorescence, Staining, Isolation, Mouse Assay

    24) 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

    25) Product Images from "Sulindac inhibits tumor cell invasion by suppressing NF-?B mediated transcription of microRNAs"

    Article Title: Sulindac inhibits tumor cell invasion by suppressing NF-?B mediated transcription of microRNAs

    Journal: Oncogene

    doi: 10.1038/onc.2011.655

    SS prevents the translocation of NF-κB through inhibiting the phosphorylation of IKKβ and IκB (A)The Western blot assay showed the phosphorylation of IKKβ is decreased in both MDA-MB-231 and HCT116 cells in response to SS treatment. (B) The Western blot assay showed that the decline of phosphorylated IκBα versus the accumulation of IκBα when MDA-MB-231 and HCT116 cells were treated by SS. TNFα (25 ng/ml for 20 min) was used to stimulated the expression of nuclear NF-κB. (C) NF-κB immunofluorescence of MDA-MB-231 and HCT116 cells. The conditions were as follows: (1) control; (2) treatment with 50 µM SS for 12 h; (3) treatment with 25 ng/ml TNFα for 20 min; and (4) both TNFα and SS treatments. The green (anti-NF-κB) indicates NF-κB distribution, and blue indicates the location of the nucleus.
    Figure Legend Snippet: SS prevents the translocation of NF-κB through inhibiting the phosphorylation of IKKβ and IκB (A)The Western blot assay showed the phosphorylation of IKKβ is decreased in both MDA-MB-231 and HCT116 cells in response to SS treatment. (B) The Western blot assay showed that the decline of phosphorylated IκBα versus the accumulation of IκBα when MDA-MB-231 and HCT116 cells were treated by SS. TNFα (25 ng/ml for 20 min) was used to stimulated the expression of nuclear NF-κB. (C) NF-κB immunofluorescence of MDA-MB-231 and HCT116 cells. The conditions were as follows: (1) control; (2) treatment with 50 µM SS for 12 h; (3) treatment with 25 ng/ml TNFα for 20 min; and (4) both TNFα and SS treatments. The green (anti-NF-κB) indicates NF-κB distribution, and blue indicates the location of the nucleus.

    Techniques Used: Translocation Assay, Western Blot, Multiple Displacement Amplification, Expressing, Immunofluorescence

    Induced NF-κB regulates the expression of the selected miRNAs at the transcriptional level After being exposed to 25ng/ml TNFα for 5h or 250µM DCA for 2h, non-treated control and treated MDA-MB-231 and HCT116 cells were harvested for RNA isolation. QRT-PCR was employed for examine the relative expression of pri-miR-10b, pri-miR-17, pri-miR-21, and pri-miR-9. A p65 construct was transiently transfected into MDA-MB-231 and HCT116 cells as a positive control for NF-κB over-expression. (T-test was used for determining statistical significance; * indicates p
    Figure Legend Snippet: Induced NF-κB regulates the expression of the selected miRNAs at the transcriptional level After being exposed to 25ng/ml TNFα for 5h or 250µM DCA for 2h, non-treated control and treated MDA-MB-231 and HCT116 cells were harvested for RNA isolation. QRT-PCR was employed for examine the relative expression of pri-miR-10b, pri-miR-17, pri-miR-21, and pri-miR-9. A p65 construct was transiently transfected into MDA-MB-231 and HCT116 cells as a positive control for NF-κB over-expression. (T-test was used for determining statistical significance; * indicates p

    Techniques Used: Expressing, Multiple Displacement Amplification, Isolation, Quantitative RT-PCR, Construct, Transfection, Positive Control, Over Expression, T-Test

    NF-κB directly binds to the selected miRNAs promoters (A) Schematic of miR-10b promoter fragments containing p65 NF-κB binding sites. DNA fragments including two putative binding sequences of p65 (W1: −1078 to −1065; W2: −379 to −365) and the corresponding mutated sequences (M1 and M2) were cloned. (B) TNFα can induce the relative luciferase activity through W2 (p
    Figure Legend Snippet: NF-κB directly binds to the selected miRNAs promoters (A) Schematic of miR-10b promoter fragments containing p65 NF-κB binding sites. DNA fragments including two putative binding sequences of p65 (W1: −1078 to −1065; W2: −379 to −365) and the corresponding mutated sequences (M1 and M2) were cloned. (B) TNFα can induce the relative luciferase activity through W2 (p

    Techniques Used: Binding Assay, Luciferase, Activity Assay

    26) Product Images from "Broad-spectrum antiviral activity of chebulagic acid and punicalagin against viruses that use glycosaminoglycans for entry"

    Article Title: Broad-spectrum antiviral activity of chebulagic acid and punicalagin against viruses that use glycosaminoglycans for entry

    Journal: BMC Microbiology

    doi: 10.1186/1471-2180-13-187

    Effects of CHLA and PUG against virus binding analyzed by ELISA. Different cell monolayers were pre-chilled at 4°C for 1 h and then inoculated with the respective viruses in the presence or absence of various concentrations of test compounds at 4°C for an additional 2 h. Following the virus binding period, the cell monolayers were washed to remove unadsorbed virus, subsequently fixed with 4% PFA, and then blocked with 5% BSA. ELISA was performed with virus-specific antibodies and HRP-conjugated IgG, followed by development with a TMB substrate kit. The absorbance was immediately determined at 450 nm and values are expressed as the fold change of absorbance relative to the mock infection control (cells + DMSO), which is indicated by the dashed line. (A) Schematic of the experiment with the virus concentration (MOI) and test compound treatment time (i) indicated for each virus in the associated table. Analyses for (B) HCMV, (C) HCV, (D) DENV-2, (E) MV, and (F) RSV are indicated in each additional panel. Results shown are means ± SEM from three independent experiments. See text for details.
    Figure Legend Snippet: Effects of CHLA and PUG against virus binding analyzed by ELISA. Different cell monolayers were pre-chilled at 4°C for 1 h and then inoculated with the respective viruses in the presence or absence of various concentrations of test compounds at 4°C for an additional 2 h. Following the virus binding period, the cell monolayers were washed to remove unadsorbed virus, subsequently fixed with 4% PFA, and then blocked with 5% BSA. ELISA was performed with virus-specific antibodies and HRP-conjugated IgG, followed by development with a TMB substrate kit. The absorbance was immediately determined at 450 nm and values are expressed as the fold change of absorbance relative to the mock infection control (cells + DMSO), which is indicated by the dashed line. (A) Schematic of the experiment with the virus concentration (MOI) and test compound treatment time (i) indicated for each virus in the associated table. Analyses for (B) HCMV, (C) HCV, (D) DENV-2, (E) MV, and (F) RSV are indicated in each additional panel. Results shown are means ± SEM from three independent experiments. See text for details.

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay, Infection, Concentration Assay

    Examination of CHLA and PUG treatment on virus cell-to-cell spread. (A) Schematic of the experiment (left) with the virus concentration (PFU/well) and step-wise incubation periods (i, ii, iii) indicated for each virus in the table on the right. Virus infections were established (i) in the different cell types by direct inoculation (HCMV, DENV-2, MV, and RSV) or electroporation of viral RNA (HCV; *), and the cell monolayers were washed with citrate buffer or PBS before being covered with an overlay medium that prevents secondary infection. Initial virus plaques were allowed to form in the subsequent infections (ii), and then the test compounds were added to the overlay medium for an additional time of incubation (iii) before analysis of viral plaque size by immune fluorescence microscopy. Five random virus-positive plaques at the endpoint of the experiment were evaluated for each treatment group of viruses, and the data was plotted as “fold change of plaque area” against the size of the initial viral plaques formed prior to test compound treatment. Analyses for (B) HCMV, (C) HCV, (D) DENV-2, (E) MV, and (F) RSV are indicated in each additional panel. The S29 cell line and the FIP inhibitor were included as controls for HCV and MV, respectively. Results shown are means ± SEM from three independent experiments and representative micrographs of the evaluated plaques are provided in Additional file 1 Figure S1, Additional file 2 Figure S2, Additional file 3 Figure S3, Additional file 4 Figure S4 and Additional file 5 Figure S5. See text for details.
    Figure Legend Snippet: Examination of CHLA and PUG treatment on virus cell-to-cell spread. (A) Schematic of the experiment (left) with the virus concentration (PFU/well) and step-wise incubation periods (i, ii, iii) indicated for each virus in the table on the right. Virus infections were established (i) in the different cell types by direct inoculation (HCMV, DENV-2, MV, and RSV) or electroporation of viral RNA (HCV; *), and the cell monolayers were washed with citrate buffer or PBS before being covered with an overlay medium that prevents secondary infection. Initial virus plaques were allowed to form in the subsequent infections (ii), and then the test compounds were added to the overlay medium for an additional time of incubation (iii) before analysis of viral plaque size by immune fluorescence microscopy. Five random virus-positive plaques at the endpoint of the experiment were evaluated for each treatment group of viruses, and the data was plotted as “fold change of plaque area” against the size of the initial viral plaques formed prior to test compound treatment. Analyses for (B) HCMV, (C) HCV, (D) DENV-2, (E) MV, and (F) RSV are indicated in each additional panel. The S29 cell line and the FIP inhibitor were included as controls for HCV and MV, respectively. Results shown are means ± SEM from three independent experiments and representative micrographs of the evaluated plaques are provided in Additional file 1 Figure S1, Additional file 2 Figure S2, Additional file 3 Figure S3, Additional file 4 Figure S4 and Additional file 5 Figure S5. See text for details.

    Techniques Used: Concentration Assay, Incubation, Electroporation, Infection, Fluorescence, Microscopy

    Dose response of CHLA and PUG treatments against multiple viruses. Host cells for each virus (HEL for HCMV; Huh-7.5 for HCV; Vero for DENV-2, CHO-SLAM for MV; HEp-2 for RSV, and A549 for VSV and ADV-5) were co-treated with viral inoculum and increasing concentrations of test compounds for 1 – 3 h before being washed, incubated, and analyzed for virus infection by plaque assays, EGFP expression analysis, or luciferase assay as described in Methods. (A) Schematic of the experiment (shown on the left) with the virus concentration (PFU/well or MOI), co-treatment time (i), and the subsequent viral incubation period (ii) indicated for each virus in the table on the right. (B) Antiviral effect of CHLA against multiple viruses. (C) Antiviral effect of PUG against multiple viruses. Results are plotted against values for the DMSO control treatment of virus infections and the data shown are means ± the standard errors of the mean (SEM) from three independent experiments. See text for details.
    Figure Legend Snippet: Dose response of CHLA and PUG treatments against multiple viruses. Host cells for each virus (HEL for HCMV; Huh-7.5 for HCV; Vero for DENV-2, CHO-SLAM for MV; HEp-2 for RSV, and A549 for VSV and ADV-5) were co-treated with viral inoculum and increasing concentrations of test compounds for 1 – 3 h before being washed, incubated, and analyzed for virus infection by plaque assays, EGFP expression analysis, or luciferase assay as described in Methods. (A) Schematic of the experiment (shown on the left) with the virus concentration (PFU/well or MOI), co-treatment time (i), and the subsequent viral incubation period (ii) indicated for each virus in the table on the right. (B) Antiviral effect of CHLA against multiple viruses. (C) Antiviral effect of PUG against multiple viruses. Results are plotted against values for the DMSO control treatment of virus infections and the data shown are means ± the standard errors of the mean (SEM) from three independent experiments. See text for details.

    Techniques Used: Incubation, Infection, Expressing, Luciferase, Concentration Assay

    Inactivation of viral infections by CHLA and PUG. Different viruses were treated with the test compounds for a long period (incubated for 1.5 – 3 h before titration; light gray bars) or short period (immediately diluted; dark gray bars) at 37°C before diluting it 50 – 100 fold to sub-therapeutic concentrations and subsequent analysis of infection on the respective host cells. (A) Schematics of the experiment (shown on the left) with the final virus concentration (PFU/well or MOI), long-term virus-drug incubation period (i), and the subsequent incubation time (ii) indicated for each virus in the table on the right. Analyses for (B) HCMV, (C) HCV, (D) DENV-2, (E) MV, and (F) RSV are indicated in each additional panel. Results are plotted against the DMSO negative control treatment for virus infection and the data shown are the means ± SEM from three independent experiments. See text for details.
    Figure Legend Snippet: Inactivation of viral infections by CHLA and PUG. Different viruses were treated with the test compounds for a long period (incubated for 1.5 – 3 h before titration; light gray bars) or short period (immediately diluted; dark gray bars) at 37°C before diluting it 50 – 100 fold to sub-therapeutic concentrations and subsequent analysis of infection on the respective host cells. (A) Schematics of the experiment (shown on the left) with the final virus concentration (PFU/well or MOI), long-term virus-drug incubation period (i), and the subsequent incubation time (ii) indicated for each virus in the table on the right. Analyses for (B) HCMV, (C) HCV, (D) DENV-2, (E) MV, and (F) RSV are indicated in each additional panel. Results are plotted against the DMSO negative control treatment for virus infection and the data shown are the means ± SEM from three independent experiments. See text for details.

    Techniques Used: Incubation, Titration, Infection, Concentration Assay, Negative Control

    Evaluation of antiviral activities of CHLA and PUG that affect virus attachment and penetration. (A) Schematics of the experiments with the virus concentration (PFU/well or MOI) and the time of addition and treatment with tannins (i, ii, iii) for each virus in the associated tables. In virus attachment analysis by Method 1 (light gray bars), monolayers of different cell types were pre-chilled at 4°C for 1 h, and then co-treated with the respective viruses and test compounds at 4°C (1.5 – 3 h; i) before washing off the inoculates and test compounds for subsequent incubation (37°C; ii) and examination of virus infection. In virus penetration analysis (dark gray bars), seeded cell monolayers were pre-chilled at 4°C for 1 h and then challenged with the respective viruses at 4°C for 1.5 – 3 h (i). Cells were then washed and treated with the test compounds for an additional incubation period (ii) during which the temperature was shifted to 37°C to facilitate viral penetration. At the end of the incubation, extracellular viruses were removed by either citrate buffer (pH 3.0) or PBS washes and the cells were further incubated (iii) for analysis of virus infection. Results for (B) HCMV, (C) HCV, (D) DENV-2, (E) MV, and (F) RSV are indicated in each additional panel. Data are plotted against the DMSO negative control treatment of virus infection and are presented as means ± SEM from three independent experiments. See text for details.
    Figure Legend Snippet: Evaluation of antiviral activities of CHLA and PUG that affect virus attachment and penetration. (A) Schematics of the experiments with the virus concentration (PFU/well or MOI) and the time of addition and treatment with tannins (i, ii, iii) for each virus in the associated tables. In virus attachment analysis by Method 1 (light gray bars), monolayers of different cell types were pre-chilled at 4°C for 1 h, and then co-treated with the respective viruses and test compounds at 4°C (1.5 – 3 h; i) before washing off the inoculates and test compounds for subsequent incubation (37°C; ii) and examination of virus infection. In virus penetration analysis (dark gray bars), seeded cell monolayers were pre-chilled at 4°C for 1 h and then challenged with the respective viruses at 4°C for 1.5 – 3 h (i). Cells were then washed and treated with the test compounds for an additional incubation period (ii) during which the temperature was shifted to 37°C to facilitate viral penetration. At the end of the incubation, extracellular viruses were removed by either citrate buffer (pH 3.0) or PBS washes and the cells were further incubated (iii) for analysis of virus infection. Results for (B) HCMV, (C) HCV, (D) DENV-2, (E) MV, and (F) RSV are indicated in each additional panel. Data are plotted against the DMSO negative control treatment of virus infection and are presented as means ± SEM from three independent experiments. See text for details.

    Techniques Used: Concentration Assay, Incubation, Infection, Negative Control

    Post-infection analysis of antiviral effects due to CHLA and PUG. Cell monolayers were inoculated with the respective viruses at 37°C to allow viral entry, then washed by citrate buffer or PBS to remove extracellular viruses, and subsequently incubated in the presence or absence of the test compounds for infection analysis. (A) Schematic of the experiment (left) with the virus concentration (PFU/well or MOI), virus infection time (i), and test compound treatment period post-infection (ii) indicated for each virus in the table shown on the right. Results for (B) HCMV, (C) HCV, (D) DENV-2, (E) MV, and (F) RSV are indicated in each additional panel. IFN-α treatment was included as control for HCV infection. Data shown are means ± SEM from three independent experiments. See text for details.
    Figure Legend Snippet: Post-infection analysis of antiviral effects due to CHLA and PUG. Cell monolayers were inoculated with the respective viruses at 37°C to allow viral entry, then washed by citrate buffer or PBS to remove extracellular viruses, and subsequently incubated in the presence or absence of the test compounds for infection analysis. (A) Schematic of the experiment (left) with the virus concentration (PFU/well or MOI), virus infection time (i), and test compound treatment period post-infection (ii) indicated for each virus in the table shown on the right. Results for (B) HCMV, (C) HCV, (D) DENV-2, (E) MV, and (F) RSV are indicated in each additional panel. IFN-α treatment was included as control for HCV infection. Data shown are means ± SEM from three independent experiments. See text for details.

    Techniques Used: Infection, Incubation, Concentration Assay

    27) Product Images from "Protection of Recombinant Mammalian Antibodies from Development-Dependent Proteolysis in Leaves of Nicotiana benthamiana"

    Article Title: Protection of Recombinant Mammalian Antibodies from Development-Dependent Proteolysis in Leaves of Nicotiana benthamiana

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0070203

    Major proteolytic activities in N. benthamiana leaves. Plants are represented as schematic diagrams where the area of each circle represents the weight of the corresponding leaf. Colour intensities indicate cathepsin L (A) and cathepsin D/E-like (B) activity levels detected for each leaf, relative to the maximum value obtained. Inner green circles represent whole-leaf extract activity, outer blue circles apoplastic extract activity. Each value is the mean of three independent activity values assayed using protease family-specific fluorogenic peptide substrates (fluorescence units/s.µg protein). Samples were taken at 6 dpi from plants infiltrated with Agrobacteria containing an empty vector, from plants infiltrated with Agrobacteria containing the C5-1 and P19 expression vectors, or from uninfiltrated plants of the same age.
    Figure Legend Snippet: Major proteolytic activities in N. benthamiana leaves. Plants are represented as schematic diagrams where the area of each circle represents the weight of the corresponding leaf. Colour intensities indicate cathepsin L (A) and cathepsin D/E-like (B) activity levels detected for each leaf, relative to the maximum value obtained. Inner green circles represent whole-leaf extract activity, outer blue circles apoplastic extract activity. Each value is the mean of three independent activity values assayed using protease family-specific fluorogenic peptide substrates (fluorescence units/s.µg protein). Samples were taken at 6 dpi from plants infiltrated with Agrobacteria containing an empty vector, from plants infiltrated with Agrobacteria containing the C5-1 and P19 expression vectors, or from uninfiltrated plants of the same age.

    Techniques Used: Activity Assay, Fluorescence, Plasmid Preparation, Expressing

    Binary vectors for C5-1, Sl CYS8 and Sl CDI expression. The constructs were devised for targeting the proteins to the apoplast. Each construct included a duplicated version of the CaMV 35S constitutive promoter (arrow), the TEV enhancer sequence (TEV), the respective protein-coding sequences ( Sl CYS8, Sl CDI, H and L for C5-1 heavy and light chains) and a CaMV 35S terminator (35S) or nopaline synthase terminator (nos) sequence. Constructs for Sl CYS8 secretion included the coding sequence of alfalfa protein disulphide isomerase signal peptide (PDI). Constructs for C5-1 and Sl CDI included the native signal peptide (SP) of these proteins.
    Figure Legend Snippet: Binary vectors for C5-1, Sl CYS8 and Sl CDI expression. The constructs were devised for targeting the proteins to the apoplast. Each construct included a duplicated version of the CaMV 35S constitutive promoter (arrow), the TEV enhancer sequence (TEV), the respective protein-coding sequences ( Sl CYS8, Sl CDI, H and L for C5-1 heavy and light chains) and a CaMV 35S terminator (35S) or nopaline synthase terminator (nos) sequence. Constructs for Sl CYS8 secretion included the coding sequence of alfalfa protein disulphide isomerase signal peptide (PDI). Constructs for C5-1 and Sl CDI included the native signal peptide (SP) of these proteins.

    Techniques Used: Expressing, Construct, Sequencing

    Time-course accumulation of biologically active C5-1 expressed alone or along with Sl CYS8. Samples (from Leaf 2, Leaf 3 and Leaf 4) were collected at 2, 4, 6, 8, 10 and 12 dpi from leaf tissue transiently expressing C5-1 alone or along with Sl CYS8. (A) The relative activity of C5-1 was measured by ELISA on a total soluble protein basis. Each value is the mean of three independent (biological) replicates ± SE. (B) Immunodetection of Sl CYS8 at different time points following SDS-PAGE of leaf proteins under reducing conditions. The cystatin was detected with polyclonal primary antibodies raised in rabbit and an alkaline phosphatase-conjugated goat anti-rabbit secondary antibody. EV, mock infiltration with an empty vector.
    Figure Legend Snippet: Time-course accumulation of biologically active C5-1 expressed alone or along with Sl CYS8. Samples (from Leaf 2, Leaf 3 and Leaf 4) were collected at 2, 4, 6, 8, 10 and 12 dpi from leaf tissue transiently expressing C5-1 alone or along with Sl CYS8. (A) The relative activity of C5-1 was measured by ELISA on a total soluble protein basis. Each value is the mean of three independent (biological) replicates ± SE. (B) Immunodetection of Sl CYS8 at different time points following SDS-PAGE of leaf proteins under reducing conditions. The cystatin was detected with polyclonal primary antibodies raised in rabbit and an alkaline phosphatase-conjugated goat anti-rabbit secondary antibody. EV, mock infiltration with an empty vector.

    Techniques Used: Expressing, Activity Assay, Enzyme-linked Immunosorbent Assay, Immunodetection, SDS Page, Plasmid Preparation

    Effect of co-expressed Sl CYS8 or Sl CDI on C5-1 accumulation. (A) Plants are represented as schematic diagrams where the area of each circle represents the weight of the corresponding leaf. Levels of grey are proportional to the amount of C5-1 on a total soluble protein basis, as determined by quantitative ELISA with a murine IgG standard and normalised to the maximum amount obtained. Leaves were infiltrated with the C5-1 expression vector alone or along with Sl CYS8 or Sl CDI expression vectors. Each value is the mean of three independent (biological) replicates. Numbers below each plant indicate mean antibody production level for the whole plant. (B) Immunodetection of C5-1 at 6 dpi in Leaf 1, Leaf 3, Leaf 5 and Leaf 7, expressed either alone or along with Sl CYS8 or Sl CDI. The proteins were detected with alkaline phosphatase-conjugated goat anti-mouse IgG, after SDS-PAGE under non-reducing conditions. (C) Immunodetection of Sl CYS8 and Sl CDI at 6 dpi in Leaves 1 to 7, following SDS-PAGE under reducing conditions. Sl CYS8 was detected with polyclonal primary antibodies raised in rabbit, Sl CDI with IgY primary antibodies raised in chicken. The primary antibodies were detected with appropriate alkaline phosphatase-conjugated secondary antibodies. EV, mock infiltration with an empty vector.
    Figure Legend Snippet: Effect of co-expressed Sl CYS8 or Sl CDI on C5-1 accumulation. (A) Plants are represented as schematic diagrams where the area of each circle represents the weight of the corresponding leaf. Levels of grey are proportional to the amount of C5-1 on a total soluble protein basis, as determined by quantitative ELISA with a murine IgG standard and normalised to the maximum amount obtained. Leaves were infiltrated with the C5-1 expression vector alone or along with Sl CYS8 or Sl CDI expression vectors. Each value is the mean of three independent (biological) replicates. Numbers below each plant indicate mean antibody production level for the whole plant. (B) Immunodetection of C5-1 at 6 dpi in Leaf 1, Leaf 3, Leaf 5 and Leaf 7, expressed either alone or along with Sl CYS8 or Sl CDI. The proteins were detected with alkaline phosphatase-conjugated goat anti-mouse IgG, after SDS-PAGE under non-reducing conditions. (C) Immunodetection of Sl CYS8 and Sl CDI at 6 dpi in Leaves 1 to 7, following SDS-PAGE under reducing conditions. Sl CYS8 was detected with polyclonal primary antibodies raised in rabbit, Sl CDI with IgY primary antibodies raised in chicken. The primary antibodies were detected with appropriate alkaline phosphatase-conjugated secondary antibodies. EV, mock infiltration with an empty vector.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Expressing, Plasmid Preparation, Immunodetection, SDS Page

    Biological activity of C5-1 expressed alone or along with Sl CYS8 or Sl CDI. (A) Plants are represented as schematic diagrams, where the area of each circle represents the weight of the corresponding leaf. Levels of grey represent relative C5-1 activities (absorbance units) on a total soluble protein basis, as measured by ELISA and normalised to the maximum amount obtained. Each value is the mean of three biological replicates. (B) Immunodetection of C5-1 activity at 6 dpi in Leaf 1, Leaf 3, Leaf 5 and Leaf 7, with the recombinant antibody expressed alone or along with Sl CYS8 or Sl CDI. Antibody activity was inferred from binding of human IgG following electroblotting of non-reduced SDS-PAGE-separated proteins onto nitrocellulose sheets. The human IgG antigen of C5-1 was then detected with a goat anti-human conjugated with alkaline phosphatase. EV, mock infiltration with an empty vector.
    Figure Legend Snippet: Biological activity of C5-1 expressed alone or along with Sl CYS8 or Sl CDI. (A) Plants are represented as schematic diagrams, where the area of each circle represents the weight of the corresponding leaf. Levels of grey represent relative C5-1 activities (absorbance units) on a total soluble protein basis, as measured by ELISA and normalised to the maximum amount obtained. Each value is the mean of three biological replicates. (B) Immunodetection of C5-1 activity at 6 dpi in Leaf 1, Leaf 3, Leaf 5 and Leaf 7, with the recombinant antibody expressed alone or along with Sl CYS8 or Sl CDI. Antibody activity was inferred from binding of human IgG following electroblotting of non-reduced SDS-PAGE-separated proteins onto nitrocellulose sheets. The human IgG antigen of C5-1 was then detected with a goat anti-human conjugated with alkaline phosphatase. EV, mock infiltration with an empty vector.

    Techniques Used: Activity Assay, Enzyme-linked Immunosorbent Assay, Immunodetection, Recombinant, Binding Assay, SDS Page, Plasmid Preparation

    28) Product Images from "IgA is Important for Clearance and Critical for Protection from Rotavirus Infection"

    Article Title: IgA is Important for Clearance and Critical for Protection from Rotavirus Infection

    Journal: Mucosal immunology

    doi: 10.1038/mi.2012.51

    IgA is not critical for clearance of rotavirus antigenemia Mice were orally inoculated with 10 3 ID 50 of EC wt rotavirus on day 0. Serum was collected either 4 or 15 days later and analyzed for the presence of the VP6 middle capsid protein by ELISA. C57BL/6 (black bar), C57BL/6 IgA −/− (white bar), BALB/c (grey bar), and BALB/c IgA −/− (hatched bar). Each bar represents the mean OD of VP6 present in 5–6 mice + SD. *, p
    Figure Legend Snippet: IgA is not critical for clearance of rotavirus antigenemia Mice were orally inoculated with 10 3 ID 50 of EC wt rotavirus on day 0. Serum was collected either 4 or 15 days later and analyzed for the presence of the VP6 middle capsid protein by ELISA. C57BL/6 (black bar), C57BL/6 IgA −/− (white bar), BALB/c (grey bar), and BALB/c IgA −/− (hatched bar). Each bar represents the mean OD of VP6 present in 5–6 mice + SD. *, p

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

    IgA is important for clearance of rotavirus infection C57BL/6 (A) and BALB/c (B) mice were orally inoculated with 10 3 ID 50 of EC wt rotavirus on day 0. Fecal pellets were collected every other day and analyzed for the presence of the VP6 middle capsid protein by ELISA. Each symbol represents the OD of VP6 present in an individual wild type (black circles) or IgA −/− (grey triangles) mouse. The mean OD values for the groups are indicated for wild type (solid line) and IgA −/− (dotted line) mice. Horizontal line indicates limit of detection of the assay. C, the mean number of time points antigen was detected in fecal samples for each group. Black bar, wild type; grey bar, IgA −/− . Each bar represents the mean for each group+SD (n=10–12 mice/group). *, p
    Figure Legend Snippet: IgA is important for clearance of rotavirus infection C57BL/6 (A) and BALB/c (B) mice were orally inoculated with 10 3 ID 50 of EC wt rotavirus on day 0. Fecal pellets were collected every other day and analyzed for the presence of the VP6 middle capsid protein by ELISA. Each symbol represents the OD of VP6 present in an individual wild type (black circles) or IgA −/− (grey triangles) mouse. The mean OD values for the groups are indicated for wild type (solid line) and IgA −/− (dotted line) mice. Horizontal line indicates limit of detection of the assay. C, the mean number of time points antigen was detected in fecal samples for each group. Black bar, wild type; grey bar, IgA −/− . Each bar represents the mean for each group+SD (n=10–12 mice/group). *, p

    Techniques Used: Infection, Mouse Assay, Enzyme-linked Immunosorbent Assay

    IgA is required for protection from a secondary rotavirus infection C57BL/6 (A) and BALB/c (B) wild type or IgA −/− mice were orally inoculated with 10 3 ID 50 of EC wt rotavirus and received a secondary inoculation with 10 3 ID 50 EC wt either six weeks or twelve weeks later. Fecal pellets were collected every other day and analyzed for the presence of the VP6 middle capsid protein by ELISA. Each symbol represents the OD obtained from an individual wild type mouse (black circles) or IgA −/− mouse at six weeks (black squares) or 12 weeks (grey triangles). The mean antigen OD values for the groups are indicated for wild type (solid line) and IgA −/− at either six weeks (large dotted line) or twelve weeks (small dotted line). Horizontal line indicates limit of detection of the assay. C, the mean number of time points antigen was detected in fecal samples for each group. White bar, wild type mice, six or twelve week challenge (no antigen shedding at either time point); black bar, IgA −/− mice, six week challenge; grey bar, IgA −/− mice, twelve week challenge. Each bar represents the mean for each group+SD (n=5–6 mice/group). *, p
    Figure Legend Snippet: IgA is required for protection from a secondary rotavirus infection C57BL/6 (A) and BALB/c (B) wild type or IgA −/− mice were orally inoculated with 10 3 ID 50 of EC wt rotavirus and received a secondary inoculation with 10 3 ID 50 EC wt either six weeks or twelve weeks later. Fecal pellets were collected every other day and analyzed for the presence of the VP6 middle capsid protein by ELISA. Each symbol represents the OD obtained from an individual wild type mouse (black circles) or IgA −/− mouse at six weeks (black squares) or 12 weeks (grey triangles). The mean antigen OD values for the groups are indicated for wild type (solid line) and IgA −/− at either six weeks (large dotted line) or twelve weeks (small dotted line). Horizontal line indicates limit of detection of the assay. C, the mean number of time points antigen was detected in fecal samples for each group. White bar, wild type mice, six or twelve week challenge (no antigen shedding at either time point); black bar, IgA −/− mice, six week challenge; grey bar, IgA −/− mice, twelve week challenge. Each bar represents the mean for each group+SD (n=5–6 mice/group). *, p

    Techniques Used: Infection, Mouse Assay, Enzyme-linked Immunosorbent Assay

    Mice lacking IgA are susceptible to multiple infections with rotavirus Naïve C57BL/6 IgA −/− (A) or BALB/c IgA −/− (B) mice were orally inoculated with 10 3 ID 50 of EC wt rotavirus (mean value, solid line) followed by an identical second inoculation six weeks (mean value, large dotted line) and third inoculation twelve weeks later (mean value, small dotted line). Fecal pellets were collected every other day and analyzed for the presence of the VP6 middle capsid protein by ELISA. Each symbol is the OD, representative of the amount of VP6 present in a sample from an individual IgA −/− mouse following the first (black circles), second (black squares, six weeks), and third (grey triangles, 12 weeks) inoculations. The mean antigen OD values for the groups are indicated following first (solid line), second (large dotted line), or third (small dotted line) inoculation. Horizontal line indicates the limit of detection of the assay. C, the mean number of time points antigen was detected in fecal samples for each group. White bars, initial infection, black bars, six week inoculation, grey bars, twelve week inoculation. Each bar represents the mean for each group+SD (n=5–6 mice per group). *, p
    Figure Legend Snippet: Mice lacking IgA are susceptible to multiple infections with rotavirus Naïve C57BL/6 IgA −/− (A) or BALB/c IgA −/− (B) mice were orally inoculated with 10 3 ID 50 of EC wt rotavirus (mean value, solid line) followed by an identical second inoculation six weeks (mean value, large dotted line) and third inoculation twelve weeks later (mean value, small dotted line). Fecal pellets were collected every other day and analyzed for the presence of the VP6 middle capsid protein by ELISA. Each symbol is the OD, representative of the amount of VP6 present in a sample from an individual IgA −/− mouse following the first (black circles), second (black squares, six weeks), and third (grey triangles, 12 weeks) inoculations. The mean antigen OD values for the groups are indicated following first (solid line), second (large dotted line), or third (small dotted line) inoculation. Horizontal line indicates the limit of detection of the assay. C, the mean number of time points antigen was detected in fecal samples for each group. White bars, initial infection, black bars, six week inoculation, grey bars, twelve week inoculation. Each bar represents the mean for each group+SD (n=5–6 mice per group). *, p

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Infection

    Mice lacking IgA do not develop detectable compensatory rotavirus-specific IgG or IgM fecal antibody Mice were orally inoculated with 10 3 ID 50 of EC wt rotavirus on day 0. Fecal pellets were analyzed for the presence of rotavirus specific total antibody (IgA, IgM, and IgG) 15 days following viral exposure by ELISA. Each bar represents the geometric mean titer 4–6 mice + SD. *, p
    Figure Legend Snippet: Mice lacking IgA do not develop detectable compensatory rotavirus-specific IgG or IgM fecal antibody Mice were orally inoculated with 10 3 ID 50 of EC wt rotavirus on day 0. Fecal pellets were analyzed for the presence of rotavirus specific total antibody (IgA, IgM, and IgG) 15 days following viral exposure by ELISA. Each bar represents the geometric mean titer 4–6 mice + SD. *, p

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

    Induction of fecal IgA coincides with clearance of rotavirus infection in mice C57BL/6 (A) and BALB/c mice (B) mice were orally inoculated with 10 3 ID 50 of EC wt rotavirus on day 0. Fecal pellets were collected every day and analyzed for the presence of the VP6 middle capsid protein by ELISA (mean values shown by solid line) or the presence of rotavirus-specific IgA (mean value shown by dotted line). Each symbol represents the OD of either antigen (black circles) or antibody (grey squares) present in the stool of an individual mouse. Horizontal line indicates limit of detection of the assay.
    Figure Legend Snippet: Induction of fecal IgA coincides with clearance of rotavirus infection in mice C57BL/6 (A) and BALB/c mice (B) mice were orally inoculated with 10 3 ID 50 of EC wt rotavirus on day 0. Fecal pellets were collected every day and analyzed for the presence of the VP6 middle capsid protein by ELISA (mean values shown by solid line) or the presence of rotavirus-specific IgA (mean value shown by dotted line). Each symbol represents the OD of either antigen (black circles) or antibody (grey squares) present in the stool of an individual mouse. Horizontal line indicates limit of detection of the assay.

    Techniques Used: Infection, Mouse Assay, Enzyme-linked Immunosorbent Assay

    29) Product Images from "Angiopoietin-Like 2 Promotes Atherogenesis in Mice"

    Article Title: Angiopoietin-Like 2 Promotes Atherogenesis in Mice

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

    doi: 10.1161/JAHA.113.000201

    Angptl2 plasma levels and angptl2 expression in atherosclerotic plaque increase with age and atherosclerosis. A, Samples corresponding to equal amounts of total protein were collected from plasma in wild‐type (WT; n=4) and LDLr −/− ; hApoB +/+ (ATX; n=4) mice at 3, 6, and 12 months of age. Low abundant proteins in the plasma samples were enriched using Bio‐Rad ProteoMiner protein enrichment kits. Following enrichment, the samples were resolved on SDS‐PAGE, transferred to nitrocellulose membranes, and probed with an antibody against angptl2. Uniform protein loading and transfer were verified by staining membranes with Ponceau red. Results (arbitrary units, AU) are presented as the mean±SEM of 4 mice in each group. * P
    Figure Legend Snippet: Angptl2 plasma levels and angptl2 expression in atherosclerotic plaque increase with age and atherosclerosis. A, Samples corresponding to equal amounts of total protein were collected from plasma in wild‐type (WT; n=4) and LDLr −/− ; hApoB +/+ (ATX; n=4) mice at 3, 6, and 12 months of age. Low abundant proteins in the plasma samples were enriched using Bio‐Rad ProteoMiner protein enrichment kits. Following enrichment, the samples were resolved on SDS‐PAGE, transferred to nitrocellulose membranes, and probed with an antibody against angptl2. Uniform protein loading and transfer were verified by staining membranes with Ponceau red. Results (arbitrary units, AU) are presented as the mean±SEM of 4 mice in each group. * P

    Techniques Used: Expressing, Mouse Assay, Protein Enrichment, SDS Page, Staining

    Plasma angptl2 levels are higher in CAD patients than in healthy volunteers. Circulating angptl2 levels were quantified in CAD patients (n=11) and in age‐matched healthy volunteers (n=6) by ELISA. Data are presented as a dot plot. * P
    Figure Legend Snippet: Plasma angptl2 levels are higher in CAD patients than in healthy volunteers. Circulating angptl2 levels were quantified in CAD patients (n=11) and in age‐matched healthy volunteers (n=6) by ELISA. Data are presented as a dot plot. * P

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Angptl2 stimulates leukocyte adhesion to the native endothelium in ATX but not WT mice. A, Expression of P‐selectin and ICAM‐1 mRNA in freshly isolated aortic ECs from WT (n=7) and LDLr −/− ; hApoB +/+ (ATX; n=6) mice was quantified by quantitative RT‐PCR and normalized to cyclophilin A after stimulation or not with purified recombinant angptl2 (100 nmol/L). Data are mean±SEM of n mice. * P
    Figure Legend Snippet: Angptl2 stimulates leukocyte adhesion to the native endothelium in ATX but not WT mice. A, Expression of P‐selectin and ICAM‐1 mRNA in freshly isolated aortic ECs from WT (n=7) and LDLr −/− ; hApoB +/+ (ATX; n=6) mice was quantified by quantitative RT‐PCR and normalized to cyclophilin A after stimulation or not with purified recombinant angptl2 (100 nmol/L). Data are mean±SEM of n mice. * P

    Techniques Used: Mouse Assay, Expressing, Isolation, Quantitative RT-PCR, Purification, Recombinant

    Angplt2 mRNA abundance and cell surface expression of cell adhesion molecules in leukocytes from both WT and ATX mice. Basal and angptl2‐induced mRNA expression of (A) CD18, (B) CD62L, and (C) CD162 in leukocytes from 3‐month‐old WT (n=6) and ATX (n=6) mice, were quantified by quantitative RT‐PCR and normalized by cyclophilin A. Cell surface protein expression of (D) CD18, (E) CD62L, and (F) CD162 was quantified in control and angptl2‐treated WT (n=6) and ATX (n=6) leukocytes by flow cytometry. Leukocytes were labeled using monoclonal anti‐CD18, anti‐CD62, and anti‐CD162L antibodies or with corresponding isotype‐matched IgG (data not shown). Data are mean±SEM of n mice. * P
    Figure Legend Snippet: Angplt2 mRNA abundance and cell surface expression of cell adhesion molecules in leukocytes from both WT and ATX mice. Basal and angptl2‐induced mRNA expression of (A) CD18, (B) CD62L, and (C) CD162 in leukocytes from 3‐month‐old WT (n=6) and ATX (n=6) mice, were quantified by quantitative RT‐PCR and normalized by cyclophilin A. Cell surface protein expression of (D) CD18, (E) CD62L, and (F) CD162 was quantified in control and angptl2‐treated WT (n=6) and ATX (n=6) leukocytes by flow cytometry. Leukocytes were labeled using monoclonal anti‐CD18, anti‐CD62, and anti‐CD162L antibodies or with corresponding isotype‐matched IgG (data not shown). Data are mean±SEM of n mice. * P

    Techniques Used: Expressing, Mouse Assay, Quantitative RT-PCR, Flow Cytometry, Cytometry, Labeling

    Aortic angptl2 immunofluorescence increases with age and the progression of atherosclerosis, with marked accumulation within atherosclerotic plaque. Immunofluorescence was used to visualize angptl2 in (A) fresh longitudinally opened aortas and (B) frozen aortic sections of WT and LDLr −/− ; hApoB +/+ (ATX) mice at 6 and 12 months of age. A, z‐stacks were acquired, deconvolved, and 3D images rendered. Angptl2 accumulates in plaque and in media. Angptl2 levels are shown in red and basal membrane in green. Nuclei are shown in blue. C, Double‐immunostaining of angptl2 (red) and CD‐31 (green) in frozen aortic sections from 6‐month‐old ATX mice, showing that angptl2 colocalizes with the endothelial marker CD‐31. Angptl2 indicates angiopoietin like‐2; WT, wild type; LDL, low‐density lipoprotein.
    Figure Legend Snippet: Aortic angptl2 immunofluorescence increases with age and the progression of atherosclerosis, with marked accumulation within atherosclerotic plaque. Immunofluorescence was used to visualize angptl2 in (A) fresh longitudinally opened aortas and (B) frozen aortic sections of WT and LDLr −/− ; hApoB +/+ (ATX) mice at 6 and 12 months of age. A, z‐stacks were acquired, deconvolved, and 3D images rendered. Angptl2 accumulates in plaque and in media. Angptl2 levels are shown in red and basal membrane in green. Nuclei are shown in blue. C, Double‐immunostaining of angptl2 (red) and CD‐31 (green) in frozen aortic sections from 6‐month‐old ATX mice, showing that angptl2 colocalizes with the endothelial marker CD‐31. Angptl2 indicates angiopoietin like‐2; WT, wild type; LDL, low‐density lipoprotein.

    Techniques Used: Immunofluorescence, Mouse Assay, Double Immunostaining, Marker

    Chronic angptl2 infusion for 1 month ATX mice accelerates atherogenesis in 3‐month‐old ATX mice. A, Infusion with angptl2 promotes the expression of inflammatory cytokines and adhesion molecules in freshly scraped ECs from the aorta evaluated by quantitative RT‐PCR and normalized by cyclophilin A. B, angptl2 accelerates the formation of atherosclerotic plaque. C, angptl2 increases total cholesterol and LDL plasma levels. Data are mean±SEM of n=5 angptl2‐infused mice and n=5 TBSE‐infused mice. * P
    Figure Legend Snippet: Chronic angptl2 infusion for 1 month ATX mice accelerates atherogenesis in 3‐month‐old ATX mice. A, Infusion with angptl2 promotes the expression of inflammatory cytokines and adhesion molecules in freshly scraped ECs from the aorta evaluated by quantitative RT‐PCR and normalized by cyclophilin A. B, angptl2 accelerates the formation of atherosclerotic plaque. C, angptl2 increases total cholesterol and LDL plasma levels. Data are mean±SEM of n=5 angptl2‐infused mice and n=5 TBSE‐infused mice. * P

    Techniques Used: Mouse Assay, Expressing, Quantitative RT-PCR

    Expression and secretion of angptl2 is greater in ECs than in VSMCs, but angptl2 binding is higher in VSMCs than in ECs. A, Western blot analysis of endogenous angptl2 secreted into the culture medium overnight (16 h) by hIMAECs, HUVECs, and VSMCs. Angptl2 protein expression was also quantified in the cell lysate. B, Endogenous angptl2 expression in the cells was detected by immunofluorescence using a confocal microscope. Scale bar represents 20 μm. C, Quantitative RT‐PCR analysis of angptl2 mRNA expression was performed in the cell lysate from cultured cells (n=3 hIMAECs, n=3 HUVECs, n=3 VSMCs). Results were normalized to GAPDH expression, and average gene expression in hIMAECs was arbitrarily set at 1. Data are mean±SEM of n=3; each experiment was performed in duplicate. * P
    Figure Legend Snippet: Expression and secretion of angptl2 is greater in ECs than in VSMCs, but angptl2 binding is higher in VSMCs than in ECs. A, Western blot analysis of endogenous angptl2 secreted into the culture medium overnight (16 h) by hIMAECs, HUVECs, and VSMCs. Angptl2 protein expression was also quantified in the cell lysate. B, Endogenous angptl2 expression in the cells was detected by immunofluorescence using a confocal microscope. Scale bar represents 20 μm. C, Quantitative RT‐PCR analysis of angptl2 mRNA expression was performed in the cell lysate from cultured cells (n=3 hIMAECs, n=3 HUVECs, n=3 VSMCs). Results were normalized to GAPDH expression, and average gene expression in hIMAECs was arbitrarily set at 1. Data are mean±SEM of n=3; each experiment was performed in duplicate. * P

    Techniques Used: Expressing, Binding Assay, Western Blot, Immunofluorescence, Microscopy, Quantitative RT-PCR, Cell Culture

    Angptl2 stimulates the expression of inflammatory markers in ECs. Quantitative RT‐PCR analysis of expression of TNF‐α mRNA (a) and IL‐6 mRNA (b) was performed in ECs freshly extracted from aortas of 3‐month‐old WT and LDLr −/− ; hApoB +/+ (ATX) mice stimulated and unstimulated (control) with purified recombinant angptl2 (100 nmol/L). Results were normalized to cyclophilin A (CycloA) expression, and average gene expression level in WT control cells was arbitrarily set at 1. Data are mean±SEM of n=4 WT and n=4 ATX mice; each experiment was performed in triplicate. * P
    Figure Legend Snippet: Angptl2 stimulates the expression of inflammatory markers in ECs. Quantitative RT‐PCR analysis of expression of TNF‐α mRNA (a) and IL‐6 mRNA (b) was performed in ECs freshly extracted from aortas of 3‐month‐old WT and LDLr −/− ; hApoB +/+ (ATX) mice stimulated and unstimulated (control) with purified recombinant angptl2 (100 nmol/L). Results were normalized to cyclophilin A (CycloA) expression, and average gene expression level in WT control cells was arbitrarily set at 1. Data are mean±SEM of n=4 WT and n=4 ATX mice; each experiment was performed in triplicate. * P

    Techniques Used: Expressing, Quantitative RT-PCR, Mouse Assay, Purification, Recombinant

    30) Product Images from "Inhibition of the HCV Core Protein on the Immune Response to HBV Surface Antigen and on HBV Gene Expression and Replication In Vivo"

    Article Title: Inhibition of the HCV Core Protein on the Immune Response to HBV Surface Antigen and on HBV Gene Expression and Replication In Vivo

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0045146

    Co-application of pC191 at the different sites cannot prevent the priming of immune responses to HBsAg. BALB/c mice (n = 6 for each group) were immunized three times by in vivo electroporation with different plasmids at different leg. Sera of immunized mice were collected on the 10 th day after second and third immunizations, serially diluted, and testd by ELISA assay. (A) Total HBsAb IgG responses. Splenocytes were collected from mice at day 10 after the third immunization and subjected to ELISPOT assay. (B) Splenocytes were re-stimulated with an HBsAg peptide (H-2L d CTL epitope aa 29–38).HBsAg specific IFN-producing cells were determined by identified by spot formation. The numbers represent the means of spot-forming cells per 2×10 5 splenocytes. The error bars represent the standard deviation. Statistically significant differences between the groups are displayed as *(p
    Figure Legend Snippet: Co-application of pC191 at the different sites cannot prevent the priming of immune responses to HBsAg. BALB/c mice (n = 6 for each group) were immunized three times by in vivo electroporation with different plasmids at different leg. Sera of immunized mice were collected on the 10 th day after second and third immunizations, serially diluted, and testd by ELISA assay. (A) Total HBsAb IgG responses. Splenocytes were collected from mice at day 10 after the third immunization and subjected to ELISPOT assay. (B) Splenocytes were re-stimulated with an HBsAg peptide (H-2L d CTL epitope aa 29–38).HBsAg specific IFN-producing cells were determined by identified by spot formation. The numbers represent the means of spot-forming cells per 2×10 5 splenocytes. The error bars represent the standard deviation. Statistically significant differences between the groups are displayed as *(p

    Techniques Used: Mouse Assay, In Vivo, Electroporation, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, CTL Assay, Standard Deviation

    The co-administration of pC191 with pHBsAg delayed HBsAg and HBV DNA clearance in immunized mice after HI. BALB/c (H-2 d ) mice (n = 6 for each group) were immunized three times by in vivo electroporation with different plasmid combinations and then challenged with pAAV/HBV1.3 by tail vein HI on day 5 after the third immunization. Sera were collected at the indicated time points for HBsAg, HBsAb, and HBV DNA detection. (A) The positive rate of serum HBsAg in mice of each group. (B) The kinetic of the HBsAg titres. (C) The kinetics of the total HBsAb IgG titres. (D) The kinetics of HBV DNA detection in mice. The results shown represent the average of all of the mice.
    Figure Legend Snippet: The co-administration of pC191 with pHBsAg delayed HBsAg and HBV DNA clearance in immunized mice after HI. BALB/c (H-2 d ) mice (n = 6 for each group) were immunized three times by in vivo electroporation with different plasmid combinations and then challenged with pAAV/HBV1.3 by tail vein HI on day 5 after the third immunization. Sera were collected at the indicated time points for HBsAg, HBsAb, and HBV DNA detection. (A) The positive rate of serum HBsAg in mice of each group. (B) The kinetic of the HBsAg titres. (C) The kinetics of the total HBsAb IgG titres. (D) The kinetics of HBV DNA detection in mice. The results shown represent the average of all of the mice.

    Techniques Used: Mouse Assay, In Vivo, Electroporation, Plasmid Preparation

    Co-administration of pC191 and rHBsAg cannot prevent priming of immune responses to recombinant HBsAg. BALB/c mice (n = 6 for each group) were immunised three times by in vivo electroporation with different combinations of plasmid and/or recombinant HBsAg (rHBsAg). Sera of immunized mice were collected on the 10th day after the first or second immunisation boost and were serially diluted and titred by ELISA. (A) Total HBsAb IgG. (B) HBsAb IgG subclasses IgG1 and IgG2a. (C) Splenocytes were collected from mice at day 10 after the third immunization and subjected to ELISPOT assay. Splenocytes were re-stimulated with an HBsAg peptide (H-2L d CTL epitope aa 29–38). HBsAg specific IFN-producing cells were determined by identified by spot formation. The numbers represent the means of spot-forming cells per 2×10 5 splenocytes. The error bars represent the standard deviation. Statistically significant differences between the boost and the boosts relative to the prime in each group are displayed as *(p
    Figure Legend Snippet: Co-administration of pC191 and rHBsAg cannot prevent priming of immune responses to recombinant HBsAg. BALB/c mice (n = 6 for each group) were immunised three times by in vivo electroporation with different combinations of plasmid and/or recombinant HBsAg (rHBsAg). Sera of immunized mice were collected on the 10th day after the first or second immunisation boost and were serially diluted and titred by ELISA. (A) Total HBsAb IgG. (B) HBsAb IgG subclasses IgG1 and IgG2a. (C) Splenocytes were collected from mice at day 10 after the third immunization and subjected to ELISPOT assay. Splenocytes were re-stimulated with an HBsAg peptide (H-2L d CTL epitope aa 29–38). HBsAg specific IFN-producing cells were determined by identified by spot formation. The numbers represent the means of spot-forming cells per 2×10 5 splenocytes. The error bars represent the standard deviation. Statistically significant differences between the boost and the boosts relative to the prime in each group are displayed as *(p

    Techniques Used: Recombinant, Mouse Assay, In Vivo, Electroporation, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, CTL Assay, Standard Deviation

    Co-application of pC191 inhibited the effective boosting of HBsAg-specific immune response. BALB/c mice (n = 6 for each group) were immunized three times by in vivo electroporation with different doses of the plasmids pCI, pHBsAg, pC191, or combinations as indicated in (A). Sera of immunized mice were collected on 10th days after the third immunization, serially diluted, and tested by ELISA assay. (B) Total HBsAb IgG responses. (C) HBsAb IgG subclasses IgG1 and IgG2a. (D) Ratios of HBsAb IgG2a/IgG1. (E) Splenocytes were collected from mice at day 10 after the third immunization and subjected to ELISPOT assay. Splenocytes were re-stimulated with a HBsAg peptide (H-2L d CTL epitope aa 29–38). HBsAg specific IFN-producing cells were determined by identified by spot formation. The numbers represent the means of spot-forming cells per 2×10 5 splenocytes. The error bars represent the standard deviation. Statistically significant differences between the groups are displayed as *(p
    Figure Legend Snippet: Co-application of pC191 inhibited the effective boosting of HBsAg-specific immune response. BALB/c mice (n = 6 for each group) were immunized three times by in vivo electroporation with different doses of the plasmids pCI, pHBsAg, pC191, or combinations as indicated in (A). Sera of immunized mice were collected on 10th days after the third immunization, serially diluted, and tested by ELISA assay. (B) Total HBsAb IgG responses. (C) HBsAb IgG subclasses IgG1 and IgG2a. (D) Ratios of HBsAb IgG2a/IgG1. (E) Splenocytes were collected from mice at day 10 after the third immunization and subjected to ELISPOT assay. Splenocytes were re-stimulated with a HBsAg peptide (H-2L d CTL epitope aa 29–38). HBsAg specific IFN-producing cells were determined by identified by spot formation. The numbers represent the means of spot-forming cells per 2×10 5 splenocytes. The error bars represent the standard deviation. Statistically significant differences between the groups are displayed as *(p

    Techniques Used: Mouse Assay, In Vivo, Electroporation, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, CTL Assay, Standard Deviation

    Co-administration of pC191 and pAAV/HBV1.3 by HI reduced HBV replication and gene expression in vivo. BALB/c (H-2 d ) mice (n = 12 for each group) were subjected to HI with different plasmids combinations. Sera were collected and subjected to HBsAg and HBV DNA detection at the indicated time points. The kinetics of HBsAb total IgG and IgG subclasse responses after HI in BALB/c (H-2 d ) mice were tested by ELISA assay. (A) Positive rate of serum HBsAg in the mice from each group. (B) The kinetics of the HBsAg titer after HI. (C) The kinetics of HBV DNA detection in the mice. The kinetics of the total HBsAb IgG (D), IgG1 (E) and IgG2a (F) titres after HI. The results shown represent the average of all of the mice. The error bars represent the standard deviations. Statistically significant differences between the groups are displayed as *(p
    Figure Legend Snippet: Co-administration of pC191 and pAAV/HBV1.3 by HI reduced HBV replication and gene expression in vivo. BALB/c (H-2 d ) mice (n = 12 for each group) were subjected to HI with different plasmids combinations. Sera were collected and subjected to HBsAg and HBV DNA detection at the indicated time points. The kinetics of HBsAb total IgG and IgG subclasse responses after HI in BALB/c (H-2 d ) mice were tested by ELISA assay. (A) Positive rate of serum HBsAg in the mice from each group. (B) The kinetics of the HBsAg titer after HI. (C) The kinetics of HBV DNA detection in the mice. The kinetics of the total HBsAb IgG (D), IgG1 (E) and IgG2a (F) titres after HI. The results shown represent the average of all of the mice. The error bars represent the standard deviations. Statistically significant differences between the groups are displayed as *(p

    Techniques Used: Expressing, In Vivo, Mouse Assay, Enzyme-linked Immunosorbent Assay, Hi-C

    The HCV core reduced HBsAg levels and promoted the HBsAb antibody response in a mouse model of persistent HBV replication. C57BL/6 (H-2 b ) mice (n = 12 for each group) were challenged with different plasmids combinations by tail vein HI. Sera were collected at indicated time points, serially diluted, and tested by ELISA assay.(A) Positive rate of serum HBsAg in the mice from each group. (B) The kinetics of the HBsAg titres after HI. (C) Positive rate of serum HBsAb in the mice from each group. (D) The kinetics of the total HBsAb IgG titres after HI. The error bars represent the standard deviation. Statistically significant differences between the groups are displayed as *(p
    Figure Legend Snippet: The HCV core reduced HBsAg levels and promoted the HBsAb antibody response in a mouse model of persistent HBV replication. C57BL/6 (H-2 b ) mice (n = 12 for each group) were challenged with different plasmids combinations by tail vein HI. Sera were collected at indicated time points, serially diluted, and tested by ELISA assay.(A) Positive rate of serum HBsAg in the mice from each group. (B) The kinetics of the HBsAg titres after HI. (C) Positive rate of serum HBsAb in the mice from each group. (D) The kinetics of the total HBsAb IgG titres after HI. The error bars represent the standard deviation. Statistically significant differences between the groups are displayed as *(p

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Hi-C, Standard Deviation

    Co-administration of pC191 and pHBsAg prevented effective priming of immune responses to HBsAg. BALB/c mice (n = 6 for each group) were immunised three times with different concentrations and different combinations of the pHBsAg, pCI-neo (pCI), pC191, or pC145 constructs by in vivo electroporation. Sera from 6 mice per group were collected on the 10 th day after the first or second immunisation boost and were serially diluted and titred by ELISA. (A) Total HBsAb IgG. (B) HBsAb IgG subclasses IgG1 and IgG2a. (C) Ratios of HBsAb IgG2a/IgG1. (D) Splenocytes were collected at day 10 after the third immunization and subjected to ELISPOT assay. Splenocytes were re-stimulated with an HBsAg peptide (H-2L d CTL epitope aa 29–38). HBsAg specific IFN-producing cells were determined by identified by spot formation. The numbers represent the means of spot-forming cells per 2×10 5 splenocytes. The error bars represent the standard deviation. Statistically significant differences between the groups are displayed as *(p
    Figure Legend Snippet: Co-administration of pC191 and pHBsAg prevented effective priming of immune responses to HBsAg. BALB/c mice (n = 6 for each group) were immunised three times with different concentrations and different combinations of the pHBsAg, pCI-neo (pCI), pC191, or pC145 constructs by in vivo electroporation. Sera from 6 mice per group were collected on the 10 th day after the first or second immunisation boost and were serially diluted and titred by ELISA. (A) Total HBsAb IgG. (B) HBsAb IgG subclasses IgG1 and IgG2a. (C) Ratios of HBsAb IgG2a/IgG1. (D) Splenocytes were collected at day 10 after the third immunization and subjected to ELISPOT assay. Splenocytes were re-stimulated with an HBsAg peptide (H-2L d CTL epitope aa 29–38). HBsAg specific IFN-producing cells were determined by identified by spot formation. The numbers represent the means of spot-forming cells per 2×10 5 splenocytes. The error bars represent the standard deviation. Statistically significant differences between the groups are displayed as *(p

    Techniques Used: Mouse Assay, Construct, In Vivo, Electroporation, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, CTL Assay, Standard Deviation

    31) Product Images from "Intramembrane proteolysis mediates shedding of a key adhesin during erythrocyte invasion by the malaria parasite"

    Article Title: Intramembrane proteolysis mediates shedding of a key adhesin during erythrocyte invasion by the malaria parasite

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200604136

    EBA-175 is shed at or around the point of erythrocyte invasion and retains region VI. (A) Schematic of EBA-175 structure, indicating signal sequence (SS), F1 and F2 DBL domains, TMD, and cytoplasmic domain (CYT). The position of region VI is shown, with a Coomassie blue–stained SDS-PAGE gel of purified recombinant region VI (inset). (B) Western blot of 3D7 schizonts, merozoites, and culture supernatant, run on nonreducing 7.5% SDS-PAGE and probed with anti–region VI antibodies. Shed EBA-175 (arrow) migrates more rapidly than the parasite forms. Molecular mass markers are indicated. (C) IFA images of an acetone-fixed segmented 3D7 schizont and free merozoites dual labeled after fixation with mAb 1E1 (anti-MSP1 19 ; red) and anti–region VI antibodies (green). The punctate, apical pattern obtained with the latter is typical of microneme staining. (D) EBA-175 is discharged onto the apical surface of free merozoites. Merozoites released from 3D7 schizonts in the presence of anti–region VI antibodies were washed, fixed, and probed with FITC anti-mouse IgG to detect bound anti–region VI antibodies (green), followed by mAb 1E1 (anti-MSP1 19 ; red). Many free merozoites exhibit a strong apical FITC signal (thin arrows), whereas residual intact schizonts containing merozoites that were not accessible to the anti–region VI antibodies show only background FITC labeling (thick arrows). No signal was associated with merozoites released in the presence of preimmune mouse antisera (unpublished data). (E) IFA images of newly invaded (≤3-h-old) ring-stage 3D7 parasites probed with anti–region VI antibodies after acetone fixation. Most rings did not react at all with the antibodies. Nuclei were stained with DAPI (blue). Identical results were obtained with the W2mef clone (unpublished data). Bars, 5 μm.
    Figure Legend Snippet: EBA-175 is shed at or around the point of erythrocyte invasion and retains region VI. (A) Schematic of EBA-175 structure, indicating signal sequence (SS), F1 and F2 DBL domains, TMD, and cytoplasmic domain (CYT). The position of region VI is shown, with a Coomassie blue–stained SDS-PAGE gel of purified recombinant region VI (inset). (B) Western blot of 3D7 schizonts, merozoites, and culture supernatant, run on nonreducing 7.5% SDS-PAGE and probed with anti–region VI antibodies. Shed EBA-175 (arrow) migrates more rapidly than the parasite forms. Molecular mass markers are indicated. (C) IFA images of an acetone-fixed segmented 3D7 schizont and free merozoites dual labeled after fixation with mAb 1E1 (anti-MSP1 19 ; red) and anti–region VI antibodies (green). The punctate, apical pattern obtained with the latter is typical of microneme staining. (D) EBA-175 is discharged onto the apical surface of free merozoites. Merozoites released from 3D7 schizonts in the presence of anti–region VI antibodies were washed, fixed, and probed with FITC anti-mouse IgG to detect bound anti–region VI antibodies (green), followed by mAb 1E1 (anti-MSP1 19 ; red). Many free merozoites exhibit a strong apical FITC signal (thin arrows), whereas residual intact schizonts containing merozoites that were not accessible to the anti–region VI antibodies show only background FITC labeling (thick arrows). No signal was associated with merozoites released in the presence of preimmune mouse antisera (unpublished data). (E) IFA images of newly invaded (≤3-h-old) ring-stage 3D7 parasites probed with anti–region VI antibodies after acetone fixation. Most rings did not react at all with the antibodies. Nuclei were stained with DAPI (blue). Identical results were obtained with the W2mef clone (unpublished data). Bars, 5 μm.

    Techniques Used: Sequencing, Staining, SDS Page, Purification, Recombinant, Western Blot, Immunofluorescence, Labeling

    32) 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

    33) Product Images from "Response of Mice and Ferrets to a Monovalent Influenza A (H7N9) Split Vaccine"

    Article Title: Response of Mice and Ferrets to a Monovalent Influenza A (H7N9) Split Vaccine

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0099322

    Antibody responses to the H7N9/PR8 split vaccine in mice. Groups of 20 BALB/c mice were immunized intramuscularly at weeks 0 and 2 with 7.5, 15, 30, or 45 µg (HA levels) of the H7N9/PR8 split vaccine. (A) HI antibody responses to the (wt) AnHui virus after vaccination as described above. Serum samples were collected on day 0; 2 weeks after priming; and 2, 4, and 8 weeks after boosting. (B) Serum IgG titers against the (wt) AnHui virus measured 2 weeks after both the first and second doses of vaccine. (C) The ratios of serum IgG1 to IgG2a titers against the (wt) AnHui virus, calculated 2 weeks after the second dose of vaccine. The data are presented as means ± SDs of the data from three experiments, each performed in duplicate. HI, hemagglutination inhibition; PBS, phosphate-buffered saline. 2WPD1: 2 weeks post-vaccination with dose 1. 2WPD2: 2 weeks post-vaccination with dose 2; 4WPD2: 4 weeks post-vaccination with dose 2; 8WPD2: 8 weeks post-vaccination with dose 2.
    Figure Legend Snippet: Antibody responses to the H7N9/PR8 split vaccine in mice. Groups of 20 BALB/c mice were immunized intramuscularly at weeks 0 and 2 with 7.5, 15, 30, or 45 µg (HA levels) of the H7N9/PR8 split vaccine. (A) HI antibody responses to the (wt) AnHui virus after vaccination as described above. Serum samples were collected on day 0; 2 weeks after priming; and 2, 4, and 8 weeks after boosting. (B) Serum IgG titers against the (wt) AnHui virus measured 2 weeks after both the first and second doses of vaccine. (C) The ratios of serum IgG1 to IgG2a titers against the (wt) AnHui virus, calculated 2 weeks after the second dose of vaccine. The data are presented as means ± SDs of the data from three experiments, each performed in duplicate. HI, hemagglutination inhibition; PBS, phosphate-buffered saline. 2WPD1: 2 weeks post-vaccination with dose 1. 2WPD2: 2 weeks post-vaccination with dose 2; 4WPD2: 4 weeks post-vaccination with dose 2; 8WPD2: 8 weeks post-vaccination with dose 2.

    Techniques Used: Mouse Assay, HI Assay

    34) Product Images from "Schwann-like cells seeded in acellular nerve grafts improve nerve regeneration"

    Article Title: Schwann-like cells seeded in acellular nerve grafts improve nerve regeneration

    Journal: BMC Musculoskeletal Disorders

    doi: 10.1186/1471-2474-15-165

    S-100 and VEGF immunostaining and IOD value analyses at 12 weeks postoperatively. S-100 immunostaining ( A-C , scale bar = 10 μm) and VEGF immunostaining ( D-F , scale bar = 20 μm) at 12 weeks postoperatively with SLCs (A and D) , BM-MSCs (B and E) and isografts (C and F) . The IOD values of the positive expression of S-100 and VEGF in the regenerated nerves (G) . The isograft and ANG + SLC groups showed a significantly higher level in both S-100 and VEGF immunostaining compared with the ANG + BM-MSC group. The data were shown as the mean ± SEM. * P
    Figure Legend Snippet: S-100 and VEGF immunostaining and IOD value analyses at 12 weeks postoperatively. S-100 immunostaining ( A-C , scale bar = 10 μm) and VEGF immunostaining ( D-F , scale bar = 20 μm) at 12 weeks postoperatively with SLCs (A and D) , BM-MSCs (B and E) and isografts (C and F) . The IOD values of the positive expression of S-100 and VEGF in the regenerated nerves (G) . The isograft and ANG + SLC groups showed a significantly higher level in both S-100 and VEGF immunostaining compared with the ANG + BM-MSC group. The data were shown as the mean ± SEM. * P

    Techniques Used: Immunostaining, Expressing

    35) Product Images from "GPI-anchored single chain Fv - an effective way to capture transiently-exposed neutralization epitopes on HIV-1 envelope spike"

    Article Title: GPI-anchored single chain Fv - an effective way to capture transiently-exposed neutralization epitopes on HIV-1 envelope spike

    Journal: Retrovirology

    doi: 10.1186/1742-4690-7-79

    Expression of secretory and GPI-anchored scFvs in transduced TZM.bl cells . A . Schematic diagram of the lentiviral vectors pRRL-scFv/hinge/his-tag/DAF and pRRL-scFv/hinge/his-tag. Single chain Fvs (scFvs) were derived from seven human monoclonal antibodies AB31, AB32, TG15, 48d, X5 and AB65; hinge: a human IgG3 hinge region; his-tag: a 6 histidine residue tag; DAF: the C-terminal 34 amino acid residues of decay accelerating factor. B . Western blot analysis of expression of scFvs (AB31, AB32, TG15, 48d, X5 and AB65) in TZM.bl cells transduced with lentiviral vectors pRRL-scFv/hinge/his-tag/DAF and pRRL-scFv/hinge/his-tag. GPI-scFv: GPI-anchored scFv; Sec-scFv: secretory scFv; anti-his: anti-his-tag antibody. C . FACS analysis of cell surface expression of scFv/hinge/histag/DAF in mock-, scFvs (AB31, AB32, TG15, 48d, X5 and AB65)/hinge/histag/DAF- or m-scFv(TG15)-transduced TZM.bl cells with or without PI-PLC treatment.
    Figure Legend Snippet: Expression of secretory and GPI-anchored scFvs in transduced TZM.bl cells . A . Schematic diagram of the lentiviral vectors pRRL-scFv/hinge/his-tag/DAF and pRRL-scFv/hinge/his-tag. Single chain Fvs (scFvs) were derived from seven human monoclonal antibodies AB31, AB32, TG15, 48d, X5 and AB65; hinge: a human IgG3 hinge region; his-tag: a 6 histidine residue tag; DAF: the C-terminal 34 amino acid residues of decay accelerating factor. B . Western blot analysis of expression of scFvs (AB31, AB32, TG15, 48d, X5 and AB65) in TZM.bl cells transduced with lentiviral vectors pRRL-scFv/hinge/his-tag/DAF and pRRL-scFv/hinge/his-tag. GPI-scFv: GPI-anchored scFv; Sec-scFv: secretory scFv; anti-his: anti-his-tag antibody. C . FACS analysis of cell surface expression of scFv/hinge/histag/DAF in mock-, scFvs (AB31, AB32, TG15, 48d, X5 and AB65)/hinge/histag/DAF- or m-scFv(TG15)-transduced TZM.bl cells with or without PI-PLC treatment.

    Techniques Used: Expressing, Derivative Assay, Western Blot, Transduction, Size-exclusion Chromatography, FACS, Planar Chromatography

    36) 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

    37) 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

    38) 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

    39) 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

    40) 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

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    Electrophoresis:

    Article Title: Novel mouse monoclonal antibodies specifically recognize Aspergillus fumigatus galactomannan
    Article Snippet: .. Supporting information Electrophoretic and western blot analyses of monoclonal antibodies 7B8 and 8G4. (A) Coomassie blue stained 12% SDS–PAAG electrophoretic analysis of purified mAb 7B8 and mAb 8G4 in reducing conditions. (B) Western blot analysis of mAb 7B8 and mAb 8G4 fractionated by 12.5% SDS-PAAG electrophoresis in reducing conditions and developed with alkaline phosphatase conjugated anti-mouse IgG (whole molecule) goat antibody (Sigma-Aldrich, USA). .. Protein molecular marker masses, in kilodaltons, are shown at the left side of the gel. (TIF) Click here for additional data file.

    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).

    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.

    Article Title: Novel mouse monoclonal antibodies specifically recognize Aspergillus fumigatus galactomannan
    Article Snippet: .. Supporting information Electrophoretic and western blot analyses of monoclonal antibodies 7B8 and 8G4. (A) Coomassie blue stained 12% SDS–PAAG electrophoretic analysis of purified mAb 7B8 and mAb 8G4 in reducing conditions. (B) Western blot analysis of mAb 7B8 and mAb 8G4 fractionated by 12.5% SDS-PAAG electrophoresis in reducing conditions and developed with alkaline phosphatase conjugated anti-mouse IgG (whole molecule) goat antibody (Sigma-Aldrich, USA). .. Protein molecular marker masses, in kilodaltons, are shown at the left side of the gel. (TIF) Click here for additional data file.

    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].

    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).

    Article Title: Novel mouse monoclonal antibodies specifically recognize Aspergillus fumigatus galactomannan
    Article Snippet: .. Supporting information Electrophoretic and western blot analyses of monoclonal antibodies 7B8 and 8G4. (A) Coomassie blue stained 12% SDS–PAAG electrophoretic analysis of purified mAb 7B8 and mAb 8G4 in reducing conditions. (B) Western blot analysis of mAb 7B8 and mAb 8G4 fractionated by 12.5% SDS-PAAG electrophoresis in reducing conditions and developed with alkaline phosphatase conjugated anti-mouse IgG (whole molecule) goat antibody (Sigma-Aldrich, USA). .. Protein molecular marker masses, in kilodaltons, are shown at the left side of the gel. (TIF) Click here for additional data file.

    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
    AT 1 R-AT 2 R heteromer expression in brain striatal sections of Parkinson’s disease (PD) rat model. a–d <t>PLA</t> assays in striatal sections from the 6-OH-dopamine PD rat model, non-lesioned ( a ), lesioned ( b ), and lesioned <t>plus</t> chronically treated with l -DOPA and either lacking ( c ) or displaying ( d ) dyskinesias. Confocal microscopy images (stacks of 3 consecutive planes) show heteroreceptor complexes as red clusters and Hoechst-stained nuclei (blue). Scale bar: 20 μm. e Bar graph showing the percentage of red dots/cell. Data are the mean S.E.M . of counts in 9–12 different fields per animal ( n = 4 per group). One-way ANOVA followed by Bonferroni’s post-hoc multiple comparison tests were used to compare the red dots/cell values. The number of clusters ( r ) was the dependent variable and the four animal groups treatments were independent variables (*** p
    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
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    88
    Millipore goat anti mouse immunoglobulin g igg horseradish peroxidase conjugate
    Virion-specific <t>IgG</t> and neutralizing-antibody responses in the DNA primed/FI-MCMV-boosted or live virus-vaccinated mice. Mice from each group vaccinated with either pc3-Ua plus PBS plus alum (two mice per group), All-U pDNA plus FI+alum (six mice
    Goat Anti Mouse Immunoglobulin G Igg Horseradish Peroxidase Conjugate, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/goat anti mouse immunoglobulin g igg horseradish peroxidase conjugate/product/Millipore
    Average 88 stars, based on 2 article reviews
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    99
    Millipore anti mouse igg whole molecule peroxidase antibody produced in goat
    Conception and applicability of autotransporter (AT)—complement receptor ligand (CRL) fusions. ( a ) Graphical abstract. ( b ) The modification of YadA to yield YFC (YadA-FLAG-C3d) and YFP (YadA-FLAG-p28); SS: signal sequence. F: FLAG tag. ( c ) Heterologous expression of plasmid-borne YadA by Ec and Ft detected by western blot with α-YadA sera followed by horseradish peroxidase <t>(HRP)-conjugated</t> secondary Ab. Strains bearing empty vector are denoted by “-”. Arrows indicate the trimers and monomers of unmodified YadA. ( d ) Collagen-coated ELISA plates were incubated with serial dilutions of intact Ft :-, Ft :YadA, and Ft :YFC; bound bacteria were detected by α- Ft LPS Ab. ( e , f ) Whole cell lysates of Ec , Kp , and Ft containing empty vector (-) or the YFP or YFC expression vectors were probed by western blot with primary Ab specific for C3d and the FLAG epitope followed sequentially by biotinylated secondary Abs and streptavidin-HRP (SA-HRP). YFC and YFP trimers and monomers are designated with black and grey arrows. The ~20 kDa bands evident in all lanes are endogenously biotinylated bacterial proteins (annotated AccB in Ec and Ft [ 40 ]) detected by SA-HRP. ( g ) Intact bacteria as indicated were incubated in solution with α-FLAG Ab. ( h ) Intact Ft as indicated were incubated in solution with the indicated Ab. IglC is primarily a cytoplasmic Ft protein. Washed bacteria in ( g , h ) were probed for <t>IgG</t> heavy chain (HC), followed by biotinylated secondary Abs and SA-HRP.
    Anti Mouse Igg Whole Molecule Peroxidase Antibody Produced In Goat, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti mouse igg whole molecule peroxidase antibody produced in goat/product/Millipore
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    Price from $9.99 to $1999.99
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    Image Search Results


    AT 1 R-AT 2 R heteromer expression in brain striatal sections of Parkinson’s disease (PD) rat model. a–d PLA assays in striatal sections from the 6-OH-dopamine PD rat model, non-lesioned ( a ), lesioned ( b ), and lesioned plus chronically treated with l -DOPA and either lacking ( c ) or displaying ( d ) dyskinesias. Confocal microscopy images (stacks of 3 consecutive planes) show heteroreceptor complexes as red clusters and Hoechst-stained nuclei (blue). Scale bar: 20 μm. e Bar graph showing the percentage of red dots/cell. Data are the mean S.E.M . of counts in 9–12 different fields per animal ( n = 4 per group). One-way ANOVA followed by Bonferroni’s post-hoc multiple comparison tests were used to compare the red dots/cell values. The number of clusters ( r ) was the dependent variable and the four animal groups treatments were independent variables (*** p

    Journal: Journal of Neuroinflammation

    Article Title: Angiotensin AT1 and AT2 receptor heteromer expression in the hemilesioned rat model of Parkinson’s disease that increases with levodopa-induced dyskinesia

    doi: 10.1186/s12974-020-01908-z

    Figure Lengend Snippet: AT 1 R-AT 2 R heteromer expression in brain striatal sections of Parkinson’s disease (PD) rat model. a–d PLA assays in striatal sections from the 6-OH-dopamine PD rat model, non-lesioned ( a ), lesioned ( b ), and lesioned plus chronically treated with l -DOPA and either lacking ( c ) or displaying ( d ) dyskinesias. Confocal microscopy images (stacks of 3 consecutive planes) show heteroreceptor complexes as red clusters and Hoechst-stained nuclei (blue). Scale bar: 20 μm. e Bar graph showing the percentage of red dots/cell. Data are the mean S.E.M . of counts in 9–12 different fields per animal ( n = 4 per group). One-way ANOVA followed by Bonferroni’s post-hoc multiple comparison tests were used to compare the red dots/cell values. The number of clusters ( r ) was the dependent variable and the four animal groups treatments were independent variables (*** p

    Article Snippet: Cells or brain sections were further processed using the proximity ligation assays (PLA) probes detecting primary antibodies (Duolink In Situ PLA probe Anti-Mouse plus and Duolink In Situ PLA probe Anti-Rabbit minus) (1/5 v:v for 1-hour at 37 °C).

    Techniques: Expressing, Proximity Ligation Assay, Confocal Microscopy, Staining

    Virion-specific IgG and neutralizing-antibody responses in the DNA primed/FI-MCMV-boosted or live virus-vaccinated mice. Mice from each group vaccinated with either pc3-Ua plus PBS plus alum (two mice per group), All-U pDNA plus FI+alum (six mice

    Journal:

    Article Title: Systemic Priming-Boosting Immunization with a Trivalent Plasmid DNA and Inactivated Murine Cytomegalovirus (MCMV) Vaccine Provides Long-Term Protection against Viral Replication following Systemic or Mucosal MCMV Challenge

    doi: 10.1128/JVI.79.1.159-175.2005

    Figure Lengend Snippet: Virion-specific IgG and neutralizing-antibody responses in the DNA primed/FI-MCMV-boosted or live virus-vaccinated mice. Mice from each group vaccinated with either pc3-Ua plus PBS plus alum (two mice per group), All-U pDNA plus FI+alum (six mice

    Article Snippet: To detect gB, blocked blots were incubated with the monoclonal antibody 2E8.12A (a gift from Lambert Loh, University of Saskatchewan, Saskatoon, Canada), and bound antibody was detected using a goat-anti-mouse immunoglobulin G (IgG) horseradish peroxidase conjugate (Calbiochem) and enhanced chemiluminescence (Supersignal West Pico; Pierce).

    Techniques: Mouse Assay

    Virion-specific IgG and neutralizing-antibody responses in vaccinated mice. On the weeks of the experiment shown in Fig. , four to eight mice per vaccine group were retroorbitally bled and sera were prepared. Arrows and numbers indicate

    Journal:

    Article Title: Systemic Priming-Boosting Immunization with a Trivalent Plasmid DNA and Inactivated Murine Cytomegalovirus (MCMV) Vaccine Provides Long-Term Protection against Viral Replication following Systemic or Mucosal MCMV Challenge

    doi: 10.1128/JVI.79.1.159-175.2005

    Figure Lengend Snippet: Virion-specific IgG and neutralizing-antibody responses in vaccinated mice. On the weeks of the experiment shown in Fig. , four to eight mice per vaccine group were retroorbitally bled and sera were prepared. Arrows and numbers indicate

    Article Snippet: To detect gB, blocked blots were incubated with the monoclonal antibody 2E8.12A (a gift from Lambert Loh, University of Saskatchewan, Saskatoon, Canada), and bound antibody was detected using a goat-anti-mouse immunoglobulin G (IgG) horseradish peroxidase conjugate (Calbiochem) and enhanced chemiluminescence (Supersignal West Pico; Pierce).

    Techniques: Mouse Assay

    Conception and applicability of autotransporter (AT)—complement receptor ligand (CRL) fusions. ( a ) Graphical abstract. ( b ) The modification of YadA to yield YFC (YadA-FLAG-C3d) and YFP (YadA-FLAG-p28); SS: signal sequence. F: FLAG tag. ( c ) Heterologous expression of plasmid-borne YadA by Ec and Ft detected by western blot with α-YadA sera followed by horseradish peroxidase (HRP)-conjugated secondary Ab. Strains bearing empty vector are denoted by “-”. Arrows indicate the trimers and monomers of unmodified YadA. ( d ) Collagen-coated ELISA plates were incubated with serial dilutions of intact Ft :-, Ft :YadA, and Ft :YFC; bound bacteria were detected by α- Ft LPS Ab. ( e , f ) Whole cell lysates of Ec , Kp , and Ft containing empty vector (-) or the YFP or YFC expression vectors were probed by western blot with primary Ab specific for C3d and the FLAG epitope followed sequentially by biotinylated secondary Abs and streptavidin-HRP (SA-HRP). YFC and YFP trimers and monomers are designated with black and grey arrows. The ~20 kDa bands evident in all lanes are endogenously biotinylated bacterial proteins (annotated AccB in Ec and Ft [ 40 ]) detected by SA-HRP. ( g ) Intact bacteria as indicated were incubated in solution with α-FLAG Ab. ( h ) Intact Ft as indicated were incubated in solution with the indicated Ab. IglC is primarily a cytoplasmic Ft protein. Washed bacteria in ( g , h ) were probed for IgG heavy chain (HC), followed by biotinylated secondary Abs and SA-HRP.

    Journal: Pathogens

    Article Title: Autotransporter-Mediated Display of Complement Receptor Ligands by Gram-Negative Bacteria Increases Antibody Responses and Limits Disease Severity

    doi: 10.3390/pathogens9050375

    Figure Lengend Snippet: Conception and applicability of autotransporter (AT)—complement receptor ligand (CRL) fusions. ( a ) Graphical abstract. ( b ) The modification of YadA to yield YFC (YadA-FLAG-C3d) and YFP (YadA-FLAG-p28); SS: signal sequence. F: FLAG tag. ( c ) Heterologous expression of plasmid-borne YadA by Ec and Ft detected by western blot with α-YadA sera followed by horseradish peroxidase (HRP)-conjugated secondary Ab. Strains bearing empty vector are denoted by “-”. Arrows indicate the trimers and monomers of unmodified YadA. ( d ) Collagen-coated ELISA plates were incubated with serial dilutions of intact Ft :-, Ft :YadA, and Ft :YFC; bound bacteria were detected by α- Ft LPS Ab. ( e , f ) Whole cell lysates of Ec , Kp , and Ft containing empty vector (-) or the YFP or YFC expression vectors were probed by western blot with primary Ab specific for C3d and the FLAG epitope followed sequentially by biotinylated secondary Abs and streptavidin-HRP (SA-HRP). YFC and YFP trimers and monomers are designated with black and grey arrows. The ~20 kDa bands evident in all lanes are endogenously biotinylated bacterial proteins (annotated AccB in Ec and Ft [ 40 ]) detected by SA-HRP. ( g ) Intact bacteria as indicated were incubated in solution with α-FLAG Ab. ( h ) Intact Ft as indicated were incubated in solution with the indicated Ab. IglC is primarily a cytoplasmic Ft protein. Washed bacteria in ( g , h ) were probed for IgG heavy chain (HC), followed by biotinylated secondary Abs and SA-HRP.

    Article Snippet: Ft -specific Ab were detected with 1:5000 of the respective goat anti-mouse HRP-conjugated secondary, incubated in blocking buffer for 1 h at room temperature.

    Techniques: Modification, Sequencing, FLAG-tag, Expressing, Plasmid Preparation, Western Blot, Enzyme-linked Immunosorbent Assay, Incubation