anti phosphotyrosine  (Thermo Fisher)


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    Structured Review

    Thermo Fisher anti phosphotyrosine
    Immunodetection of phosphorylation and O -GlcNAcylation of OBP isoforms from S . scrofa . Each well contains a normalized quantity (5 μg) of the four HPLC fractions. HPLC-purified short-size VEG was used as negative control. Anti-P-Ser : anti-phosphoserine antibodies (1:500 dilution); Anti-P-Tyr : <t>anti-phosphotyrosine</t> antibodies (1:2,000); Anti-P-Thr : anti-phosphothreonine antibodies (1:500); RL2 (1:2,000), CTD110.6 (1:4,000). Secondary antibodies for anti-phosphorylation antibodies at 1:40,000 dilution (rabbit IgG-HRP linked whole antibodies), ECL Plus detection (11 min exposure). Secondary antibodies for O -GlcNAc detection: goat anti-mouse IgG-HRP linked (1:30,000) for RL2, and rabbit anti-mouse IgM-HRP linked (1:30,000) for CTD110.6, ECL Plus detection (30 s exposure).
    Anti Phosphotyrosine, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 2845 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti phosphotyrosine/product/Thermo Fisher
    Average 92 stars, based on 2845 article reviews
    Price from $9.99 to $1999.99
    anti phosphotyrosine - by Bioz Stars, 2020-08
    92/100 stars

    Images

    1) Product Images from "Binding Specificity of Native Odorant-Binding Protein Isoforms Is Driven by Phosphorylation and O-N-Acetylglucosaminylation in the Pig Sus scrofa"

    Article Title: Binding Specificity of Native Odorant-Binding Protein Isoforms Is Driven by Phosphorylation and O-N-Acetylglucosaminylation in the Pig Sus scrofa

    Journal: Frontiers in Endocrinology

    doi: 10.3389/fendo.2018.00816

    Immunodetection of phosphorylation and O -GlcNAcylation of OBP isoforms from S . scrofa . Each well contains a normalized quantity (5 μg) of the four HPLC fractions. HPLC-purified short-size VEG was used as negative control. Anti-P-Ser : anti-phosphoserine antibodies (1:500 dilution); Anti-P-Tyr : anti-phosphotyrosine antibodies (1:2,000); Anti-P-Thr : anti-phosphothreonine antibodies (1:500); RL2 (1:2,000), CTD110.6 (1:4,000). Secondary antibodies for anti-phosphorylation antibodies at 1:40,000 dilution (rabbit IgG-HRP linked whole antibodies), ECL Plus detection (11 min exposure). Secondary antibodies for O -GlcNAc detection: goat anti-mouse IgG-HRP linked (1:30,000) for RL2, and rabbit anti-mouse IgM-HRP linked (1:30,000) for CTD110.6, ECL Plus detection (30 s exposure).
    Figure Legend Snippet: Immunodetection of phosphorylation and O -GlcNAcylation of OBP isoforms from S . scrofa . Each well contains a normalized quantity (5 μg) of the four HPLC fractions. HPLC-purified short-size VEG was used as negative control. Anti-P-Ser : anti-phosphoserine antibodies (1:500 dilution); Anti-P-Tyr : anti-phosphotyrosine antibodies (1:2,000); Anti-P-Thr : anti-phosphothreonine antibodies (1:500); RL2 (1:2,000), CTD110.6 (1:4,000). Secondary antibodies for anti-phosphorylation antibodies at 1:40,000 dilution (rabbit IgG-HRP linked whole antibodies), ECL Plus detection (11 min exposure). Secondary antibodies for O -GlcNAc detection: goat anti-mouse IgG-HRP linked (1:30,000) for RL2, and rabbit anti-mouse IgM-HRP linked (1:30,000) for CTD110.6, ECL Plus detection (30 s exposure).

    Techniques Used: Immunodetection, High Performance Liquid Chromatography, Purification, Negative Control

    2) Product Images from "Constitutive activation of signal transducer and activator of transcription 3 regulates expression of vascular endothelial growth factor in human meningioma differentiation"

    Article Title: Constitutive activation of signal transducer and activator of transcription 3 regulates expression of vascular endothelial growth factor in human meningioma differentiation

    Journal: Journal of Cancer Research and Clinical Oncology

    doi: 10.1007/s00432-009-0743-9

    Plots of gene expression from RT-PCR and Western blot analysis in normal dura and meningioma tissues normalized to that of Beta-actin. Relative mRNA expression of JAK1, STAT3, and VEGFa in a normal dura tissue, b grade I tissue, and c grade II tissue. Relative protein expression of JAK1, p-JAK1, STAT3, p-STAT3, and VEGF in d normal dura tissue, e grade I tissue, and f grade II tissue
    Figure Legend Snippet: Plots of gene expression from RT-PCR and Western blot analysis in normal dura and meningioma tissues normalized to that of Beta-actin. Relative mRNA expression of JAK1, STAT3, and VEGFa in a normal dura tissue, b grade I tissue, and c grade II tissue. Relative protein expression of JAK1, p-JAK1, STAT3, p-STAT3, and VEGF in d normal dura tissue, e grade I tissue, and f grade II tissue

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

    Western blot analysis of protein expression of JAK1, p-JAK1, STAT3, p-STAT3, and VEGF. a Protein level of JAK1, p-JAK1, STAT3, p-STAT3, and VEGF in normal dura and tumor tissue. Beta-actin was a loading control. b Relative protein expression of JAK1, p-JAK1, STAT3, p-STAT3, and VEGF normalized to that of Beta-actin. * P
    Figure Legend Snippet: Western blot analysis of protein expression of JAK1, p-JAK1, STAT3, p-STAT3, and VEGF. a Protein level of JAK1, p-JAK1, STAT3, p-STAT3, and VEGF in normal dura and tumor tissue. Beta-actin was a loading control. b Relative protein expression of JAK1, p-JAK1, STAT3, p-STAT3, and VEGF normalized to that of Beta-actin. * P

    Techniques Used: Western Blot, Expressing

    RT–PCR analysis of mRNA expression of JAK1, STAT3, and VEGFa in normal dura and meningioma tissues. a mRNA levels of JAK1, STAT3, VEGFa. Beta-actin is a loading control. b Quantification of mRNA levels of JAK1, STAT3, and VEGFa normalized to that of Beta-actin. * P
    Figure Legend Snippet: RT–PCR analysis of mRNA expression of JAK1, STAT3, and VEGFa in normal dura and meningioma tissues. a mRNA levels of JAK1, STAT3, VEGFa. Beta-actin is a loading control. b Quantification of mRNA levels of JAK1, STAT3, and VEGFa normalized to that of Beta-actin. * P

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing

    3) Product Images from "Learning-induced and stathmin-dependent changes in microtubule stability are critical for memory and disrupted in ageing"

    Article Title: Learning-induced and stathmin-dependent changes in microtubule stability are critical for memory and disrupted in ageing

    Journal: Nature communications

    doi: 10.1038/ncomms5389

    Stathmin regulates learning-dependent dendritic transport of GluA2 ( a–c ) Immunoblot estimation of the level of GluA2 in synaptosomal ( a , n = 8 per group, pooled tissues from 3–4 mice per sample whole cell extract ( b, n = 4 per group) and microtubule ( c , n = 4 per group, pooled tissues from 3–4 mice per sample) fractions of the DG in mice injected with AAV-GFP or AAV-Stat4A-IRES-GFP in naïve and 8 h after training conditions. * p
    Figure Legend Snippet: Stathmin regulates learning-dependent dendritic transport of GluA2 ( a–c ) Immunoblot estimation of the level of GluA2 in synaptosomal ( a , n = 8 per group, pooled tissues from 3–4 mice per sample whole cell extract ( b, n = 4 per group) and microtubule ( c , n = 4 per group, pooled tissues from 3–4 mice per sample) fractions of the DG in mice injected with AAV-GFP or AAV-Stat4A-IRES-GFP in naïve and 8 h after training conditions. * p

    Techniques Used: Mouse Assay, Injection

    4) Product Images from "The CD20 homologue MS4A4 directs trafficking of KIT toward clathrin-independent endocytosis pathways and thus regulates receptor signaling and recycling"

    Article Title: The CD20 homologue MS4A4 directs trafficking of KIT toward clathrin-independent endocytosis pathways and thus regulates receptor signaling and recycling

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E14-07-1221

    Silencing MS4A4 alters KIT colocalization with caveolin-1 but not clathrin. (A) Immunofluorescence confocal microscopy of scramble shRNA–treated (left) and shMS4A4- treated (right) LAD-2 cells stained with rabbit anti-clathrin HC (secondary, AF488; green) and mouse anti-KIT (APC, red) left unstimulated or stimulated with SCF for 2 min. Scale bars, 5 μm. (B) Manders coefficient of colocalization of KIT and clathrin HC at times indicated (minutes) after SCF stimulation. (C) Immunofluorescence confocal microscopy of scramble shRNA–treated (left) and shMS4A4-treated (right) LAD-2 cells stained with rabbit anti–caveolin-1 (secondary, AF488; green) and mouse anti-KIT (APC, red) left unstimulated or stimulated with SCF for 2 min. Scale bars, 5 μm. (D) Manders coefficient of colocalization of KIT and caveolin-1 with SCF stimulation time course. For B and D, bars are mean + SEM from the volume of 16 stacks of images from two separate experiments. ** p
    Figure Legend Snippet: Silencing MS4A4 alters KIT colocalization with caveolin-1 but not clathrin. (A) Immunofluorescence confocal microscopy of scramble shRNA–treated (left) and shMS4A4- treated (right) LAD-2 cells stained with rabbit anti-clathrin HC (secondary, AF488; green) and mouse anti-KIT (APC, red) left unstimulated or stimulated with SCF for 2 min. Scale bars, 5 μm. (B) Manders coefficient of colocalization of KIT and clathrin HC at times indicated (minutes) after SCF stimulation. (C) Immunofluorescence confocal microscopy of scramble shRNA–treated (left) and shMS4A4-treated (right) LAD-2 cells stained with rabbit anti–caveolin-1 (secondary, AF488; green) and mouse anti-KIT (APC, red) left unstimulated or stimulated with SCF for 2 min. Scale bars, 5 μm. (D) Manders coefficient of colocalization of KIT and caveolin-1 with SCF stimulation time course. For B and D, bars are mean + SEM from the volume of 16 stacks of images from two separate experiments. ** p

    Techniques Used: Immunofluorescence, Confocal Microscopy, shRNA, Staining

    MS4A4 colocalizes with KIT and EEA1 in LAD-2 cells. (A) Confocal micrographs of LAD-2 cells transfected with MS4A4:EGFP chimeric protein (green) immunostained for KIT (APC) (red) in the absence (top) or after stimulation with SCF (bottom). (B) Manders coefficient of colocalization for MS4A4 and KIT. (C) Confocal micrographs of LAD-2 cells transfected with MS4A4:EGFP (left) or cells immunostained with anti-MS4A4 followed by AF488-labeled anti-mouse secondary (right). (D) Immunofluorescence confocal micrographs of LAD-2 cells immunostained for mouse anti-MS4A4 (secondary, AF488) and rabbit anti-EEA1 (secondary, AF594) in the absence of SCF (top) or with SCF stimulation for 10 min (bottom). Scale bars, 5 μm. (E) Manders coefficient of colocalization for MS4A4 and EEA1. For B and E, bars are the mean + SEM from the volume of 16 stacks of images from two separate experiments). * p
    Figure Legend Snippet: MS4A4 colocalizes with KIT and EEA1 in LAD-2 cells. (A) Confocal micrographs of LAD-2 cells transfected with MS4A4:EGFP chimeric protein (green) immunostained for KIT (APC) (red) in the absence (top) or after stimulation with SCF (bottom). (B) Manders coefficient of colocalization for MS4A4 and KIT. (C) Confocal micrographs of LAD-2 cells transfected with MS4A4:EGFP (left) or cells immunostained with anti-MS4A4 followed by AF488-labeled anti-mouse secondary (right). (D) Immunofluorescence confocal micrographs of LAD-2 cells immunostained for mouse anti-MS4A4 (secondary, AF488) and rabbit anti-EEA1 (secondary, AF594) in the absence of SCF (top) or with SCF stimulation for 10 min (bottom). Scale bars, 5 μm. (E) Manders coefficient of colocalization for MS4A4 and EEA1. For B and E, bars are the mean + SEM from the volume of 16 stacks of images from two separate experiments). * p

    Techniques Used: Transfection, Labeling, Immunofluorescence

    Silencing MS4A4 alters endocytic KIT trafficking. (A) Immunofluorescence confocal microscopy of scramble shRNA–treated (top) and shMS4A4-treated (bottom) LAD-2 cells stained with rabbit anti-EEA1 (secondary AF488; green) and mouse anti-KIT (APC; red) in the absence of SCF. White arrowhead shows occasional enlarged endosomes in shMS4A4-treated cells. Right, superimposed z -projection of the stack of images. Scale bars, 10 μm. (B) Immunofluorescence confocal microscopy after stimulation with SCF for 10 min. Yellow arrowheads show enlarged endosomes positive for KIT. Orange arrowheads show very large endosomes in shMS4A4-treated cells that are negative for KIT immunofluorescence. Scale bars, 10 μm. (C–E). Manders coefficient of colocalization of KIT and Rab5 (C), KIT and EEA1 (D), and KIT and Rab9 (E) before and after stimulation with SCF. Scramble shRNA, blue bars; shMS4A4, red bars. (Bars are mean + SEM from the volume of 16 stacks of images from two separate experiments.) (F). Endosome size from the volume of image stacks calculated using Imaris software. Two observers obtained comparable measure­ments. Scramble shRNA, blue circles; shMS4A4, red circles. For C–F, * p
    Figure Legend Snippet: Silencing MS4A4 alters endocytic KIT trafficking. (A) Immunofluorescence confocal microscopy of scramble shRNA–treated (top) and shMS4A4-treated (bottom) LAD-2 cells stained with rabbit anti-EEA1 (secondary AF488; green) and mouse anti-KIT (APC; red) in the absence of SCF. White arrowhead shows occasional enlarged endosomes in shMS4A4-treated cells. Right, superimposed z -projection of the stack of images. Scale bars, 10 μm. (B) Immunofluorescence confocal microscopy after stimulation with SCF for 10 min. Yellow arrowheads show enlarged endosomes positive for KIT. Orange arrowheads show very large endosomes in shMS4A4-treated cells that are negative for KIT immunofluorescence. Scale bars, 10 μm. (C–E). Manders coefficient of colocalization of KIT and Rab5 (C), KIT and EEA1 (D), and KIT and Rab9 (E) before and after stimulation with SCF. Scramble shRNA, blue bars; shMS4A4, red bars. (Bars are mean + SEM from the volume of 16 stacks of images from two separate experiments.) (F). Endosome size from the volume of image stacks calculated using Imaris software. Two observers obtained comparable measure­ments. Scramble shRNA, blue circles; shMS4A4, red circles. For C–F, * p

    Techniques Used: Immunofluorescence, Confocal Microscopy, shRNA, Staining, Software

    MS4A4 colocalizes preferentially with caveolin-1 over clathrin after stimulation with SCF promoting PLCγ1 phosphorylation. (A) LAD-2 human mast cells immunostained with mouse anti-MS4A4 and rabbit anti-clathrin HC, followed by anti-mouse AF488 and anti-rabbit AF594 before (top) and after SCF stimulation (bottom). No increase in colocalization was observed with stimulation. (B) Manders coefficient of colocalization of MS4A4 and clathrin HC with SCF stimulation time course. (C) LAD-2 human mast cells immunostained with mouse anti-MS4A4 and rabbit anti–caveolin-1 demonstrated an increase in colocalization with SCF stimulation (bottom) compared with untreated cells (top). Scale bars, 5 μm (A, C). (D) Manders coefficient of colocalization of MS4A4 and caveolin-1 with SCF stimulation time course. For B and D, bars are the mean + SEM from the volume of 15 stacks of images from two separate experiments. ** p
    Figure Legend Snippet: MS4A4 colocalizes preferentially with caveolin-1 over clathrin after stimulation with SCF promoting PLCγ1 phosphorylation. (A) LAD-2 human mast cells immunostained with mouse anti-MS4A4 and rabbit anti-clathrin HC, followed by anti-mouse AF488 and anti-rabbit AF594 before (top) and after SCF stimulation (bottom). No increase in colocalization was observed with stimulation. (B) Manders coefficient of colocalization of MS4A4 and clathrin HC with SCF stimulation time course. (C) LAD-2 human mast cells immunostained with mouse anti-MS4A4 and rabbit anti–caveolin-1 demonstrated an increase in colocalization with SCF stimulation (bottom) compared with untreated cells (top). Scale bars, 5 μm (A, C). (D) Manders coefficient of colocalization of MS4A4 and caveolin-1 with SCF stimulation time course. For B and D, bars are the mean + SEM from the volume of 15 stacks of images from two separate experiments. ** p

    Techniques Used:

    5) Product Images from "Human NUMB6 Induces Epithelial-Mesenchymal Transition and Enhances Breast Cancer Cells Migration and Invasion"

    Article Title: Human NUMB6 Induces Epithelial-Mesenchymal Transition and Enhances Breast Cancer Cells Migration and Invasion

    Journal: Journal of cellular biochemistry

    doi: 10.1002/jcb.25628

    Knockdown of Slug in NUMB6-GFP DB-7 cells modulates expression of MMP9 and vimentin. ( A ) NUMB6-induced MMP9 activity was decreased with Slug-siRNA treatment in DB-7 cells. MMP-9 activity was assessed by zymography in conditioned media collected after 24 hr post-transfection of NUMB4-GFP, NUMB-GFP or vector control GFP DB-7 cells with Slug-siRNA or control siRNA. ( B ) Slug knockdown reduces Mmp9 expression in NUMB6-GFP DB-7 cells. siRNA treatment with 2.5uM or 5uM Slug siRNA reduced Mmp9 mRNA levels. The mRNA levels of Mmp9 were measured by quantitative RT-PCR analysis of total RNA extracted from NUMB4-GFP, NUMB6-GFP or control vector GFP DB-7 cells treated with Slug-siRNA or control siRNA for 48hr. The mRNA levels of Mmp9 are expressed relative to β-actin transcripts. Each experiment was performed in triplicate and repeated three times. Error bars represent SEM. ( C ) Expression level of MMP9 protein was decreased in Slug-depleted NUMB6-GFP DB-7 cells. NUMB4-GFP, NUMB6-GFP and control vector GFP DB-7 cells were transfected with control siRNA or Slug 5uM siRNAs for 48hr. Expression levels of MMP9 and Slug were analyzed by immunoblotting. β-actin was used as loading control. ( D ) Decreased expression of vimentin resulted from Slug knockdown of NUMB6-GFP DB-7 cells was confirmed by immunofluorescence staining. Alexa-Fluor 594 conjugated anti-mouse IgG was used as a secondary antibody against vimentin (red). Blue is staining for nuclei. Bars=15um. ( E ) Western blot shows that vimentin protein was reduced in Slug-depleted NUMB6-GFP DB-7 cells. NUMB4-GFP, NUMB6-GFP and control vector GFP DB-7 cells were transfected with control siRNA or Slug 5μM siRNAs for 48hr. β-actin was used as loading control.
    Figure Legend Snippet: Knockdown of Slug in NUMB6-GFP DB-7 cells modulates expression of MMP9 and vimentin. ( A ) NUMB6-induced MMP9 activity was decreased with Slug-siRNA treatment in DB-7 cells. MMP-9 activity was assessed by zymography in conditioned media collected after 24 hr post-transfection of NUMB4-GFP, NUMB-GFP or vector control GFP DB-7 cells with Slug-siRNA or control siRNA. ( B ) Slug knockdown reduces Mmp9 expression in NUMB6-GFP DB-7 cells. siRNA treatment with 2.5uM or 5uM Slug siRNA reduced Mmp9 mRNA levels. The mRNA levels of Mmp9 were measured by quantitative RT-PCR analysis of total RNA extracted from NUMB4-GFP, NUMB6-GFP or control vector GFP DB-7 cells treated with Slug-siRNA or control siRNA for 48hr. The mRNA levels of Mmp9 are expressed relative to β-actin transcripts. Each experiment was performed in triplicate and repeated three times. Error bars represent SEM. ( C ) Expression level of MMP9 protein was decreased in Slug-depleted NUMB6-GFP DB-7 cells. NUMB4-GFP, NUMB6-GFP and control vector GFP DB-7 cells were transfected with control siRNA or Slug 5uM siRNAs for 48hr. Expression levels of MMP9 and Slug were analyzed by immunoblotting. β-actin was used as loading control. ( D ) Decreased expression of vimentin resulted from Slug knockdown of NUMB6-GFP DB-7 cells was confirmed by immunofluorescence staining. Alexa-Fluor 594 conjugated anti-mouse IgG was used as a secondary antibody against vimentin (red). Blue is staining for nuclei. Bars=15um. ( E ) Western blot shows that vimentin protein was reduced in Slug-depleted NUMB6-GFP DB-7 cells. NUMB4-GFP, NUMB6-GFP and control vector GFP DB-7 cells were transfected with control siRNA or Slug 5μM siRNAs for 48hr. β-actin was used as loading control.

    Techniques Used: Expressing, Activity Assay, Zymography, Transfection, Plasmid Preparation, Quantitative RT-PCR, Immunofluorescence, Staining, Western Blot

    Slug knockdown partially restores E-cadherin expression in NUMB6-GFP DB-7 cells. Western blot analysis, real-time PCR and immunostaining assays were carried out to evaluate the expression of E-cadherin in DB-7 cells overexpressing control vector with GFP or NUMB6-GFP. ( A ) Control vector GFP and Numb-6 GFP DB-7 cells were transfected with control siRNA or Slug siRNA for 48 h and then E-cadherin levels were evaluated by qPCR. NUMB6-induced loss of E-cadherin mRNA expression was recovered when cells were treated with Slug siRNA. ( B ) Control GFP, NUMB4-GFP or NUMB6-GFP DB-7 cells were transfected with control siRNA or Slug siRNA, respectively. Immunostaining was performed in 48 hours later to assess the expression levels of E-cadherin; red is Alexa Fluor-546 for E-cadherin; nuclei were stained with DAPI. Bars=15um ( C ) Control GFP, or NUMB6-GFP DB-7 cells were transfected with control siRNA or Slug siRNA, and E-cadherin expression was evaluated by Western Blot.
    Figure Legend Snippet: Slug knockdown partially restores E-cadherin expression in NUMB6-GFP DB-7 cells. Western blot analysis, real-time PCR and immunostaining assays were carried out to evaluate the expression of E-cadherin in DB-7 cells overexpressing control vector with GFP or NUMB6-GFP. ( A ) Control vector GFP and Numb-6 GFP DB-7 cells were transfected with control siRNA or Slug siRNA for 48 h and then E-cadherin levels were evaluated by qPCR. NUMB6-induced loss of E-cadherin mRNA expression was recovered when cells were treated with Slug siRNA. ( B ) Control GFP, NUMB4-GFP or NUMB6-GFP DB-7 cells were transfected with control siRNA or Slug siRNA, respectively. Immunostaining was performed in 48 hours later to assess the expression levels of E-cadherin; red is Alexa Fluor-546 for E-cadherin; nuclei were stained with DAPI. Bars=15um ( C ) Control GFP, or NUMB6-GFP DB-7 cells were transfected with control siRNA or Slug siRNA, and E-cadherin expression was evaluated by Western Blot.

    Techniques Used: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Immunostaining, Plasmid Preparation, Transfection, Staining

    6) Product Images from "Binding Specificity of Native Odorant-Binding Protein Isoforms Is Driven by Phosphorylation and O-N-Acetylglucosaminylation in the Pig Sus scrofa"

    Article Title: Binding Specificity of Native Odorant-Binding Protein Isoforms Is Driven by Phosphorylation and O-N-Acetylglucosaminylation in the Pig Sus scrofa

    Journal: Frontiers in Endocrinology

    doi: 10.3389/fendo.2018.00816

    Immunodetection of phosphorylation and O -GlcNAcylation of OBP isoforms from S . scrofa . Each well contains a normalized quantity (5 μg) of the four HPLC fractions. HPLC-purified short-size VEG was used as negative control. Anti-P-Ser : anti-phosphoserine antibodies (1:500 dilution); Anti-P-Tyr : anti-phosphotyrosine antibodies (1:2,000); Anti-P-Thr : anti-phosphothreonine antibodies (1:500); RL2 (1:2,000), CTD110.6 (1:4,000). Secondary antibodies for anti-phosphorylation antibodies at 1:40,000 dilution (rabbit IgG-HRP linked whole antibodies), ECL Plus detection (11 min exposure). Secondary antibodies for O -GlcNAc detection: goat anti-mouse IgG-HRP linked (1:30,000) for RL2, and rabbit anti-mouse IgM-HRP linked (1:30,000) for CTD110.6, ECL Plus detection (30 s exposure).
    Figure Legend Snippet: Immunodetection of phosphorylation and O -GlcNAcylation of OBP isoforms from S . scrofa . Each well contains a normalized quantity (5 μg) of the four HPLC fractions. HPLC-purified short-size VEG was used as negative control. Anti-P-Ser : anti-phosphoserine antibodies (1:500 dilution); Anti-P-Tyr : anti-phosphotyrosine antibodies (1:2,000); Anti-P-Thr : anti-phosphothreonine antibodies (1:500); RL2 (1:2,000), CTD110.6 (1:4,000). Secondary antibodies for anti-phosphorylation antibodies at 1:40,000 dilution (rabbit IgG-HRP linked whole antibodies), ECL Plus detection (11 min exposure). Secondary antibodies for O -GlcNAc detection: goat anti-mouse IgG-HRP linked (1:30,000) for RL2, and rabbit anti-mouse IgM-HRP linked (1:30,000) for CTD110.6, ECL Plus detection (30 s exposure).

    Techniques Used: Immunodetection, High Performance Liquid Chromatography, Purification, Negative Control

    7) Product Images from "Expression of claudins, occludin, junction adhesion molecule A and zona occludens 1 in canine organs"

    Article Title: Expression of claudins, occludin, junction adhesion molecule A and zona occludens 1 in canine organs

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2016.5725

    Organ-specific mRNA and protein expression levels of OCLN, JAM-A and ZO-1 in canine duodenum, lung, liver and kidney. mRNA expression levels of (A) OCLN, (B) JAM-A and (C) ZO-1 were greatest in the duodenum, moderate in the lung and kidney, and low in the liver. Protein expression levels of (D) OCLN, (E) JAM-A and (F) ZO-1 followed a similar pattern to mRNA expression levels. mRNA and protein expression were normalized to β-actin. Data are presented as the mean ± standard deviation. OCLN, occludin; JAM-A, junction adhesion molecule A; ZO-1, zona occludens 1; ACTB, β-actin; D, duodenum; Lu, lung; Li, liver; K, kidney.
    Figure Legend Snippet: Organ-specific mRNA and protein expression levels of OCLN, JAM-A and ZO-1 in canine duodenum, lung, liver and kidney. mRNA expression levels of (A) OCLN, (B) JAM-A and (C) ZO-1 were greatest in the duodenum, moderate in the lung and kidney, and low in the liver. Protein expression levels of (D) OCLN, (E) JAM-A and (F) ZO-1 followed a similar pattern to mRNA expression levels. mRNA and protein expression were normalized to β-actin. Data are presented as the mean ± standard deviation. OCLN, occludin; JAM-A, junction adhesion molecule A; ZO-1, zona occludens 1; ACTB, β-actin; D, duodenum; Lu, lung; Li, liver; K, kidney.

    Techniques Used: Expressing, Standard Deviation

    Organ-specific localization of OCLN, JAM-A and ZO-1 in canine duodenum, lung, liver, and kidney, as detected by immunohistochemistry. (A) OCLN, (B) JAM-A and (C) ZO-1 immunostaining was detected in all organs examined. Boxes in the upper panels (magnification, ×200) are areas magnified in the lower panels (magnification, ×400). Arrows indicate positive staining. OCLN, occludin; JAM-A, junction adhesion molecule A; ZO-1, zona occludens 1.
    Figure Legend Snippet: Organ-specific localization of OCLN, JAM-A and ZO-1 in canine duodenum, lung, liver, and kidney, as detected by immunohistochemistry. (A) OCLN, (B) JAM-A and (C) ZO-1 immunostaining was detected in all organs examined. Boxes in the upper panels (magnification, ×200) are areas magnified in the lower panels (magnification, ×400). Arrows indicate positive staining. OCLN, occludin; JAM-A, junction adhesion molecule A; ZO-1, zona occludens 1.

    Techniques Used: Immunohistochemistry, Immunostaining, Staining

    8) Product Images from "Activation-coupled membrane-type 1 matrix metalloproteinase membrane trafficking"

    Article Title: Activation-coupled membrane-type 1 matrix metalloproteinase membrane trafficking

    Journal: The Biochemical Journal

    doi: 10.1042/BJ20070552

    α1-PI MT1 induces perinuclear accumulation of endogenous MT1-MMP in MDA-MB-231 cells ( A ) Cells were transfected with α1-PI WT and after 24 h, the cells were fixed with 3.7% (v/v) formalin and permeablized using 0.1% (v/v) Triton X-100. and immunostained using a rabbit anti-[MT1-MMP (hinge region)] antibody and a monoclonal mouse anti-EEA1 (early endosome antigen 1) antibody ( B ) Cells were stably transfected with α1-PI WT or α1-PI MT1 and, after 24 h, the cells were fixed with 3.7% (v/v) formalin and permeablized using 0.1% (v/v) Triton X-100. The cells were immunostained using a rabbit anti-[MT1-MMP (hinge region)] antibody and a monoclonal mouse anti-p58 (Golgi marker) monoclonal antibody. In both ( A ) and ( B ), the primary immunostaining was followed by Alexa Fluor® 488 -conjugated goat anti-(rabbit IgG) and Alexa Fluor® 546-conjugated goat anti-(mouse IgG) secondary antibodies. The images were acquired using a Zeiss LSM510 laser-scanning confocal microscope. Overlay images (MERGE) were generated using Adobe Photoshop software. Scale bar, 20 μm.
    Figure Legend Snippet: α1-PI MT1 induces perinuclear accumulation of endogenous MT1-MMP in MDA-MB-231 cells ( A ) Cells were transfected with α1-PI WT and after 24 h, the cells were fixed with 3.7% (v/v) formalin and permeablized using 0.1% (v/v) Triton X-100. and immunostained using a rabbit anti-[MT1-MMP (hinge region)] antibody and a monoclonal mouse anti-EEA1 (early endosome antigen 1) antibody ( B ) Cells were stably transfected with α1-PI WT or α1-PI MT1 and, after 24 h, the cells were fixed with 3.7% (v/v) formalin and permeablized using 0.1% (v/v) Triton X-100. The cells were immunostained using a rabbit anti-[MT1-MMP (hinge region)] antibody and a monoclonal mouse anti-p58 (Golgi marker) monoclonal antibody. In both ( A ) and ( B ), the primary immunostaining was followed by Alexa Fluor® 488 -conjugated goat anti-(rabbit IgG) and Alexa Fluor® 546-conjugated goat anti-(mouse IgG) secondary antibodies. The images were acquired using a Zeiss LSM510 laser-scanning confocal microscope. Overlay images (MERGE) were generated using Adobe Photoshop software. Scale bar, 20 μm.

    Techniques Used: Multiple Displacement Amplification, Transfection, Stable Transfection, Marker, Immunostaining, Microscopy, Generated, Software

    9) Product Images from "Synthesis, Maturation, and Trafficking of Human Na+-Dicarboxylate Cotransporter NaDC1 Requires the Chaperone Activity of Cyclophilin B *"

    Article Title: Synthesis, Maturation, and Trafficking of Human Na+-Dicarboxylate Cotransporter NaDC1 Requires the Chaperone Activity of Cyclophilin B *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.171728

    Co-localization and co-immunoprecipitation of cyclophilin B with NaDC1. A , subcellular localization of CypB and NaDC1 in non-transfected HEK293 cells treated ± 200 ng/ml brefeldin A , Western analyses of the last seven fractions out of 10 (numbered 1–7), in which GM130 ( cis -Golgi Matrix protein 130; cis -Golgi marker) and PDI (ER marker) were immunodetected. In non-treated cells, CypB co-localized with immature NaDC1 in the ER (fractions 6 and 7). In brefeldin A-treated cells, the expression of the immature NaDC1 form and CypB was increased in the ER (fractions 6 and 7). B , co-immunoprecipitation of CypB with NaDC1: HA-tagged CypB was co-injected with either FLAG-tagged NaDC1 or wt TRPV6 (positive control). Non-injected oocytes were used as negative control. NaDC1 and TRPV6 were immunoprecipitated (IP) with rabbit polyclonal anti-FLAG and chicken polyclonal anti-huTRPV6 antibodies, respectively. Western membranes were immunoblotted (IB) with the mouse monoclononal anti-HA. A CypB immunoreactive band (∼20 kDa) was detected in all co-immunoprecipitation studies.
    Figure Legend Snippet: Co-localization and co-immunoprecipitation of cyclophilin B with NaDC1. A , subcellular localization of CypB and NaDC1 in non-transfected HEK293 cells treated ± 200 ng/ml brefeldin A , Western analyses of the last seven fractions out of 10 (numbered 1–7), in which GM130 ( cis -Golgi Matrix protein 130; cis -Golgi marker) and PDI (ER marker) were immunodetected. In non-treated cells, CypB co-localized with immature NaDC1 in the ER (fractions 6 and 7). In brefeldin A-treated cells, the expression of the immature NaDC1 form and CypB was increased in the ER (fractions 6 and 7). B , co-immunoprecipitation of CypB with NaDC1: HA-tagged CypB was co-injected with either FLAG-tagged NaDC1 or wt TRPV6 (positive control). Non-injected oocytes were used as negative control. NaDC1 and TRPV6 were immunoprecipitated (IP) with rabbit polyclonal anti-FLAG and chicken polyclonal anti-huTRPV6 antibodies, respectively. Western membranes were immunoblotted (IB) with the mouse monoclononal anti-HA. A CypB immunoreactive band (∼20 kDa) was detected in all co-immunoprecipitation studies.

    Techniques Used: Immunoprecipitation, Transfection, Western Blot, Marker, Expressing, Injection, Positive Control, Negative Control

    10) Product Images from "Predictable, Tunable Protein Production in Salmonella for Studying Host-Pathogen Interactions"

    Article Title: Predictable, Tunable Protein Production in Salmonella for Studying Host-Pathogen Interactions

    Journal: Frontiers in Cellular and Infection Microbiology

    doi: 10.3389/fcimb.2017.00475

    Tunable constitutive expression of the T3SS1 regulator, HilA. (A) Bacteria were grown in 125 mL flasks and OD600 measurements were taken every 30 min. Growth curves for bacteria harboring the indicated plasmids. Shown is mean OD600 of three independent experiments. (B) Late log phase samples were solubilized and processed for immunoblotting using antibodies to detect HA and DnaK. Representative immunoblots are shown (top panels) along with quantification of three experiments by densitometry analysis (bottom panels). Shown is the ratio of HA signal to DnaK signal (mean ± SD, n = 3). WT Salmonella (the chromosomal hilA is not HA-tagged) were used as a negative control for HA detection. (C) Invasion assay in HeLa cells. Values were normalized to WT (Mean ± SD, n = 3). np, no plasmid; low, low copy plasmid; med, medium copy plasmid. * Expression of hilA under ProC resulted in inconsistent growth and protein levels between experiments.
    Figure Legend Snippet: Tunable constitutive expression of the T3SS1 regulator, HilA. (A) Bacteria were grown in 125 mL flasks and OD600 measurements were taken every 30 min. Growth curves for bacteria harboring the indicated plasmids. Shown is mean OD600 of three independent experiments. (B) Late log phase samples were solubilized and processed for immunoblotting using antibodies to detect HA and DnaK. Representative immunoblots are shown (top panels) along with quantification of three experiments by densitometry analysis (bottom panels). Shown is the ratio of HA signal to DnaK signal (mean ± SD, n = 3). WT Salmonella (the chromosomal hilA is not HA-tagged) were used as a negative control for HA detection. (C) Invasion assay in HeLa cells. Values were normalized to WT (Mean ± SD, n = 3). np, no plasmid; low, low copy plasmid; med, medium copy plasmid. * Expression of hilA under ProC resulted in inconsistent growth and protein levels between experiments.

    Techniques Used: Expressing, Western Blot, Negative Control, Invasion Assay, Plasmid Preparation

    Tunable constitutive expression of the T3SS1 effector, SopB. (A) Δ sopB bacteria harboring the indicated plasmids were grown to late-log phase in LB-Miller broth with aeration. Samples were solubilized and processed for immunoblotting using antibodies to detect HA and DnaK. Representative immunoblots (top panel) are shown along with quantification of three experiments by densitometry analysis (bottom panel). Shown is the ratio of HA signal to DnaK signal (mean ± SD ). (B) Infected HeLa cells were solubilized at 60 min pi and processed for immunoblotting using antibodies to detect phospho-Akt and total Akt. Representative immunoblots (top panel) are shown along with quantification of three experiments by densitometry analysis (bottom panel). Shown is the ratio of phospho-Akt signal to total Akt signal (mean ± SD ). * Signifies P -value ≤ 0.05.
    Figure Legend Snippet: Tunable constitutive expression of the T3SS1 effector, SopB. (A) Δ sopB bacteria harboring the indicated plasmids were grown to late-log phase in LB-Miller broth with aeration. Samples were solubilized and processed for immunoblotting using antibodies to detect HA and DnaK. Representative immunoblots (top panel) are shown along with quantification of three experiments by densitometry analysis (bottom panel). Shown is the ratio of HA signal to DnaK signal (mean ± SD ). (B) Infected HeLa cells were solubilized at 60 min pi and processed for immunoblotting using antibodies to detect phospho-Akt and total Akt. Representative immunoblots (top panel) are shown along with quantification of three experiments by densitometry analysis (bottom panel). Shown is the ratio of phospho-Akt signal to total Akt signal (mean ± SD ). * Signifies P -value ≤ 0.05.

    Techniques Used: Expressing, Western Blot, Infection

    Evaluating synthetic promoter activity in Salmonella Typhimurium. (A,B) Bacteria harboring the indicated plasmids were grown to late-log phase in LB-Miller broth with aeration. Samples were solubilized and processed for immunoblotting using antibodies to detect GFP (A) or mCherry (B) . DnaK was used as a loading control. Representative immunoblots are shown (top panels) along with quantification of three experiments by densitometry analysis (bottom panels). Shown is the ratio of GFP or mCherry signal to DnaK signal (mean ± SD ). (C,D) Bacteria were grown in 96-well plates and fluorescence and OD600 measurements were taken every 15 min. Growth curves for strains harboring GFP constructs (C) or mCherry constructs (D) . Shown is the mean OD600 of three independent experiments. Log phase is observed between 1 h and 3 h post inoculation (dotted lines). (E,F) . Relative promoter units normalized to ProA (RPU A ) were calculated using fluorescence of GFP (E) or mCherry (F) at 1.5 and 2.5 h time points. The fluorescence intensity for pCON-ProC. gfp and pCON-ProD. gfp or pCON-ProC. mCherry and pCON-ProD. mCherry during log phase is plotted against OD600 in the insets (Mean ± SD, n = 3).
    Figure Legend Snippet: Evaluating synthetic promoter activity in Salmonella Typhimurium. (A,B) Bacteria harboring the indicated plasmids were grown to late-log phase in LB-Miller broth with aeration. Samples were solubilized and processed for immunoblotting using antibodies to detect GFP (A) or mCherry (B) . DnaK was used as a loading control. Representative immunoblots are shown (top panels) along with quantification of three experiments by densitometry analysis (bottom panels). Shown is the ratio of GFP or mCherry signal to DnaK signal (mean ± SD ). (C,D) Bacteria were grown in 96-well plates and fluorescence and OD600 measurements were taken every 15 min. Growth curves for strains harboring GFP constructs (C) or mCherry constructs (D) . Shown is the mean OD600 of three independent experiments. Log phase is observed between 1 h and 3 h post inoculation (dotted lines). (E,F) . Relative promoter units normalized to ProA (RPU A ) were calculated using fluorescence of GFP (E) or mCherry (F) at 1.5 and 2.5 h time points. The fluorescence intensity for pCON-ProC. gfp and pCON-ProD. gfp or pCON-ProC. mCherry and pCON-ProD. mCherry during log phase is plotted against OD600 in the insets (Mean ± SD, n = 3).

    Techniques Used: Activity Assay, Western Blot, Fluorescence, Construct

    11) Product Images from "Androgen receptor and chemokine receptors 4 and 7 form a signaling axis to regulate CXCL12-dependent cellular motility"

    Article Title: Androgen receptor and chemokine receptors 4 and 7 form a signaling axis to regulate CXCL12-dependent cellular motility

    Journal: BMC Cancer

    doi: 10.1186/s12885-015-1201-5

    CXCR7 functionally interacts and colocalizes with AR. (A) Western blot of LNCaP cells transfected with the indicated siRNA combinations: control (50 nM), AR (50 nM), CXCR7 (50 nM), AR/control (25 nM/25 nM), CXCR7/control (25 nM/25 nM), or AR/CXCR7 (25 nM/25 nM) for 72 hrs. Western blot was performed using AR, CXCR7, and PSA antibodies. Silver staining demonstrates equivalent loading across the samples. The densitometry values were normalized to control siRNA transfected cells and labeled below the blots. (B) Immunofluorescence analysis of CXCR7 and AR in LNCaP cells under AR or CXCR7 knockdown conditions. Cells were transfected with control (I-I to I-III), AR (II-I to II-III), or CXCR7 (III-I to III-III) siRNA, stained with antibodies against AR and CXCR7, and treated with DAPI.
    Figure Legend Snippet: CXCR7 functionally interacts and colocalizes with AR. (A) Western blot of LNCaP cells transfected with the indicated siRNA combinations: control (50 nM), AR (50 nM), CXCR7 (50 nM), AR/control (25 nM/25 nM), CXCR7/control (25 nM/25 nM), or AR/CXCR7 (25 nM/25 nM) for 72 hrs. Western blot was performed using AR, CXCR7, and PSA antibodies. Silver staining demonstrates equivalent loading across the samples. The densitometry values were normalized to control siRNA transfected cells and labeled below the blots. (B) Immunofluorescence analysis of CXCR7 and AR in LNCaP cells under AR or CXCR7 knockdown conditions. Cells were transfected with control (I-I to I-III), AR (II-I to II-III), or CXCR7 (III-I to III-III) siRNA, stained with antibodies against AR and CXCR7, and treated with DAPI.

    Techniques Used: Western Blot, Transfection, Silver Staining, Labeling, Immunofluorescence, Staining

    CXCR7 subcellular localization in prostate-cancer cells. (A) Western blot of proteins from cytosolic, membrane, nuclear, and chromatin fractions isolated from normal, AD -, and AS -LNCaP cells with antibodies to pAbCXCR7 (top left panel), Hsp90, EEA1, AR, or histone H3 (right panels, with blank lanes in lanes 5 and 10). Silver-stained gel demonstrated equal protein loading across samples (bottom left panel). (B) Upper panels: AD -LNCaP cells. Left panel: Image of a single optical section (section 17 out of 32), with “Cut View” analysis of the entire Z-stack shown in margins. Green = CXCR7, Red = EEA1, Blue = DAPI. Right top panel: Reconstruction of entire Z-stack of sample shown in left. Scale bar = 20 μm. Right bottom panel: Similar reconstruction of cells stained in the presence of competing peptide. Scale bar in lower right = 20 μm and is for top and bottom right panels. Lower panels: Androgen stimulated LNCaP cells. Left panel: Image of a single optical section (section 12 out of 32), with “Cut View” analysis of the entire Z-stack shown in margins. Green = CXCR7, Red = EEA1, Blue = DAPI. Scale bar = 20 μm. Right top panel: Reconstruction of entire Z-stack of sample shown in left. Right bottom panel: Similar reconstruction of cells stained in the presence of competing peptide. Scale bar in lower right = 20 μm and is for top and bottom right panels.
    Figure Legend Snippet: CXCR7 subcellular localization in prostate-cancer cells. (A) Western blot of proteins from cytosolic, membrane, nuclear, and chromatin fractions isolated from normal, AD -, and AS -LNCaP cells with antibodies to pAbCXCR7 (top left panel), Hsp90, EEA1, AR, or histone H3 (right panels, with blank lanes in lanes 5 and 10). Silver-stained gel demonstrated equal protein loading across samples (bottom left panel). (B) Upper panels: AD -LNCaP cells. Left panel: Image of a single optical section (section 17 out of 32), with “Cut View” analysis of the entire Z-stack shown in margins. Green = CXCR7, Red = EEA1, Blue = DAPI. Right top panel: Reconstruction of entire Z-stack of sample shown in left. Scale bar = 20 μm. Right bottom panel: Similar reconstruction of cells stained in the presence of competing peptide. Scale bar in lower right = 20 μm and is for top and bottom right panels. Lower panels: Androgen stimulated LNCaP cells. Left panel: Image of a single optical section (section 12 out of 32), with “Cut View” analysis of the entire Z-stack shown in margins. Green = CXCR7, Red = EEA1, Blue = DAPI. Scale bar = 20 μm. Right top panel: Reconstruction of entire Z-stack of sample shown in left. Right bottom panel: Similar reconstruction of cells stained in the presence of competing peptide. Scale bar in lower right = 20 μm and is for top and bottom right panels.

    Techniques Used: Western Blot, Isolation, Staining

    Speculative models for CXCR7 signaling in prostate cancer cells. (A) CXCR7 and AR protein levels are modulated by AR and CXCR7 respectively, through post-transcriptional mechanisms ( e.g., protein stability). (B) Reciprocal feedback loop regulating the expression of CXCR4 and CXCR7 in LNCaP prostate cancer cells, including protein trafficking pathways that could account for the transport of CXCR7 into the nuclear compartment. Question mark indicates that AR may directly or indirectly interact with CXCR7. (I) CXCR7 gains access to the nucleus through the nucleoplasmic reticulum ( e.g. invaginations of the nuclear envelope) [ 73 , 77 ]. (II) CXCR7 gains access to the nucleus through the nuclear pore complex ( e.g., transportin-dependent), as shown for other GPCRs and more recently for CXCR4 [ 69 , 70 ].
    Figure Legend Snippet: Speculative models for CXCR7 signaling in prostate cancer cells. (A) CXCR7 and AR protein levels are modulated by AR and CXCR7 respectively, through post-transcriptional mechanisms ( e.g., protein stability). (B) Reciprocal feedback loop regulating the expression of CXCR4 and CXCR7 in LNCaP prostate cancer cells, including protein trafficking pathways that could account for the transport of CXCR7 into the nuclear compartment. Question mark indicates that AR may directly or indirectly interact with CXCR7. (I) CXCR7 gains access to the nucleus through the nucleoplasmic reticulum ( e.g. invaginations of the nuclear envelope) [ 73 , 77 ]. (II) CXCR7 gains access to the nucleus through the nuclear pore complex ( e.g., transportin-dependent), as shown for other GPCRs and more recently for CXCR4 [ 69 , 70 ].

    Techniques Used: Expressing

    CXCR7 modulates androgen-mediated cell motility through CXCR4. (A) Transwell assay assessing the effects of androgens on LNCaP migration. ANOVA was used to determine significant differences between vehicle (ethanol) and androgen (R1881) treated cells (* p ≤ 0.05, n = 3). (B-D) Western blot analysis of LNCaP membrane glycoproteins enriched from cells grown in androgen-depleted medium for 72 hrs and treated with 0, 0.1, 1, or 10 nM R1881 for 24 hrs with antibodies to (B) PSA, PSMA, (C) CXCR7, and (D) CXCR4. Red asterisks = putative glycosylated CXCR4 and CXCR7 isoforms. The densitometry values were normalized to vehicle-treated lysates and labeled below the blots. (E) Silver staining demonstrates equivalent loading across the samples. (F) Immunofluorescence staining of CXCR4 and CXCR7 in semi-permeabilized AD -LNCaP cells treated with 0, 0.1, 1, or 10 nM R1881. The nuclei are labeled with DAPI.
    Figure Legend Snippet: CXCR7 modulates androgen-mediated cell motility through CXCR4. (A) Transwell assay assessing the effects of androgens on LNCaP migration. ANOVA was used to determine significant differences between vehicle (ethanol) and androgen (R1881) treated cells (* p ≤ 0.05, n = 3). (B-D) Western blot analysis of LNCaP membrane glycoproteins enriched from cells grown in androgen-depleted medium for 72 hrs and treated with 0, 0.1, 1, or 10 nM R1881 for 24 hrs with antibodies to (B) PSA, PSMA, (C) CXCR7, and (D) CXCR4. Red asterisks = putative glycosylated CXCR4 and CXCR7 isoforms. The densitometry values were normalized to vehicle-treated lysates and labeled below the blots. (E) Silver staining demonstrates equivalent loading across the samples. (F) Immunofluorescence staining of CXCR4 and CXCR7 in semi-permeabilized AD -LNCaP cells treated with 0, 0.1, 1, or 10 nM R1881. The nuclei are labeled with DAPI.

    Techniques Used: Transwell Assay, Migration, Western Blot, Labeling, Silver Staining, Immunofluorescence, Staining

    CXCR7 knockdown leads to a reduction in CXCR4 protein levels in LNCaP cells. (A) Transwell assay assessing the effects of CXCL12 on LNCaP migration. ANOVA was used to determine significant differences between vehicle (0.1% BSA) and CXCL12-treated cells (* p ≤ 0.05, n = 3). (B) Transwell assay assessing the effects of CXCR4 and CXCR7 knockdown on CXCL12-induced LNCaP cell migration. LNCaP cells transfected with 100 nM scrambled control, CXCR4, or CXCR7 siRNAs were seeded to the top chamber of the insert. The bottom chamber contained medium with 1% CS serum with 1 nM R1881 and CXCL12 at 0, 0.003, 0.03, or 0.3 nM concentrations. Data was normalized to control siRNA transfected, vehicle-treated cells. ANOVA was used to determine significant differences (*p ≤ 0.05, n = 3) between control and experimental cells. (C-E) Western blots to test the effects of CXCR7 and CXCR4 knockdown on AR signaling in LNCaP cells. Cells were transfected with control, CXCR7 or CXCR4 siRNA for 72 hrs and probed with antibodies against (C) CXCR7, (D) CXCR4, (E) AR, and PSA. The densitometry values were normalized to control siRNA transfected cells and labeled below the blots. (F) Silver staining demonstrates equivalent loading across the samples.
    Figure Legend Snippet: CXCR7 knockdown leads to a reduction in CXCR4 protein levels in LNCaP cells. (A) Transwell assay assessing the effects of CXCL12 on LNCaP migration. ANOVA was used to determine significant differences between vehicle (0.1% BSA) and CXCL12-treated cells (* p ≤ 0.05, n = 3). (B) Transwell assay assessing the effects of CXCR4 and CXCR7 knockdown on CXCL12-induced LNCaP cell migration. LNCaP cells transfected with 100 nM scrambled control, CXCR4, or CXCR7 siRNAs were seeded to the top chamber of the insert. The bottom chamber contained medium with 1% CS serum with 1 nM R1881 and CXCL12 at 0, 0.003, 0.03, or 0.3 nM concentrations. Data was normalized to control siRNA transfected, vehicle-treated cells. ANOVA was used to determine significant differences (*p ≤ 0.05, n = 3) between control and experimental cells. (C-E) Western blots to test the effects of CXCR7 and CXCR4 knockdown on AR signaling in LNCaP cells. Cells were transfected with control, CXCR7 or CXCR4 siRNA for 72 hrs and probed with antibodies against (C) CXCR7, (D) CXCR4, (E) AR, and PSA. The densitometry values were normalized to control siRNA transfected cells and labeled below the blots. (F) Silver staining demonstrates equivalent loading across the samples.

    Techniques Used: Transwell Assay, Migration, Transfection, Western Blot, Labeling, Silver Staining

    CXCR7 expression and localization are modulated by CXCL11 and CXCL12. (A) Immunofluorescence staining of CXCR7 in AD -LNCaP cells treated with vehicle (0.1% BSA), CXCL11 (100 nM), or CXCL12 (100 nM) for 30 min. Nuclei and F-actin are labeled with DAPI and Texas-red phalloidin, respectively. (B) Western blot of cytosolic, membrane, and nuclear protein fractions isolated from LNCaP cells, cultured as described in (A) with pAbCXCR7 antibody. (C) Immunofluorescence staining of CXCR4 in AD -LNCaP cells as described in (A) . (D-E) Western blot of cytosolic, membrane, and nuclear protein fractions isolated from LNCaP cells, cultured as described in (A), with antibodies to (D) CXCR4, and (E) AR, GM130, and histone H3. Silver staining demonstrates equivalent loading across samples. The densitometry values were normalized to BSA-treated samples for each subcellular compartment and labeled below the blots.
    Figure Legend Snippet: CXCR7 expression and localization are modulated by CXCL11 and CXCL12. (A) Immunofluorescence staining of CXCR7 in AD -LNCaP cells treated with vehicle (0.1% BSA), CXCL11 (100 nM), or CXCL12 (100 nM) for 30 min. Nuclei and F-actin are labeled with DAPI and Texas-red phalloidin, respectively. (B) Western blot of cytosolic, membrane, and nuclear protein fractions isolated from LNCaP cells, cultured as described in (A) with pAbCXCR7 antibody. (C) Immunofluorescence staining of CXCR4 in AD -LNCaP cells as described in (A) . (D-E) Western blot of cytosolic, membrane, and nuclear protein fractions isolated from LNCaP cells, cultured as described in (A), with antibodies to (D) CXCR4, and (E) AR, GM130, and histone H3. Silver staining demonstrates equivalent loading across samples. The densitometry values were normalized to BSA-treated samples for each subcellular compartment and labeled below the blots.

    Techniques Used: Expressing, Immunofluorescence, Staining, Labeling, Western Blot, Isolation, Cell Culture, Silver Staining

    CXCR7 colocalizes and physically interacts with AR in LNCaP prostate-tumor cells. (A) Immunofluorescence staining of CXCR7 and AR in semi-permeabilized AD -LNCaP cells treated with vehicle (0.1% BSA), 0.1, 1, or 10 nM R1881 for 96 hrs. The nuclei are labeled with DAPI. (B) Western blot analysis of crude cytosolic, membrane, and nuclear proteins isolated from LNCaP cells stably expressing streptavidin binding peptide tag (SBP) or CXCR7 with a SBP-tag on the C-terminus (C7-SBP) using antibodies to GM130 and Histone H3 (top panel). Silver-stained gel demonstrated equal protein loading across samples (bottom panel). (C) Western blot analysis of streptavidin affinity purified samples from detergent-solubilized microsomal protein fraction of SBP or C7-SBP cells using the SBP, and (D) AR antibody. Equal proportions (10 μl) of input, flow-through (void), and 5% (10 ul) of the biotin elution were loaded.
    Figure Legend Snippet: CXCR7 colocalizes and physically interacts with AR in LNCaP prostate-tumor cells. (A) Immunofluorescence staining of CXCR7 and AR in semi-permeabilized AD -LNCaP cells treated with vehicle (0.1% BSA), 0.1, 1, or 10 nM R1881 for 96 hrs. The nuclei are labeled with DAPI. (B) Western blot analysis of crude cytosolic, membrane, and nuclear proteins isolated from LNCaP cells stably expressing streptavidin binding peptide tag (SBP) or CXCR7 with a SBP-tag on the C-terminus (C7-SBP) using antibodies to GM130 and Histone H3 (top panel). Silver-stained gel demonstrated equal protein loading across samples (bottom panel). (C) Western blot analysis of streptavidin affinity purified samples from detergent-solubilized microsomal protein fraction of SBP or C7-SBP cells using the SBP, and (D) AR antibody. Equal proportions (10 μl) of input, flow-through (void), and 5% (10 ul) of the biotin elution were loaded.

    Techniques Used: Immunofluorescence, Staining, Labeling, Western Blot, Isolation, Stable Transfection, Expressing, Binding Assay, Affinity Purification, Flow Cytometry

    CXCR7 expression in prostate-cancer cells. (A) Western blot of 1, 2, and 4 μg of LNCaP total lysate with pAbCXCR7 antibody in the presence of the non-competitive AR peptide (a.a. 299–315, left panel) or the CXCR7 blocking peptide (a.a. 348–362, right panel) as detailed in Materials and Methods section. The CXCR7 bands are indicated by arrowheads. (B) AR , CXCR7 , CXCR4 , and PSA gene expressions in LNCaP cells transfected with AR, CXCR7, or scrambled control siRNA. RNA was isolated 72 hrs post-transfection and measured by qPCR. Student’s t -test was used to calculate significant differences (* p ≤ 0.05, n = 3) between control and experimental cells. (C) Western blot (left panel) of whole cell lysates from LNCaP cells transfected with control or two experimentally-validated CXCR7 siRNAs (CXCR7 #1 or #2) for 72 hrs using antibodies to pAbCXCR7 and GAPDH. Silver-stained gel demonstrated equal protein loading across samples (right panel). The densitometry values were labeled below the blot and normalized to the control transfected cells loaded with the same amount of total proteins. (D) Light microscopy of LNCaP cells transfected with control or CXCR7 siRNA for 72 hrs.
    Figure Legend Snippet: CXCR7 expression in prostate-cancer cells. (A) Western blot of 1, 2, and 4 μg of LNCaP total lysate with pAbCXCR7 antibody in the presence of the non-competitive AR peptide (a.a. 299–315, left panel) or the CXCR7 blocking peptide (a.a. 348–362, right panel) as detailed in Materials and Methods section. The CXCR7 bands are indicated by arrowheads. (B) AR , CXCR7 , CXCR4 , and PSA gene expressions in LNCaP cells transfected with AR, CXCR7, or scrambled control siRNA. RNA was isolated 72 hrs post-transfection and measured by qPCR. Student’s t -test was used to calculate significant differences (* p ≤ 0.05, n = 3) between control and experimental cells. (C) Western blot (left panel) of whole cell lysates from LNCaP cells transfected with control or two experimentally-validated CXCR7 siRNAs (CXCR7 #1 or #2) for 72 hrs using antibodies to pAbCXCR7 and GAPDH. Silver-stained gel demonstrated equal protein loading across samples (right panel). The densitometry values were labeled below the blot and normalized to the control transfected cells loaded with the same amount of total proteins. (D) Light microscopy of LNCaP cells transfected with control or CXCR7 siRNA for 72 hrs.

    Techniques Used: Expressing, Western Blot, Blocking Assay, Transfection, Isolation, Real-time Polymerase Chain Reaction, Staining, Labeling, Light Microscopy

    CXCR7 modulates AR transcriptional activity. (A) Luciferase assay testing the effects of CXCR7 overexpression on the AR-target promoter probasin in LNCaP cells. LNCaP cells were co-transfected with the pGL4.10-Luc2- probasin and pRLSV40 Renilla vectors along with increasing amounts (30 ng experimental + 270 ng pcDNA3, 100 ng experimental + 200 ng pcDNA3, 300 ng experimental + 0 ng pcDNA3) of CXCR7 or ACTN4 cDNA mammalian expression vectors. The maximal amount (300 ng) of the pcDNA3 mammalian expression vector served as the positive control. Cells were subsequently treated with androgen (1 nM R1881) or vehicle (ethanol) and tested for dual luciferase activity. Student’s t -test was used to calculate significant differences (* p ≤ 0.05, n = 3) between control and experimental cells within the androgen-treatment group. (B) Luciferase assay testing the effects of CXCR7 siRNA knockdown and treatment with CXCR7 ligand on the AR-target promoter probasin . LNCaP cells were co-transfected with the pGL4.10-Luc2- probasin and pRLSV40- renilla vectors, along with control or experimental siRNAs (50 nM). Next, cells were pre-treated with indicated ligands (BSA, CXCL11, or CXCL12) for 30 min. Cells were then subsequently treated with androgen (1 nM R1881) or vehicle (ethanol) for 18 hrs and tested for dual luciferase activity. Student’s t -test was used to calculate significant differences (* p ≤ 0.05, n = 3) between control cells and experimental cells within the androgen-treatment group. (C) RNA isolated from LNCaP cells treated with vehicle (0.1% BSA), CXCL11 (10 nM), or CXCL12 (10 nM) for 30 min and subsequently treated with vehicle or androgen (1 nM R1881) for 18 hrs were subjected to qPCR analysis for AR , FASN , NKX3.1 , PSA , and TMPRSS2 gene expressions. Student’s t -test was used to calculate significant differences (* p ≤ 0.05, n = 3) between control and chemokine ligand-treated cells.
    Figure Legend Snippet: CXCR7 modulates AR transcriptional activity. (A) Luciferase assay testing the effects of CXCR7 overexpression on the AR-target promoter probasin in LNCaP cells. LNCaP cells were co-transfected with the pGL4.10-Luc2- probasin and pRLSV40 Renilla vectors along with increasing amounts (30 ng experimental + 270 ng pcDNA3, 100 ng experimental + 200 ng pcDNA3, 300 ng experimental + 0 ng pcDNA3) of CXCR7 or ACTN4 cDNA mammalian expression vectors. The maximal amount (300 ng) of the pcDNA3 mammalian expression vector served as the positive control. Cells were subsequently treated with androgen (1 nM R1881) or vehicle (ethanol) and tested for dual luciferase activity. Student’s t -test was used to calculate significant differences (* p ≤ 0.05, n = 3) between control and experimental cells within the androgen-treatment group. (B) Luciferase assay testing the effects of CXCR7 siRNA knockdown and treatment with CXCR7 ligand on the AR-target promoter probasin . LNCaP cells were co-transfected with the pGL4.10-Luc2- probasin and pRLSV40- renilla vectors, along with control or experimental siRNAs (50 nM). Next, cells were pre-treated with indicated ligands (BSA, CXCL11, or CXCL12) for 30 min. Cells were then subsequently treated with androgen (1 nM R1881) or vehicle (ethanol) for 18 hrs and tested for dual luciferase activity. Student’s t -test was used to calculate significant differences (* p ≤ 0.05, n = 3) between control cells and experimental cells within the androgen-treatment group. (C) RNA isolated from LNCaP cells treated with vehicle (0.1% BSA), CXCL11 (10 nM), or CXCL12 (10 nM) for 30 min and subsequently treated with vehicle or androgen (1 nM R1881) for 18 hrs were subjected to qPCR analysis for AR , FASN , NKX3.1 , PSA , and TMPRSS2 gene expressions. Student’s t -test was used to calculate significant differences (* p ≤ 0.05, n = 3) between control and chemokine ligand-treated cells.

    Techniques Used: Activity Assay, Luciferase, Over Expression, Transfection, Expressing, Plasmid Preparation, Positive Control, Isolation, Real-time Polymerase Chain Reaction

    12) Product Images from "Polybasic Trafficking Signal Mediates Golgi Export, ER Retention or ER Export and Retrieval Based on Membrane-Proximity"

    Article Title: Polybasic Trafficking Signal Mediates Golgi Export, ER Retention or ER Export and Retrieval Based on Membrane-Proximity

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0094194

    Membrane-distal and -proximal PBMs alter ER-Golgi p14 trafficking. (A) QM5 cells transfected with p14-V9T or p14extPB in a p14-V9T backbone (see Fig. 1 ) were surface stained at 24 h post-transfection using anti-p14ecto antiserum and Alexa-647 secondary antibody, and analyzed by flow cytometry. Percent cell surface fluorescence relative to p14-V9T is presented as mean ± SEM from three independent experiments in triplicate. Statistical significance from student t-test is shown relative to p14 (***p
    Figure Legend Snippet: Membrane-distal and -proximal PBMs alter ER-Golgi p14 trafficking. (A) QM5 cells transfected with p14-V9T or p14extPB in a p14-V9T backbone (see Fig. 1 ) were surface stained at 24 h post-transfection using anti-p14ecto antiserum and Alexa-647 secondary antibody, and analyzed by flow cytometry. Percent cell surface fluorescence relative to p14-V9T is presented as mean ± SEM from three independent experiments in triplicate. Statistical significance from student t-test is shown relative to p14 (***p

    Techniques Used: Transfection, Staining, Flow Cytometry, Cytometry, Fluorescence

    An internal PBM does not alter p14 trafficking to the plasma membrane. (A) QM5 cells transfected with p14-G2A or the indicated p14 mutants in a p14-G2A backbone (see Fig. 1 ) were surface stained at 24 h post-transfection using anti-p14ecto antiserum and Alexa-647 secondary antibody, and analyzed by flow cytometry. Percent cell surface fluorescence relative to p14 are presented as mean ± SEM from three independent experiments in triplicate. Statistical significance by one-way ANNOVA and Tukey post-test is shown relative to p14 (***p
    Figure Legend Snippet: An internal PBM does not alter p14 trafficking to the plasma membrane. (A) QM5 cells transfected with p14-G2A or the indicated p14 mutants in a p14-G2A backbone (see Fig. 1 ) were surface stained at 24 h post-transfection using anti-p14ecto antiserum and Alexa-647 secondary antibody, and analyzed by flow cytometry. Percent cell surface fluorescence relative to p14 are presented as mean ± SEM from three independent experiments in triplicate. Statistical significance by one-way ANNOVA and Tukey post-test is shown relative to p14 (***p

    Techniques Used: Transfection, Staining, Flow Cytometry, Cytometry, Fluorescence

    13) Product Images from "S-Adenosylmethionine regulates apoptosis and autophagy in MCF-7 breast cancer cells through the modulation of specific microRNAs"

    Article Title: S-Adenosylmethionine regulates apoptosis and autophagy in MCF-7 breast cancer cells through the modulation of specific microRNAs

    Journal: Cancer Cell International

    doi: 10.1186/s12935-018-0697-6

    MiRNA expression pattern in AdoMet-treated MCF-7 cells. Cells treated with AdoMet 500 µM for 72 h, were subjected to miRNA expression profiling using a 384-well TaqMan Array CARD. Twenty-eight miRNAs were differentially expressed in AdoMet-treated cells if compared to control samples. Log2 fold change is graphically represented. The analysis was repeated at least three times and always gave similar results
    Figure Legend Snippet: MiRNA expression pattern in AdoMet-treated MCF-7 cells. Cells treated with AdoMet 500 µM for 72 h, were subjected to miRNA expression profiling using a 384-well TaqMan Array CARD. Twenty-eight miRNAs were differentially expressed in AdoMet-treated cells if compared to control samples. Log2 fold change is graphically represented. The analysis was repeated at least three times and always gave similar results

    Techniques Used: Expressing

    14) Product Images from "Expression of Tas1 Taste Receptors in Mammalian Spermatozoa: Functional Role of Tas1r1 in Regulating Basal Ca2+ and cAMP Concentrations in Spermatozoa"

    Article Title: Expression of Tas1 Taste Receptors in Mammalian Spermatozoa: Functional Role of Tas1r1 in Regulating Basal Ca2+ and cAMP Concentrations in Spermatozoa

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0032354

    Tas1r1 expression in mammalian spermatozoa. [ A ] Extrusion of the mCherry protein during sperm maturation in the epididymis. Cryosections of the caput of the epididymis of a Tas1r1/mCherry reporter mouse were incubated with an anti-mCherry antiserum (red; [ mCherry ]) and counterstained with the nuclear dye TO-PRO-3 (blue; [ TOPRO ]). ([ mCherry+TOPRO ], inset, arrowhead). [ B ] mCherry fluorescence is not detectable in mature epididymal sperm. Isolated sperm of the mutant mouse line were fixed with PFA and counterstained with the FITC-coupled acrosomal marker PNA (middle panel; arrow; [ PNA ]). Imaging sperm for mCherry fluorescence revealed that the fluorescent protein was completely lost during epididymal maturation (left panel [ mCherry ]). Insets in the right panels show higher magnification of the tubule's lumen [ A ] or a sperm's acrosome [ B ], respectively. [ C ] Expression of Tas1r1 in human spermatozoa. Ejaculated human sperm were incubated with a human specific Tas1r1 antiserum; bound primary antiserum was visualized applying a FITC-conjugated anti-rabbit IgG. The two representative confocal micrographs document that the anti-Tas1r1 IgG ([ Tas1r1 ]) showed a staining in the flagellum (arrow) and in the post-acrosomal region as well as at the equatorial segment (arrowheads). Immunostaining in both subcellular compartments was extinguished upon neutralizing the primary antiserum with an excess of the corresponding immunogenic peptide (lower panels; [ Tas1r1+BP ]), thus confirming specificity of the detected immunolabeling. Negative controls, in which the primary antiserum was omitted, did not show any labeling (data not shown). Confocal images were produced by an overlay of corresponding fluorescence channels (propidium iodide, [red]; FITC-conjugated secondary antiserum, [green]) and the transmission channel. Boxes indicate regions that are magnified in insets in the right panels. Experiments were repeated with at least three independent sperm preparations from different donors, showing comparable results.
    Figure Legend Snippet: Tas1r1 expression in mammalian spermatozoa. [ A ] Extrusion of the mCherry protein during sperm maturation in the epididymis. Cryosections of the caput of the epididymis of a Tas1r1/mCherry reporter mouse were incubated with an anti-mCherry antiserum (red; [ mCherry ]) and counterstained with the nuclear dye TO-PRO-3 (blue; [ TOPRO ]). ([ mCherry+TOPRO ], inset, arrowhead). [ B ] mCherry fluorescence is not detectable in mature epididymal sperm. Isolated sperm of the mutant mouse line were fixed with PFA and counterstained with the FITC-coupled acrosomal marker PNA (middle panel; arrow; [ PNA ]). Imaging sperm for mCherry fluorescence revealed that the fluorescent protein was completely lost during epididymal maturation (left panel [ mCherry ]). Insets in the right panels show higher magnification of the tubule's lumen [ A ] or a sperm's acrosome [ B ], respectively. [ C ] Expression of Tas1r1 in human spermatozoa. Ejaculated human sperm were incubated with a human specific Tas1r1 antiserum; bound primary antiserum was visualized applying a FITC-conjugated anti-rabbit IgG. The two representative confocal micrographs document that the anti-Tas1r1 IgG ([ Tas1r1 ]) showed a staining in the flagellum (arrow) and in the post-acrosomal region as well as at the equatorial segment (arrowheads). Immunostaining in both subcellular compartments was extinguished upon neutralizing the primary antiserum with an excess of the corresponding immunogenic peptide (lower panels; [ Tas1r1+BP ]), thus confirming specificity of the detected immunolabeling. Negative controls, in which the primary antiserum was omitted, did not show any labeling (data not shown). Confocal images were produced by an overlay of corresponding fluorescence channels (propidium iodide, [red]; FITC-conjugated secondary antiserum, [green]) and the transmission channel. Boxes indicate regions that are magnified in insets in the right panels. Experiments were repeated with at least three independent sperm preparations from different donors, showing comparable results.

    Techniques Used: Expressing, Incubation, Fluorescence, Isolation, Mutagenesis, Marker, Imaging, Staining, Immunostaining, Immunolabeling, Labeling, Produced, Transmission Assay

    15) Product Images from "MERS-CoV 4b protein interferes with the NF-κB-dependent innate immune response during infection"

    Article Title: MERS-CoV 4b protein interferes with the NF-κB-dependent innate immune response during infection

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006838

    Generation of MERS-CoV-Δ4a and Δ4b deletion mutants. (A) Schematic representation of MERS-CoV WT genome. Essential viral genes (ORF1a, ORF1b, S, E, M, N) and accessory genes (3, 4a, 4b and 5) are indicated. L, leader sequence; A n , poly(A) tail; CS, conserved sequence included in transcription-regulating sequences. Genes 4a and 4b were deleted from the MERS-CoV infectious cDNA clone by PCR-directed mutagenesis as described in Materials and Methods. Deleted regions within 4a and 4b genes are illustrated as light grey boxes. The size of both deleted and remaining regions in 4a and 4b genes is indicated by arrows. The patterned boxes in Δ4a and Δ4b mutants indicate frameshift mutations in 4a and 4b sequence caused by the deletions. (B) Growth kinetics of Δ4a and Δ4b deletion mutants in Huh-7 cells at the indicated MOIs. Supernatants were collected at 24, 48 and 72 hpi and titrated by plaque assay. (C) NF-κB-dependent cytokine response of Huh-7 cells either mock-infected or infected with WT, Δ4ab, Δ4a or Δ4b mutants (MOI = 1 PFU/cell). At 24 hpi, mRNA levels of IL-6, IL-8 and TNF-α were quantified by RT-qPCR and compared to those in WT-infected cells, using the ΔΔCt method for calculation and HMBS as a reference endogenous gene. Shown are means with standard deviations, which were analyzed using an unpaired t-test against the wild-type (**, p
    Figure Legend Snippet: Generation of MERS-CoV-Δ4a and Δ4b deletion mutants. (A) Schematic representation of MERS-CoV WT genome. Essential viral genes (ORF1a, ORF1b, S, E, M, N) and accessory genes (3, 4a, 4b and 5) are indicated. L, leader sequence; A n , poly(A) tail; CS, conserved sequence included in transcription-regulating sequences. Genes 4a and 4b were deleted from the MERS-CoV infectious cDNA clone by PCR-directed mutagenesis as described in Materials and Methods. Deleted regions within 4a and 4b genes are illustrated as light grey boxes. The size of both deleted and remaining regions in 4a and 4b genes is indicated by arrows. The patterned boxes in Δ4a and Δ4b mutants indicate frameshift mutations in 4a and 4b sequence caused by the deletions. (B) Growth kinetics of Δ4a and Δ4b deletion mutants in Huh-7 cells at the indicated MOIs. Supernatants were collected at 24, 48 and 72 hpi and titrated by plaque assay. (C) NF-κB-dependent cytokine response of Huh-7 cells either mock-infected or infected with WT, Δ4ab, Δ4a or Δ4b mutants (MOI = 1 PFU/cell). At 24 hpi, mRNA levels of IL-6, IL-8 and TNF-α were quantified by RT-qPCR and compared to those in WT-infected cells, using the ΔΔCt method for calculation and HMBS as a reference endogenous gene. Shown are means with standard deviations, which were analyzed using an unpaired t-test against the wild-type (**, p

    Techniques Used: Sequencing, Polymerase Chain Reaction, Mutagenesis, Plaque Assay, Infection, Quantitative RT-PCR

    Interference of MERS-CoV 4b protein in NF-κB-karyopherin 4 binding during infection. Huh-7 cells were transfected with a plasmid encoding karyopherin 4 (KPNA4)-FLAG. At 24 hpt, cells were infected (MOI 0.1 PFU/cell) with WT, Δ4b or 4b-NLS mutants. At 20 hpi, cells lysates were immunoprecipitated with anti-FLAG antibodies. Cell lysates (CL) and eluted proteins were analyzed by immunoblotting with indicated antibodies.
    Figure Legend Snippet: Interference of MERS-CoV 4b protein in NF-κB-karyopherin 4 binding during infection. Huh-7 cells were transfected with a plasmid encoding karyopherin 4 (KPNA4)-FLAG. At 24 hpt, cells were infected (MOI 0.1 PFU/cell) with WT, Δ4b or 4b-NLS mutants. At 20 hpi, cells lysates were immunoprecipitated with anti-FLAG antibodies. Cell lysates (CL) and eluted proteins were analyzed by immunoblotting with indicated antibodies.

    Techniques Used: Binding Assay, Infection, Transfection, Plasmid Preparation, Immunoprecipitation

    Proposed mechanism of 4b action in MERS-CoV infection. In WT MERS-CoV infection, 4b protein with an intact NLS binds KPNA4 for nuclear translocation thereby outcompeting NF-κB binding to KPNA4, which retains NF-κB in the cytoplasm and inhibits the expression of NF-κB-dependent pro-inflammatory cytokines. In contrast, in the absence of 4b (MERS-CoV-Δ4b) or in the presence of cytoplasmic 4b NLS-mutants (MERS-CoV-4b-mNLS), which do not bind KPNA4, NF-κB is imported into the nucleus by KPNA4, where it binds to DNA κB sites to promote the expression of pro-inflammatory cytokines (TNF-α, IL-6 and IL-8).
    Figure Legend Snippet: Proposed mechanism of 4b action in MERS-CoV infection. In WT MERS-CoV infection, 4b protein with an intact NLS binds KPNA4 for nuclear translocation thereby outcompeting NF-κB binding to KPNA4, which retains NF-κB in the cytoplasm and inhibits the expression of NF-κB-dependent pro-inflammatory cytokines. In contrast, in the absence of 4b (MERS-CoV-Δ4b) or in the presence of cytoplasmic 4b NLS-mutants (MERS-CoV-4b-mNLS), which do not bind KPNA4, NF-κB is imported into the nucleus by KPNA4, where it binds to DNA κB sites to promote the expression of pro-inflammatory cytokines (TNF-α, IL-6 and IL-8).

    Techniques Used: Infection, Translocation Assay, Binding Assay, Expressing

    NF-κB-dependent cytokine response during infection with MERS-CoV-4b-NLS mutants (MOI = 1 PFU/cell, 24 hpi). (A) The mRNA expression levels of IL-6, IL-8 and TNF-α were quantified by RT-qPCR in Huh-7 cells either mock-treated or treated with the NF-κB inhibitor parthenolide (12 μM), as described in Fig 1 . Error bars represent SD. (B) Viral titers in the supernatant of Huh-7 cells infected with MERS-CoV-4b-NLS mutants either mock-treated or treated with parthenolide. (C) The mRNA expression levels of IL6, IL8, TNF-α and IFNB1 were quantified by RT-qPCR in Calu-3 cells infected with MERS-CoV-4b-NLS mutants as described in S1 Fig . (D) Analysis by Western-blot of NF-κB p65 levels at 24 hpi in Huh-7 cells infected with 4b-NLS mutants (MOI = 1 PFU/cell). (E) Analysis by Western-blot of TNF-α induced IκBα degradation in Huh-7 cells either mock-infected or infected with MERS.CoV-4b-NLS mutants (MOI = 1 PFU/cell). At 14 hpi, cell supernatant was replaced by fresh medium containing TNF-α (50 ng/ml). After 30 min treatment, cell lysates were prepared for immunoblotting with anti IκBα antibody. Actin was used as a loading control. Shown are means with standard deviations, which were analyzed using an unpaired t-test against the wild-type (*, p
    Figure Legend Snippet: NF-κB-dependent cytokine response during infection with MERS-CoV-4b-NLS mutants (MOI = 1 PFU/cell, 24 hpi). (A) The mRNA expression levels of IL-6, IL-8 and TNF-α were quantified by RT-qPCR in Huh-7 cells either mock-treated or treated with the NF-κB inhibitor parthenolide (12 μM), as described in Fig 1 . Error bars represent SD. (B) Viral titers in the supernatant of Huh-7 cells infected with MERS-CoV-4b-NLS mutants either mock-treated or treated with parthenolide. (C) The mRNA expression levels of IL6, IL8, TNF-α and IFNB1 were quantified by RT-qPCR in Calu-3 cells infected with MERS-CoV-4b-NLS mutants as described in S1 Fig . (D) Analysis by Western-blot of NF-κB p65 levels at 24 hpi in Huh-7 cells infected with 4b-NLS mutants (MOI = 1 PFU/cell). (E) Analysis by Western-blot of TNF-α induced IκBα degradation in Huh-7 cells either mock-infected or infected with MERS.CoV-4b-NLS mutants (MOI = 1 PFU/cell). At 14 hpi, cell supernatant was replaced by fresh medium containing TNF-α (50 ng/ml). After 30 min treatment, cell lysates were prepared for immunoblotting with anti IκBα antibody. Actin was used as a loading control. Shown are means with standard deviations, which were analyzed using an unpaired t-test against the wild-type (*, p

    Techniques Used: Infection, Expressing, Quantitative RT-PCR, Western Blot

    MERS-CoV 4b protein interacts with importin-α2 family karyopherins. (A-B). 4b interacts with KPNA3 and KPNA4 when expressed in isolation. Huh-7 cells were transfected with plasmids expressing NSP15-3XFLAG or 4b-3XFLAG (A) or KPNA4-FLAG and 4a-HA or 4b-HA (B). Cells were collected 48 hours later and cell lysates were immunoprecipitated with anti-FLAG monoclonal antibody. Cell lysates (CL) and eluted proteins were analyzed by immunoblotting with indicated antibodies. (C) 4b interacts with KPNA4 during infection. Huh-7 cells were transfected with plasmids expressing GFP or KPNA4-FLAG (K4-FL). 48 hours later cells were infected with MERS-CoV at an MOI of 0.1 PFU/cell. Cells were collected at 20 hpi and cell lysates were immunoprecipitated and analyzed as described above. (D) 4b requires both NLS Site 1 and Site 2 for efficient interaction with KPNA4. Huh-7 cells were transfected with plasmids expressing sMacro-3XFLAG (C-), 4b-3XFLAG (4b), or the 4b-NLS mutants 4b-mNLS-S1–3XFLAG (NLS-S1), or 4b-mNLS-S2–3XFLAG (NLS-S2). Cells were collected 48 hours later and cell lysates were immunoprecipitated and analyzed as described above. Viral protein 4a and cell proteins actin and GAPDH have been used as controls for non-specific binding to KPNA4-FLAG or to FLAG-antibody coated beads. GFP has been used as a control for 4b non-nonspecific binding to the FLAG-antibody coated beads in the absence of karyopherin protein.
    Figure Legend Snippet: MERS-CoV 4b protein interacts with importin-α2 family karyopherins. (A-B). 4b interacts with KPNA3 and KPNA4 when expressed in isolation. Huh-7 cells were transfected with plasmids expressing NSP15-3XFLAG or 4b-3XFLAG (A) or KPNA4-FLAG and 4a-HA or 4b-HA (B). Cells were collected 48 hours later and cell lysates were immunoprecipitated with anti-FLAG monoclonal antibody. Cell lysates (CL) and eluted proteins were analyzed by immunoblotting with indicated antibodies. (C) 4b interacts with KPNA4 during infection. Huh-7 cells were transfected with plasmids expressing GFP or KPNA4-FLAG (K4-FL). 48 hours later cells were infected with MERS-CoV at an MOI of 0.1 PFU/cell. Cells were collected at 20 hpi and cell lysates were immunoprecipitated and analyzed as described above. (D) 4b requires both NLS Site 1 and Site 2 for efficient interaction with KPNA4. Huh-7 cells were transfected with plasmids expressing sMacro-3XFLAG (C-), 4b-3XFLAG (4b), or the 4b-NLS mutants 4b-mNLS-S1–3XFLAG (NLS-S1), or 4b-mNLS-S2–3XFLAG (NLS-S2). Cells were collected 48 hours later and cell lysates were immunoprecipitated and analyzed as described above. Viral protein 4a and cell proteins actin and GAPDH have been used as controls for non-specific binding to KPNA4-FLAG or to FLAG-antibody coated beads. GFP has been used as a control for 4b non-nonspecific binding to the FLAG-antibody coated beads in the absence of karyopherin protein.

    Techniques Used: Isolation, Transfection, Expressing, Immunoprecipitation, Infection, Binding Assay

    Generation of MERS-CoV-4b-NLS mutants. (A) Schematic representation of 4a and 4b overlapping sequences in MERS-CoV WT (top) and 4b-NLS mutants (bottom). Nuclear localization signals in 4b protein (NLS-S1 and NLS-S2), consisting of positively charged amino acids lysine (K) and arginine (R) are indicated. The 189 nt-sequence duplicated in 4b-NLS mutants is indicated by the shadowed area and the arrows. DUP, mutant including WT duplicated 4a-4b sequences, used as a control; DUP-mNLS-S1, mutant with 4b NLS Site 1 mutated to alanine; DUP-mNLS-S2, mutant with Site 2 and two close Lys residues mutated to alanine, as described in Materials and Methods. The light blue boxes in DUP, DUP-mNLS-S1 and DUP-mNLS-S2 mutants indicate duplicated 4a sequences. PacI and NheI restriction sites used for the assembly of infectious cDNA clones and their genomic positions (first nucleotide of the recognition sequence) are indicated. (B) Analysis by confocal microscopy of 4b subcellular localization in Huh-7 cells either mock-infected or infected with MERS-CoV-4b-NLS mutants (MOI = 0.1 PFU/cell, 24 hpi). 4b protein (green) and dsRNA (red) were detected with specific antibodies, while nuclei (blue) were stained with DAPI. (C) Growth kinetics of MERS-CoV-4b-NLS mutants in Huh-7 and Calu-3 cells at an MOI of 0.1. Supernatants were collected at 24, 48 and 72 hpi and titrated by plaque assay. Error bars represent SD.
    Figure Legend Snippet: Generation of MERS-CoV-4b-NLS mutants. (A) Schematic representation of 4a and 4b overlapping sequences in MERS-CoV WT (top) and 4b-NLS mutants (bottom). Nuclear localization signals in 4b protein (NLS-S1 and NLS-S2), consisting of positively charged amino acids lysine (K) and arginine (R) are indicated. The 189 nt-sequence duplicated in 4b-NLS mutants is indicated by the shadowed area and the arrows. DUP, mutant including WT duplicated 4a-4b sequences, used as a control; DUP-mNLS-S1, mutant with 4b NLS Site 1 mutated to alanine; DUP-mNLS-S2, mutant with Site 2 and two close Lys residues mutated to alanine, as described in Materials and Methods. The light blue boxes in DUP, DUP-mNLS-S1 and DUP-mNLS-S2 mutants indicate duplicated 4a sequences. PacI and NheI restriction sites used for the assembly of infectious cDNA clones and their genomic positions (first nucleotide of the recognition sequence) are indicated. (B) Analysis by confocal microscopy of 4b subcellular localization in Huh-7 cells either mock-infected or infected with MERS-CoV-4b-NLS mutants (MOI = 0.1 PFU/cell, 24 hpi). 4b protein (green) and dsRNA (red) were detected with specific antibodies, while nuclei (blue) were stained with DAPI. (C) Growth kinetics of MERS-CoV-4b-NLS mutants in Huh-7 and Calu-3 cells at an MOI of 0.1. Supernatants were collected at 24, 48 and 72 hpi and titrated by plaque assay. Error bars represent SD.

    Techniques Used: Sequencing, Mutagenesis, Clone Assay, Confocal Microscopy, Infection, Staining, Plaque Assay

    Subcellular localization of NF-κB during infection with MERS-CoV 4b-NLS mutants. Huh-7 (A) or Calu-3 (B) cells were mock-infected or infected (MOI = 0.1 PFU/cell) with WT, Δ4b or 4b-NLS mutants. At 18 hpi, cells were fractionated into cytoplasmic (C) and nuclear (N) fractions and analyzed by Western-blot for 4b and p65 detection. GAPDH and histone H3 were used as cytoplasmic and nuclear markers, respectively. Huh-7 (C) or Calu-3 (D) cells were infected with WT, Δ4b or 4b-NLS mutants (MOI 0.1 PFU/cell). At 24 hpi, cells were fixed and stained with antibodies against 4b (green) and p65 (red). Cell nuclei were stained with DAPI (blue).
    Figure Legend Snippet: Subcellular localization of NF-κB during infection with MERS-CoV 4b-NLS mutants. Huh-7 (A) or Calu-3 (B) cells were mock-infected or infected (MOI = 0.1 PFU/cell) with WT, Δ4b or 4b-NLS mutants. At 18 hpi, cells were fractionated into cytoplasmic (C) and nuclear (N) fractions and analyzed by Western-blot for 4b and p65 detection. GAPDH and histone H3 were used as cytoplasmic and nuclear markers, respectively. Huh-7 (C) or Calu-3 (D) cells were infected with WT, Δ4b or 4b-NLS mutants (MOI 0.1 PFU/cell). At 24 hpi, cells were fixed and stained with antibodies against 4b (green) and p65 (red). Cell nuclei were stained with DAPI (blue).

    Techniques Used: Infection, Western Blot, Staining

    MERS-CoV 4b has higher affinity for interaction with karyopherin-α4 than other karyopherins. (A) Huh-7 cells were co-transfected with plasmids expressing 4b-HA and empty vector (EV) or Control-3XFLAG (C-) or KPNA-FLAG proteins KPNA1-FLAG (K1), KPNA2-FLAG (K2), KPNA3-FLAG (K3), or KPNA4-FLAG (K4). Cells were collected 48 hours later and cell lysates (CL) were immunoprecipitated with anti-FLAG monoclonal antibody. Cell lysates and eluted proteins were analyzed by immunoblotting with indicated antibodies. (B) Quantification of 4b-karyopherin interactions. The relative binding of 4b to each karyopherin was determined by measuring the HA signal and dividing by the FLAG signal, derived using Image Studio Software. The ratio of 4b-HA to KPNA1-FLAG was set to 1. Data represent the combined results of 2 independent experiments. Error bars represent SD. (C) Huh-7 cells were transfected with plasmids expressing GFP-3XFLAG (C-) or KPNA-FLAG proteins KPNA1-FL (K1), KPNA2-FL (K2), KPNA3-FL (K3), or KPNA4-FL (K4). 48 hours later, cells were infected with WT MERS-CoV at a MOI of 0.1 PFU/cell. Cells were collected at 20 hpi and cell lysates were immunoprecipitated with anti-FLAG monoclonal antibody and analyzed as described in (A). Cell protein RuvBL1 has been used as a control for non-specific binding to KPNA4-FLAG or to FLAG-antibody coated beads. GFP and C- have been used as controls for 4b non-nonspecific binding to the FLAG-antibody coated beads in the absence of karyopherin protein. (D) The relative binding of 4b to each karyopherin during infection was determined as described in (B). These data represent the combined results of 2 independent experiments. Error bars represent SD.
    Figure Legend Snippet: MERS-CoV 4b has higher affinity for interaction with karyopherin-α4 than other karyopherins. (A) Huh-7 cells were co-transfected with plasmids expressing 4b-HA and empty vector (EV) or Control-3XFLAG (C-) or KPNA-FLAG proteins KPNA1-FLAG (K1), KPNA2-FLAG (K2), KPNA3-FLAG (K3), or KPNA4-FLAG (K4). Cells were collected 48 hours later and cell lysates (CL) were immunoprecipitated with anti-FLAG monoclonal antibody. Cell lysates and eluted proteins were analyzed by immunoblotting with indicated antibodies. (B) Quantification of 4b-karyopherin interactions. The relative binding of 4b to each karyopherin was determined by measuring the HA signal and dividing by the FLAG signal, derived using Image Studio Software. The ratio of 4b-HA to KPNA1-FLAG was set to 1. Data represent the combined results of 2 independent experiments. Error bars represent SD. (C) Huh-7 cells were transfected with plasmids expressing GFP-3XFLAG (C-) or KPNA-FLAG proteins KPNA1-FL (K1), KPNA2-FL (K2), KPNA3-FL (K3), or KPNA4-FL (K4). 48 hours later, cells were infected with WT MERS-CoV at a MOI of 0.1 PFU/cell. Cells were collected at 20 hpi and cell lysates were immunoprecipitated with anti-FLAG monoclonal antibody and analyzed as described in (A). Cell protein RuvBL1 has been used as a control for non-specific binding to KPNA4-FLAG or to FLAG-antibody coated beads. GFP and C- have been used as controls for 4b non-nonspecific binding to the FLAG-antibody coated beads in the absence of karyopherin protein. (D) The relative binding of 4b to each karyopherin during infection was determined as described in (B). These data represent the combined results of 2 independent experiments. Error bars represent SD.

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Binding Assay, Derivative Assay, Software, Infection

    16) Product Images from "Host Acyl Coenzyme A Binding Protein Regulates Replication Complex Assembly and Activity of a Positive-Strand RNA Virus"

    Article Title: Host Acyl Coenzyme A Binding Protein Regulates Replication Complex Assembly and Activity of a Positive-Strand RNA Virus

    Journal: Journal of Virology

    doi: 10.1128/JVI.06701-11

    Formation of aberrant spherules that are smaller but 4-fold more abundant in cells lacking ACB1. Yeast cells expressing BMV 1a were examined by electron microscopy following osmium staining. (A) Spherules formed in wt cells. (B to E) Smaller (38.9 ± 11.0 nm) but more abundant (23.9 ± 7.8/cell section) spherules were formed in acb1 Δ cells. Labels indicate nucleus (Nuc) and cytoplasm (Cyto).
    Figure Legend Snippet: Formation of aberrant spherules that are smaller but 4-fold more abundant in cells lacking ACB1. Yeast cells expressing BMV 1a were examined by electron microscopy following osmium staining. (A) Spherules formed in wt cells. (B to E) Smaller (38.9 ± 11.0 nm) but more abundant (23.9 ± 7.8/cell section) spherules were formed in acb1 Δ cells. Labels indicate nucleus (Nuc) and cytoplasm (Cyto).

    Techniques Used: Expressing, Electron Microscopy, Staining

    17) Product Images from "Tubulin Polymerization-promoting Protein (TPPP/p25?) Promotes Unconventional Secretion of ?-Synuclein through Exophagy by Impairing Autophagosome-Lysosome Fusion *"

    Article Title: Tubulin Polymerization-promoting Protein (TPPP/p25?) Promotes Unconventional Secretion of ?-Synuclein through Exophagy by Impairing Autophagosome-Lysosome Fusion *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.401174

    Secretion of α-synuclein is mediated by compartments with late endosomal/amphisomal characteristics. A, p25α-expressing PC12 cells were incubated with Alexa 568-conjugated annexin-V on ice before fixation and indirect immunofluorescence
    Figure Legend Snippet: Secretion of α-synuclein is mediated by compartments with late endosomal/amphisomal characteristics. A, p25α-expressing PC12 cells were incubated with Alexa 568-conjugated annexin-V on ice before fixation and indirect immunofluorescence

    Techniques Used: Expressing, Incubation, Immunofluorescence

    18) Product Images from "Akt determines cell fate through the negative regulation of the PERK-eIF2? phosphorylation pathway"

    Article Title: Akt determines cell fate through the negative regulation of the PERK-eIF2? phosphorylation pathway

    Journal: Science signaling

    doi: 10.1126/scisignal.2001630

    Akt inactivates PERK by phosphorylation at threonine 799 ( A ) Sequence alignment of putative Akt phosphorylation sites in mouse PERK. (B) MS2 spectrum of SREGpTSSSIVFEDSGCGNASSK phosphopeptide of mouse PERK phosphorylated by Akt1 subjected to in-gel digestion with trypsin. The analysis was performed on a LTQ linear ion trap mass spectrometer. MS/MS spectra were assigned with MASCOT as described in Materials and Methods. Scaffold was used to filter MS/MS based identification and annotate MS/MS spectra. The spectrum shows a peptide, which contains the phosphothreonine 799 residue identified by the analysis of the peptide’s y and b fragments. An intense neutral loss of phosphoric acid (−98) was observed from the precursor ion (peaks at 1122.63). Alignment of the amino acid sequence surrounding the T799 phosphorylation site indicates its conservation in mouse, human and rat PERK orthologs. ( C ) Cos-1 cells (5×10 5 ) were transfected with 5 μg of pcDNA-empty vector DNA (lane 1) or 5 μg of pcDNA-vector containing either Myc-tagged mouse PERK wild type (WT) cDNA (lane 2) or Myc-tagged mouse PERK T799A cDNA (lane 3). Forty-eight hours after transfection, protein extracts (500 μg) were subjected to immunoprecipitation with anti-Myc antibody followed by immunoblotting with anti-phospho S/T Akt substrate antibody (top panel) or anti-Myc antibody (bottom panel). ( D ) Cos1 cells (5×10 5 ) were transfected with 2.5 μg of pcDNA vector containing Myc-Akt1 cDNA (lanes 1–3) and 2.5 μg of pcDNA-vector containing Myc-PERK WT cDNA (lane 1), Myc-PERK T799A cDNA (lane 2) or pcDNA vector alone (lane 3). Forty-eight hours after transfection, protein extracts (50 μg) were immunoblotted for the indicated proteins. The ratio of phosphorylated Myc-PERK to total Myc-PERK as well as eIF2αP to total eIF2α for each lane is indicated. Panels C and D are representative of three independent experiments.
    Figure Legend Snippet: Akt inactivates PERK by phosphorylation at threonine 799 ( A ) Sequence alignment of putative Akt phosphorylation sites in mouse PERK. (B) MS2 spectrum of SREGpTSSSIVFEDSGCGNASSK phosphopeptide of mouse PERK phosphorylated by Akt1 subjected to in-gel digestion with trypsin. The analysis was performed on a LTQ linear ion trap mass spectrometer. MS/MS spectra were assigned with MASCOT as described in Materials and Methods. Scaffold was used to filter MS/MS based identification and annotate MS/MS spectra. The spectrum shows a peptide, which contains the phosphothreonine 799 residue identified by the analysis of the peptide’s y and b fragments. An intense neutral loss of phosphoric acid (−98) was observed from the precursor ion (peaks at 1122.63). Alignment of the amino acid sequence surrounding the T799 phosphorylation site indicates its conservation in mouse, human and rat PERK orthologs. ( C ) Cos-1 cells (5×10 5 ) were transfected with 5 μg of pcDNA-empty vector DNA (lane 1) or 5 μg of pcDNA-vector containing either Myc-tagged mouse PERK wild type (WT) cDNA (lane 2) or Myc-tagged mouse PERK T799A cDNA (lane 3). Forty-eight hours after transfection, protein extracts (500 μg) were subjected to immunoprecipitation with anti-Myc antibody followed by immunoblotting with anti-phospho S/T Akt substrate antibody (top panel) or anti-Myc antibody (bottom panel). ( D ) Cos1 cells (5×10 5 ) were transfected with 2.5 μg of pcDNA vector containing Myc-Akt1 cDNA (lanes 1–3) and 2.5 μg of pcDNA-vector containing Myc-PERK WT cDNA (lane 1), Myc-PERK T799A cDNA (lane 2) or pcDNA vector alone (lane 3). Forty-eight hours after transfection, protein extracts (50 μg) were immunoblotted for the indicated proteins. The ratio of phosphorylated Myc-PERK to total Myc-PERK as well as eIF2αP to total eIF2α for each lane is indicated. Panels C and D are representative of three independent experiments.

    Techniques Used: Sequencing, Mass Spectrometry, Transfection, Plasmid Preparation, Immunoprecipitation

    Akt downregulates PERK activity and eIF2αP in mouse mammary gland tumors in vivo Mammary tumors from transgenic mice expressing either NDL 2–5 alone (NDL-CON; #8109, #8398) or NDL 2–5 together with hyperactive Akt1-DD (NDL-Akt1DD; #1098, #1302) subjected to IHC analysis for PERK phosphorylated at T980 (PERK-pT980), Akt phsophorylated at S473 (Akt-pS473) as well as hematoxylin and eosin (H E) staining (left panel). Lysates of mammary tumors from NDL-CON or NDL-Akt1DD mice were immunoblotted for the indicated proteins. The ratio of phosphorylated PERK to total PERK, phosphorylated Akt to total Akt and eIF2αP to total eIF2α for each lane is indicated (right panel).
    Figure Legend Snippet: Akt downregulates PERK activity and eIF2αP in mouse mammary gland tumors in vivo Mammary tumors from transgenic mice expressing either NDL 2–5 alone (NDL-CON; #8109, #8398) or NDL 2–5 together with hyperactive Akt1-DD (NDL-Akt1DD; #1098, #1302) subjected to IHC analysis for PERK phosphorylated at T980 (PERK-pT980), Akt phsophorylated at S473 (Akt-pS473) as well as hematoxylin and eosin (H E) staining (left panel). Lysates of mammary tumors from NDL-CON or NDL-Akt1DD mice were immunoblotted for the indicated proteins. The ratio of phosphorylated PERK to total PERK, phosphorylated Akt to total Akt and eIF2αP to total eIF2α for each lane is indicated (right panel).

    Techniques Used: Activity Assay, In Vivo, Transgenic Assay, Mouse Assay, Expressing, Immunohistochemistry, Staining

    19) Product Images from "PBF509, an Adenosine A2A Receptor Antagonist With Efficacy in Rodent Models of Movement Disorders"

    Article Title: PBF509, an Adenosine A2A Receptor Antagonist With Efficacy in Rodent Models of Movement Disorders

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2018.01200

    Immunoreactivity of A 2A R in the striatum of 6-OHDA-lesioned rats. (A) Immunoblot analysis of TH and A 2A R density in striatal membranes from control (R) and 6-OHDA lesioned (L) striatal hemisphere. Striatal membranes were analyzed by SDS–PAGE and immunoblotted using a rabbit anti-TH polyclonal antibody (1 μg/ml), a mouse anti-A 2A R monoclonal antibody (1 μg/ml) and a rabbit anti-α-actinin polyclonal antibody (1 μg/ml). A HRP-conjugated anti-rabbit or anti-mouse IgG (1/30,000) was used as a secondary antibody. The immunoreactive bands were visualized by chemiluminescence. Load control used for quantitating was α-actinin. A representative blot for three different lesioned animals is shown. (B) Quantification of TH and A 2A R density in striatal membranes from control (R) and 6-OHDA lesioned (L) striatal hemisphere. The immunoreactive bands corresponding to TH and A 2A R in each striatal hemisphere were normalized by α-actinin immunoreactivity. Data are expressed as percentage of the control (R) TH or A 2A R density ± SEM of three independent experiments. Asterisks indicate data significantly different from the control condition: ∗∗∗ P
    Figure Legend Snippet: Immunoreactivity of A 2A R in the striatum of 6-OHDA-lesioned rats. (A) Immunoblot analysis of TH and A 2A R density in striatal membranes from control (R) and 6-OHDA lesioned (L) striatal hemisphere. Striatal membranes were analyzed by SDS–PAGE and immunoblotted using a rabbit anti-TH polyclonal antibody (1 μg/ml), a mouse anti-A 2A R monoclonal antibody (1 μg/ml) and a rabbit anti-α-actinin polyclonal antibody (1 μg/ml). A HRP-conjugated anti-rabbit or anti-mouse IgG (1/30,000) was used as a secondary antibody. The immunoreactive bands were visualized by chemiluminescence. Load control used for quantitating was α-actinin. A representative blot for three different lesioned animals is shown. (B) Quantification of TH and A 2A R density in striatal membranes from control (R) and 6-OHDA lesioned (L) striatal hemisphere. The immunoreactive bands corresponding to TH and A 2A R in each striatal hemisphere were normalized by α-actinin immunoreactivity. Data are expressed as percentage of the control (R) TH or A 2A R density ± SEM of three independent experiments. Asterisks indicate data significantly different from the control condition: ∗∗∗ P

    Techniques Used: SDS Page

    20) Product Images from "GGA1 regulates signal-dependent sorting of BACE1 to recycling endosomes, which moderates Aβ production"

    Article Title: GGA1 regulates signal-dependent sorting of BACE1 to recycling endosomes, which moderates Aβ production

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E17-05-0270

    BACE1 phospho-mutants show differences in steady-state distribution and cell-surface expression in HeLa cells. (A) Schematic representation of BACE1 showing the luminal, transmembrane, and cytoplasmic domains. Ser498 in BACE1 was substituted with either an alanine or aspartate to mimic an unphosphorylated (green) or phosphorylated (blue) form of BACE1. (B) Immunoblotting of cell extracts of HeLa cells transfected with either wtBACE1 or BACE1 phosphomutants for 24 h and probed with rabbit anti-pSer498 BACE1 antibodies, rabbit anti-BACE1 antibodies, and mouse anti–α-tubulin antibodies, using a chemiluminescence detection system. (C, D) Confocal microscopic images of fixed and permeabilized HeLa cells transfected with either wtBACE1 or BACE1 phosphomutants and stained with rabbit polyclonal anti-human BACE1 antibodies (red) and mouse monoclonal antibodies to (C) Rab 11 or (D) EEA1 (green). Higher magnifications of the merge images are also shown. Bars represent 10 µm. (E–H) Percentage of BACE1 at the early endosomes, recycling endosomes, late endosomes, or the TGN was calculated from the percentage of total BACE1 pixels that overlapped with (E) Rab11, (F) EEA1, (G) CD63, or (H) golgin97, respectively. All calculations were performed using the OBCOL plug-in on ImageJ ( n = 15 for each marker from three independent experiments). (I) PulSA analyses. HeLa cells transfected with either wtBACE1 or BACE1 phosphomutants were harvested, fixed, and permeabilized; stained with rabbit polyclonal anti-human BACE1 antibodies; and analzyed by flow cytometry (FACS) for the pulse width of the fluorescent signal. Histograms show the mean pulse width and SEM from three independent experiments. (J) Cell-surface expression of HeLa cells transfected with either wtBACE1 or BACE1 phosphomutants. Viable cells in suspension were incubated with anti-BACE1 antibodies on ice for 30 min, fixed in 4% PFA, stained with Alexa488-conjugated IgG, and analyzed by FACS. Histograms shows the mean fluorescence intensity of cell-surface BACE1 normalized for the total BACE1 protein level for each BACE1 variant. Shown is the mean and SEM for three independent experiments. Bars represent 10 µm. (E–J) * p
    Figure Legend Snippet: BACE1 phospho-mutants show differences in steady-state distribution and cell-surface expression in HeLa cells. (A) Schematic representation of BACE1 showing the luminal, transmembrane, and cytoplasmic domains. Ser498 in BACE1 was substituted with either an alanine or aspartate to mimic an unphosphorylated (green) or phosphorylated (blue) form of BACE1. (B) Immunoblotting of cell extracts of HeLa cells transfected with either wtBACE1 or BACE1 phosphomutants for 24 h and probed with rabbit anti-pSer498 BACE1 antibodies, rabbit anti-BACE1 antibodies, and mouse anti–α-tubulin antibodies, using a chemiluminescence detection system. (C, D) Confocal microscopic images of fixed and permeabilized HeLa cells transfected with either wtBACE1 or BACE1 phosphomutants and stained with rabbit polyclonal anti-human BACE1 antibodies (red) and mouse monoclonal antibodies to (C) Rab 11 or (D) EEA1 (green). Higher magnifications of the merge images are also shown. Bars represent 10 µm. (E–H) Percentage of BACE1 at the early endosomes, recycling endosomes, late endosomes, or the TGN was calculated from the percentage of total BACE1 pixels that overlapped with (E) Rab11, (F) EEA1, (G) CD63, or (H) golgin97, respectively. All calculations were performed using the OBCOL plug-in on ImageJ ( n = 15 for each marker from three independent experiments). (I) PulSA analyses. HeLa cells transfected with either wtBACE1 or BACE1 phosphomutants were harvested, fixed, and permeabilized; stained with rabbit polyclonal anti-human BACE1 antibodies; and analzyed by flow cytometry (FACS) for the pulse width of the fluorescent signal. Histograms show the mean pulse width and SEM from three independent experiments. (J) Cell-surface expression of HeLa cells transfected with either wtBACE1 or BACE1 phosphomutants. Viable cells in suspension were incubated with anti-BACE1 antibodies on ice for 30 min, fixed in 4% PFA, stained with Alexa488-conjugated IgG, and analyzed by FACS. Histograms shows the mean fluorescence intensity of cell-surface BACE1 normalized for the total BACE1 protein level for each BACE1 variant. Shown is the mean and SEM for three independent experiments. Bars represent 10 µm. (E–J) * p

    Techniques Used: Expressing, Transfection, Staining, Marker, Flow Cytometry, Cytometry, FACS, Incubation, Fluorescence, Variant Assay

    21) Product Images from "The endocytic recycling compartment maintains cargo segregation acquired upon exit from the sorting endosome"

    Article Title: The endocytic recycling compartment maintains cargo segregation acquired upon exit from the sorting endosome

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E15-07-0514

    Segregation of CME and CIE cargoes in the ERC. (A–C) HeLa cells were serum starved for 1 h and then incubated with anti-CD59 antibody (A) and Alexa 568–conjugated transferrin for 30 min at 37°C (B) and then acid stripped. The cells were then incubated in fresh medium for 30 min before fixation. CD59 was visualized with Alexa 488–conjugated mouse secondary antibody (A). The cells were subjected to SIM analysis. Dashed boxes denote the perinuclear area where CD59 and TfR were highly concentrated. Insets, magnified images of the boxed areas. Scale bars, 10 μm. (D, E) HeLa cells plated on glass-bottom MatTek dishes were serum starved for 1 h, followed by incubation of anti-CD59 antibody, as well as of Alexa 647–conjugated transferrin, for 30 min at 37°C. Then the cells were acid stripped and either fixed (D; periphery) or chased in fresh medium for 30 min before fixation (E; perinuclear area). The plates were then incubated with Atto 488–conjugated anti-mouse antibody. dSTORM was performed by acquiring  > 1 × 10 6  localizations during  > 20,000 frames obtained every 50 ms. The single-molecule localizations were reconstructed into a normalized Gaussian image at 10-nm pixel size using the ThunderSTORM plug-in in ImageJ. Scale bar, 500 nm.
    Figure Legend Snippet: Segregation of CME and CIE cargoes in the ERC. (A–C) HeLa cells were serum starved for 1 h and then incubated with anti-CD59 antibody (A) and Alexa 568–conjugated transferrin for 30 min at 37°C (B) and then acid stripped. The cells were then incubated in fresh medium for 30 min before fixation. CD59 was visualized with Alexa 488–conjugated mouse secondary antibody (A). The cells were subjected to SIM analysis. Dashed boxes denote the perinuclear area where CD59 and TfR were highly concentrated. Insets, magnified images of the boxed areas. Scale bars, 10 μm. (D, E) HeLa cells plated on glass-bottom MatTek dishes were serum starved for 1 h, followed by incubation of anti-CD59 antibody, as well as of Alexa 647–conjugated transferrin, for 30 min at 37°C. Then the cells were acid stripped and either fixed (D; periphery) or chased in fresh medium for 30 min before fixation (E; perinuclear area). The plates were then incubated with Atto 488–conjugated anti-mouse antibody. dSTORM was performed by acquiring > 1 × 10 6 localizations during > 20,000 frames obtained every 50 ms. The single-molecule localizations were reconstructed into a normalized Gaussian image at 10-nm pixel size using the ThunderSTORM plug-in in ImageJ. Scale bar, 500 nm.

    Techniques Used: Incubation, Mass Spectrometry

    Linkage between recycling endosomes in the ERC via “membrane bridges” as observed by SIM and 3D dSTORM imaging. (A–E) HeLa cells on coverslips were transiently transfected with GFP-cellubrevin for 18 h, serum starved for 1 h, and then allowed to internalize Alexa 568–conjugated transferrin for 15 min at 37°C, followed by a 15-min incubation in fresh medium before fixation. The sample was subjected to SIM imaging. (F–H) Endogenous Rab11a in the perinuclear area is depicted by epifluorescence microscopy (F) or a reconstructed 3D dSTORM image (G). HeLa cells plated on glass-bottom MatTek dishes were fixed and stained with anti-Rab11a antibody, followed by Alexa 647–conjugated anti-rabbit antibody. The 3D dSTORM was performed by acquiring  > 9 × 10 6  localizations during 40,000 frames obtained every 50 ms. The single-molecule localizations were reconstructed into a normalized Gaussian image at 10-nm pixel size using the ThunderSTORM plug-in in ImageJ. Enlarged images of the dashed box areas in G are shown in (1) and (2). A histogram (H) representing the calculated uncertainty was plotted on frequency distribution graphs with 27 bins, and the mode of the distribution was taken as the lateral precision. (I) The Nyquist resolution.
    Figure Legend Snippet: Linkage between recycling endosomes in the ERC via “membrane bridges” as observed by SIM and 3D dSTORM imaging. (A–E) HeLa cells on coverslips were transiently transfected with GFP-cellubrevin for 18 h, serum starved for 1 h, and then allowed to internalize Alexa 568–conjugated transferrin for 15 min at 37°C, followed by a 15-min incubation in fresh medium before fixation. The sample was subjected to SIM imaging. (F–H) Endogenous Rab11a in the perinuclear area is depicted by epifluorescence microscopy (F) or a reconstructed 3D dSTORM image (G). HeLa cells plated on glass-bottom MatTek dishes were fixed and stained with anti-Rab11a antibody, followed by Alexa 647–conjugated anti-rabbit antibody. The 3D dSTORM was performed by acquiring > 9 × 10 6 localizations during 40,000 frames obtained every 50 ms. The single-molecule localizations were reconstructed into a normalized Gaussian image at 10-nm pixel size using the ThunderSTORM plug-in in ImageJ. Enlarged images of the dashed box areas in G are shown in (1) and (2). A histogram (H) representing the calculated uncertainty was plotted on frequency distribution graphs with 27 bins, and the mode of the distribution was taken as the lateral precision. (I) The Nyquist resolution.

    Techniques Used: Imaging, Transfection, Incubation, Epifluorescence Microscopy, Staining, Mass Spectrometry

    TREs selectively transport cargoes from SE. (A–F) HeLa cells on coverslips were transiently transfected with HA-MICAL-L1 for 18 h and incubated with anti-CD59 antibody for 9 min at 37°C (A–C) or pulsed for 3 min with Alexa 568–conjugated transferrin (D–F). Surface-bound CD59 antibody was removed by acid strip before fixation. Internal CD59 was visualized with Alexa 568–conjugated anti-mouse antibody (B) in the presence of saponin. MICAL-L1 was visualized with rabbit anti-HA antibody and Alexa 488–conjugated anti-rabbit antibody (A). Scale bar, 10 μm. (G–N) HeLa cells transfected with GFP–MICAL-L1 were treated for 30 min with PLD inhibitors and then either incubated with mouse anti-CD59 antibody for 10 min at 37°C, followed by staining with Alexa 568–conjugated anti-mouse antibody (G–J), or incubated with Alexa 568–conjugated transferrin for 10 min at 37°C. Rabenosyn-5 endosomes were detected by rabbit anti–Rabenosyn-5, followed by Alexa 647–conjugated anti-rabbit secondary antibody. All images are maximal projections of ∼20 SIM slices separated by 110 nm in the  Z -axis. Arrows in K–N depict TfR in Rabenosyn-5–containing endosomes that are absent from TREs. Scale bar, 1 μm. The degree of colocalization between different cargoes and Rabenosyn-5 or MICAL-L1 is quantified and summarized in Supplemental Figure S6, D and E.
    Figure Legend Snippet: TREs selectively transport cargoes from SE. (A–F) HeLa cells on coverslips were transiently transfected with HA-MICAL-L1 for 18 h and incubated with anti-CD59 antibody for 9 min at 37°C (A–C) or pulsed for 3 min with Alexa 568–conjugated transferrin (D–F). Surface-bound CD59 antibody was removed by acid strip before fixation. Internal CD59 was visualized with Alexa 568–conjugated anti-mouse antibody (B) in the presence of saponin. MICAL-L1 was visualized with rabbit anti-HA antibody and Alexa 488–conjugated anti-rabbit antibody (A). Scale bar, 10 μm. (G–N) HeLa cells transfected with GFP–MICAL-L1 were treated for 30 min with PLD inhibitors and then either incubated with mouse anti-CD59 antibody for 10 min at 37°C, followed by staining with Alexa 568–conjugated anti-mouse antibody (G–J), or incubated with Alexa 568–conjugated transferrin for 10 min at 37°C. Rabenosyn-5 endosomes were detected by rabbit anti–Rabenosyn-5, followed by Alexa 647–conjugated anti-rabbit secondary antibody. All images are maximal projections of ∼20 SIM slices separated by 110 nm in the Z -axis. Arrows in K–N depict TfR in Rabenosyn-5–containing endosomes that are absent from TREs. Scale bar, 1 μm. The degree of colocalization between different cargoes and Rabenosyn-5 or MICAL-L1 is quantified and summarized in Supplemental Figure S6, D and E.

    Techniques Used: Transfection, Incubation, Stripping Membranes, Staining

    SIM and dSTORM visualization of Rab11a and MICAL-L1–labeled TREs. (A–C) HeLa cells were stained with anti-Rab11a and anti-MICAL-L1 together with the corresponding secondary antibody and imaged by SIM. Scale bar, 10 μm. (D) Reconstructed dSTORM image showing Rab11a and MICAL-L1 in the perinuclear area (a–a′′) and in the periphery (b–b′′). HeLa cells plated on glass-bottom MatTek dishes were fixed and stained with anti-Rab11a and anti–MICAL-L1 antibodies, followed by Atto 488–conjugated anti-rabbit and Alexa 647–conjugated anti-mouse antibody. dSTORM was performed by acquiring  > 1 × 10 6  localizations during  > 20,000 frames obtained every 50 ms. The single-molecule localizations were reconstructed into a normalized Gaussian image with a pixel size of 10 nm using the ThunderSTORM plug-in in ImageJ. The entire cell from where these images are cropped is shown in D (dashed rectangles a and b).
    Figure Legend Snippet: SIM and dSTORM visualization of Rab11a and MICAL-L1–labeled TREs. (A–C) HeLa cells were stained with anti-Rab11a and anti-MICAL-L1 together with the corresponding secondary antibody and imaged by SIM. Scale bar, 10 μm. (D) Reconstructed dSTORM image showing Rab11a and MICAL-L1 in the perinuclear area (a–a′′) and in the periphery (b–b′′). HeLa cells plated on glass-bottom MatTek dishes were fixed and stained with anti-Rab11a and anti–MICAL-L1 antibodies, followed by Atto 488–conjugated anti-rabbit and Alexa 647–conjugated anti-mouse antibody. dSTORM was performed by acquiring > 1 × 10 6 localizations during > 20,000 frames obtained every 50 ms. The single-molecule localizations were reconstructed into a normalized Gaussian image with a pixel size of 10 nm using the ThunderSTORM plug-in in ImageJ. The entire cell from where these images are cropped is shown in D (dashed rectangles a and b).

    Techniques Used: Labeling, Staining, Mass Spectrometry

    22) Product Images from "Wnt/β-catenin signaling determines the vasculogenic fate of post-natal mesenchymal stem cells"

    Article Title: Wnt/β-catenin signaling determines the vasculogenic fate of post-natal mesenchymal stem cells

    Journal: Stem cells (Dayton, Ohio)

    doi: 10.1002/stem.2334

    GSK-3β inhibition is sufficient to induce vasculogenic differentiation of dental pulp stem cells. (A): Western blots for Fzd-6, P-GSK-3β, and GSK-3β from DPSC treated with 10 µM CHIR99021 for up to 24 hours. Alternatively,
    Figure Legend Snippet: GSK-3β inhibition is sufficient to induce vasculogenic differentiation of dental pulp stem cells. (A): Western blots for Fzd-6, P-GSK-3β, and GSK-3β from DPSC treated with 10 µM CHIR99021 for up to 24 hours. Alternatively,

    Techniques Used: Inhibition, Western Blot

    23) Product Images from "Tetraspanin CD82 interaction with cholesterol promotes extracellular vesicle–mediated release of ezrin to inhibit tumour cell movement"

    Article Title: Tetraspanin CD82 interaction with cholesterol promotes extracellular vesicle–mediated release of ezrin to inhibit tumour cell movement

    Journal: Journal of Extracellular Vesicles

    doi: 10.1080/20013078.2019.1692417

    CD82 and its cholesterol-binding differentially regulate cellular release of EVs. (a) Extracellular staining by filipin and Alexa488-conjugated CTxb in Du145 transfectants. Equal number of the cells were cultured on glass coverslips for 2 days, then fixed and labelled with filipin or Alexa488-conjugated CTxb. For filipin staining, intercellular regions were imaged. For CTxb staining, pericellular regions were imaged. Scale bar: 10 µm. (b) Distributions of Annexin V and Annexin A2 in Du145 transfectant cells. Alexa488-conjugated recombinant Annexin V was used for phosphatidylserine labelling, while Annexin-A2 Ab was used for Annexin-A2 staining. Scale bar: 10 μm. (c) Colocalization of Ezrin with GM1 or Annexin A2 in EVs. For Ezrin and GM1 co-staining, the cells were labelled with the Abs, Alexa488-conjugated CTxB and DAPI. For Ezrin and Annexin A2 co-staining, the cells were incubated sequentially with the primary Abs, Cy3-conjugated donkey anti-goat IgG, normal goat IgG and Alexa594-conjugated goat anti-mouse IgG. Images were obtained by confocal microscopy. Scale bar: 10 µm. (d) The cells were seeded in six-well plate at 50% confluence and cultured in DMEM containing 1% exosome-depleted FBS for 2 – 3 days. The culture supernatants were collected, spun at 2000 × g for 10 min to remove cell debris, and then analysed with NanoSight instrument for EV number and size. Data are presented as mean ± SD (n = 3 individual experiments). *: p
    Figure Legend Snippet: CD82 and its cholesterol-binding differentially regulate cellular release of EVs. (a) Extracellular staining by filipin and Alexa488-conjugated CTxb in Du145 transfectants. Equal number of the cells were cultured on glass coverslips for 2 days, then fixed and labelled with filipin or Alexa488-conjugated CTxb. For filipin staining, intercellular regions were imaged. For CTxb staining, pericellular regions were imaged. Scale bar: 10 µm. (b) Distributions of Annexin V and Annexin A2 in Du145 transfectant cells. Alexa488-conjugated recombinant Annexin V was used for phosphatidylserine labelling, while Annexin-A2 Ab was used for Annexin-A2 staining. Scale bar: 10 μm. (c) Colocalization of Ezrin with GM1 or Annexin A2 in EVs. For Ezrin and GM1 co-staining, the cells were labelled with the Abs, Alexa488-conjugated CTxB and DAPI. For Ezrin and Annexin A2 co-staining, the cells were incubated sequentially with the primary Abs, Cy3-conjugated donkey anti-goat IgG, normal goat IgG and Alexa594-conjugated goat anti-mouse IgG. Images were obtained by confocal microscopy. Scale bar: 10 µm. (d) The cells were seeded in six-well plate at 50% confluence and cultured in DMEM containing 1% exosome-depleted FBS for 2 – 3 days. The culture supernatants were collected, spun at 2000 × g for 10 min to remove cell debris, and then analysed with NanoSight instrument for EV number and size. Data are presented as mean ± SD (n = 3 individual experiments). *: p

    Techniques Used: Binding Assay, Staining, Cell Culture, Transfection, Recombinant, Incubation, Confocal Microscopy

    24) Product Images from "Intersection of the Multivesicular Body Pathway and Lipid Homeostasis in RNA Replication by a Positive-Strand RNA Virus ▿"

    Article Title: Intersection of the Multivesicular Body Pathway and Lipid Homeostasis in RNA Replication by a Positive-Strand RNA Virus ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.02031-10

    Supplemented ubiquitin complements BMV RNA replication defect in doa4 Δ cells. (A) Reduced levels of free Ub are complemented by supplemented Ub. Total protein extraction and Western blotting were done as in with monoclonal anti-Ub or anti-Pgk1p
    Figure Legend Snippet: Supplemented ubiquitin complements BMV RNA replication defect in doa4 Δ cells. (A) Reduced levels of free Ub are complemented by supplemented Ub. Total protein extraction and Western blotting were done as in with monoclonal anti-Ub or anti-Pgk1p

    Techniques Used: Protein Extraction, Western Blot

    25) Product Images from "Developmental stage-dependent regulation of spine formation by calcium-calmodulin-dependent protein kinase IIα and Rap1"

    Article Title: Developmental stage-dependent regulation of spine formation by calcium-calmodulin-dependent protein kinase IIα and Rap1

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-13728-y

    Model for the regulation of spine density by CaMKIIα activity. In the initial stage of spine formation, CaMKIIα has not yet been recruited to spines and Rap1 is not activated. In the subsequent step (arrow 1), glutamate receptors and CaMKIIα molecules begin to accumulate in spines, leading to the phosphorylation of synGAP, the dispersion of synGAP from postsynaptic sites, and the enhancement of local Rap1 activity. Increased Rap1 activity negatively regulates spine formation, possibly by destabilizing F-actin in spines (arrow 2). At a later stage, LTP-like spine-promoting mechanisms begin to operate (arrow 3).
    Figure Legend Snippet: Model for the regulation of spine density by CaMKIIα activity. In the initial stage of spine formation, CaMKIIα has not yet been recruited to spines and Rap1 is not activated. In the subsequent step (arrow 1), glutamate receptors and CaMKIIα molecules begin to accumulate in spines, leading to the phosphorylation of synGAP, the dispersion of synGAP from postsynaptic sites, and the enhancement of local Rap1 activity. Increased Rap1 activity negatively regulates spine formation, possibly by destabilizing F-actin in spines (arrow 2). At a later stage, LTP-like spine-promoting mechanisms begin to operate (arrow 3).

    Techniques Used: Activity Assay

    26) Product Images from "Importance of Promyelocytic Leukema Protein (PML) for Kaposi’s Sarcoma-Associated Herpesvirus Lytic Replication"

    Article Title: Importance of Promyelocytic Leukema Protein (PML) for Kaposi’s Sarcoma-Associated Herpesvirus Lytic Replication

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.02324

    Induction of KSHV lytic replication by TPA and NaB treatment. The cells were treated with TPA (25 ng/mL) and NaB (0.6 mM) and incubated for 48 h. The collected cells were then stained with antibodies against the indicated specific proteins followed by an Alexa Fluor ® 488 conjugated IgG (green) and analyzed by a confocal microscope. Different panels show the staining of RTA (A) , K8 (K-bZIP) (B) , and ORF59 (C) with or without TPA/NaB treatment. The cell nuclei were stained with DAPI (blue). The experiment was performed at least three times independently and one representative result is shown.
    Figure Legend Snippet: Induction of KSHV lytic replication by TPA and NaB treatment. The cells were treated with TPA (25 ng/mL) and NaB (0.6 mM) and incubated for 48 h. The collected cells were then stained with antibodies against the indicated specific proteins followed by an Alexa Fluor ® 488 conjugated IgG (green) and analyzed by a confocal microscope. Different panels show the staining of RTA (A) , K8 (K-bZIP) (B) , and ORF59 (C) with or without TPA/NaB treatment. The cell nuclei were stained with DAPI (blue). The experiment was performed at least three times independently and one representative result is shown.

    Techniques Used: Incubation, Staining, Microscopy

    Subcellular distribution of K8 (K-bZIP) in wild-type, PML-knockout, and PML-overexpressing BC3 cells. The cells were collected 48 h after treatment with TPA (25 ng/mL) and NaB (0.6 mM). Then the cells were fixed and stained with an anti-K8 (K-bZIP) Ab (mouse) and an anti-PML (rabbit) followed by Alexa Fluor ® 488 conjugated mouse IgG (green) and an Alexa Fluor ® 548 conjugated rabbit IgG (red), respectively. The cell nuclei were highlighted with DAPI. (A) Percentage of PML dots colocalized with K8 (K-bZIP) in wild-type and PML-overexpressing BC3 cells. The results showed the percentages of K8 (K-bZIP) and PML colocalized dots. PML dots were counted in the lytic induced BC3 and BC3 PML cells. The data are presented as an average of three replicates with error bars representing SEs. ∗ Statistically significant; P
    Figure Legend Snippet: Subcellular distribution of K8 (K-bZIP) in wild-type, PML-knockout, and PML-overexpressing BC3 cells. The cells were collected 48 h after treatment with TPA (25 ng/mL) and NaB (0.6 mM). Then the cells were fixed and stained with an anti-K8 (K-bZIP) Ab (mouse) and an anti-PML (rabbit) followed by Alexa Fluor ® 488 conjugated mouse IgG (green) and an Alexa Fluor ® 548 conjugated rabbit IgG (red), respectively. The cell nuclei were highlighted with DAPI. (A) Percentage of PML dots colocalized with K8 (K-bZIP) in wild-type and PML-overexpressing BC3 cells. The results showed the percentages of K8 (K-bZIP) and PML colocalized dots. PML dots were counted in the lytic induced BC3 and BC3 PML cells. The data are presented as an average of three replicates with error bars representing SEs. ∗ Statistically significant; P

    Techniques Used: Knock-Out, Staining

    Overexpression of PML in BC3 cells. Wild-type BC3 cells were transduced with a halo-tagged PML-encoding retrovirus, and stable PML-expressing cells were established after hygromycin selection. (A) Total protein from the cells were extracted and immunoblotted with an anti (α)-Halo and an α-PML Ab for detection of PML. β-tubulin was used as a loading control. (B) Cells stably expressing PML were fixed and stained with a mouse α-Halo and a rabbit α-PML Ab and signals were visualized via immunofluorescence using an Alexa Fluor ® 488 (green) conjugated anti-mouse IgG and an Alexa Fluor ® 548 (red) conjugated anti-rabbit IgG, respectively. The cell nuclei were stained with DAPI (blue).
    Figure Legend Snippet: Overexpression of PML in BC3 cells. Wild-type BC3 cells were transduced with a halo-tagged PML-encoding retrovirus, and stable PML-expressing cells were established after hygromycin selection. (A) Total protein from the cells were extracted and immunoblotted with an anti (α)-Halo and an α-PML Ab for detection of PML. β-tubulin was used as a loading control. (B) Cells stably expressing PML were fixed and stained with a mouse α-Halo and a rabbit α-PML Ab and signals were visualized via immunofluorescence using an Alexa Fluor ® 488 (green) conjugated anti-mouse IgG and an Alexa Fluor ® 548 (red) conjugated anti-rabbit IgG, respectively. The cell nuclei were stained with DAPI (blue).

    Techniques Used: Over Expression, Transduction, Expressing, Selection, Stable Transfection, Staining, Immunofluorescence

    Establishment of stable PML-knockout BC3 cells. Stable PML-knockout BC3 cells (BC3-PML KO #1 and BC3-PML KO #2) and the control one (BC3-Scrambled KO ) were generated from wild-type BC3 cells by puromycin selection after transduction with lentivirus as described in the Materials and Methods. (A) Western blot analysis of PML expression. Extracted total lysates from wild-type, control, and PML-knockout cells were separated on an SDS–PAGE and immunoblotted with antibodies to PML, DAXX and SP100. Specific proteins were visualized by using HRP conjugated secondary Abs. β-tubulin was used as a loading control. (B) Immunofluorescence analysis. The cells were fixed, permeabilized and stained with a mouse anti-PML Ab followed by an Alexa Fluor ® 546 conjugated anti-mouse IgG (red). The cell nuclei were stained with DAPI (blue).
    Figure Legend Snippet: Establishment of stable PML-knockout BC3 cells. Stable PML-knockout BC3 cells (BC3-PML KO #1 and BC3-PML KO #2) and the control one (BC3-Scrambled KO ) were generated from wild-type BC3 cells by puromycin selection after transduction with lentivirus as described in the Materials and Methods. (A) Western blot analysis of PML expression. Extracted total lysates from wild-type, control, and PML-knockout cells were separated on an SDS–PAGE and immunoblotted with antibodies to PML, DAXX and SP100. Specific proteins were visualized by using HRP conjugated secondary Abs. β-tubulin was used as a loading control. (B) Immunofluorescence analysis. The cells were fixed, permeabilized and stained with a mouse anti-PML Ab followed by an Alexa Fluor ® 546 conjugated anti-mouse IgG (red). The cell nuclei were stained with DAPI (blue).

    Techniques Used: Knock-Out, Generated, Selection, Transduction, Western Blot, Expressing, SDS Page, Immunofluorescence, Staining

    Induction of KSHV lytic replication in BC3 cells stably overexpressing PML. Wild-type and PML-overexpressing cells were treated with TPA (25 ng/mL) and NaB (0.6 mM) and incubated up to 72 h. (A) Immunofluorescence analysis. The cells at 0 or 48 h post-induction were collected and stained with antibodies against the indicated specific proteins followed by an Alexa Fluor ® 488 conjugated IgG (green) and analyzed by a confocal microscope. The cell nuclei were stained with DAPI (blue). (B) Western blot analysis. Total protein was extracted from the cells collected at the indicated time points and immunoblotted with the antibodies against the indicated specific proteins. β-tubulin was used as a loading control.
    Figure Legend Snippet: Induction of KSHV lytic replication in BC3 cells stably overexpressing PML. Wild-type and PML-overexpressing cells were treated with TPA (25 ng/mL) and NaB (0.6 mM) and incubated up to 72 h. (A) Immunofluorescence analysis. The cells at 0 or 48 h post-induction were collected and stained with antibodies against the indicated specific proteins followed by an Alexa Fluor ® 488 conjugated IgG (green) and analyzed by a confocal microscope. The cell nuclei were stained with DAPI (blue). (B) Western blot analysis. Total protein was extracted from the cells collected at the indicated time points and immunoblotted with the antibodies against the indicated specific proteins. β-tubulin was used as a loading control.

    Techniques Used: Stable Transfection, Incubation, Immunofluorescence, Staining, Microscopy, Western Blot

    27) Product Images from "Epstein-Barr virus protein EB2 stimulates cytoplasmic mRNA accumulation by counteracting the deleterious effects of SRp20 on viral mRNAs"

    Article Title: Epstein-Barr virus protein EB2 stimulates cytoplasmic mRNA accumulation by counteracting the deleterious effects of SRp20 on viral mRNAs

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks319

    SRp20, 9G8 and SF2/ASF interacts with the C-terminal half of EB2 in vitro . ( A ) Schematic representation of the EB2 protein. The light gray box represents the nuclear export domain (NES) previously identified in the protein, the dark gray box, the RNA-binding domain (RBD), the white box, the REF interacting domain and the two vertical bars the Nuclear Localization Signals (NLS). Below, the C-terminal and N-terminal deletion mutants (EB2Nter and EB2Cter respectively) are represented. ( B ) 35 S-labelled EB2, EB2Nter or EB2Cter were incubated with purified GST, GST–9G8, GST–SF2/ASF or GST–SRp20 bound to glutathione sepharose beads. The bound proteins were analysed by SDS–PAGE and visualized by autoradiography. In lane 1, the equivalent of one-fifth of the EB2-, EB2Nter- or EB2Cter-expressing rabbit reticulocyte lysate used in each assay was loaded onto the gel. ( C ) 35 S-labelled 9G8, SF2/ASF or SRp20 were incubated with purified GST, GST–EB2Nter or GST–EB2Cter bound to glutathione sepharose beads. The bound proteins were analysed by SDS–PAGE and visualized by autoradiography. In lane 1, the equivalent of one-fifth of the 9G8-, SF2/ASF- or SRp20-expressing rabbit reticulocyte lysate used in each assay was loaded onto the gel.
    Figure Legend Snippet: SRp20, 9G8 and SF2/ASF interacts with the C-terminal half of EB2 in vitro . ( A ) Schematic representation of the EB2 protein. The light gray box represents the nuclear export domain (NES) previously identified in the protein, the dark gray box, the RNA-binding domain (RBD), the white box, the REF interacting domain and the two vertical bars the Nuclear Localization Signals (NLS). Below, the C-terminal and N-terminal deletion mutants (EB2Nter and EB2Cter respectively) are represented. ( B ) 35 S-labelled EB2, EB2Nter or EB2Cter were incubated with purified GST, GST–9G8, GST–SF2/ASF or GST–SRp20 bound to glutathione sepharose beads. The bound proteins were analysed by SDS–PAGE and visualized by autoradiography. In lane 1, the equivalent of one-fifth of the EB2-, EB2Nter- or EB2Cter-expressing rabbit reticulocyte lysate used in each assay was loaded onto the gel. ( C ) 35 S-labelled 9G8, SF2/ASF or SRp20 were incubated with purified GST, GST–EB2Nter or GST–EB2Cter bound to glutathione sepharose beads. The bound proteins were analysed by SDS–PAGE and visualized by autoradiography. In lane 1, the equivalent of one-fifth of the 9G8-, SF2/ASF- or SRp20-expressing rabbit reticulocyte lysate used in each assay was loaded onto the gel.

    Techniques Used: In Vitro, RNA Binding Assay, Incubation, Purification, SDS Page, Autoradiography, Expressing

    SRp20, 9G8 and SF2/ASF coimmunoprecipitate with EB2. V5-tagged 9G8 ( A ), V5-tagged SF2/ASF ( B ), V5-tagged SRp20 ( C ) or c-Myc-tagged NXF1 ( D ) were expressed by transient transfection in HEK293T cells either alone or together with Flag-tagged EB2. Lysates were prepared and immunoprecipitations performed using an M2 anti-Flag mAb affinity gel in presence or absence of RNAse. The immunoprecipitated complexes were then analysed by western blotting using an anti-Flag polyclonal antibody to visualize EB2, an anti-V5 polyclonal antibody to visualize 9G8, SF2/ASF and SRp20 or an anti-c-Myc monoclonal antibody for NXF1. The ‘input’ corresponds to 15% of the material present in the immunoprecipitated lysates.
    Figure Legend Snippet: SRp20, 9G8 and SF2/ASF coimmunoprecipitate with EB2. V5-tagged 9G8 ( A ), V5-tagged SF2/ASF ( B ), V5-tagged SRp20 ( C ) or c-Myc-tagged NXF1 ( D ) were expressed by transient transfection in HEK293T cells either alone or together with Flag-tagged EB2. Lysates were prepared and immunoprecipitations performed using an M2 anti-Flag mAb affinity gel in presence or absence of RNAse. The immunoprecipitated complexes were then analysed by western blotting using an anti-Flag polyclonal antibody to visualize EB2, an anti-V5 polyclonal antibody to visualize 9G8, SF2/ASF and SRp20 or an anti-c-Myc monoclonal antibody for NXF1. The ‘input’ corresponds to 15% of the material present in the immunoprecipitated lysates.

    Techniques Used: Transfection, Immunoprecipitation, Western Blot

    SRp20 is required for the EB2-dependent cytoplasmic accumulation of Renilla luciferase mRNA. ( A ) Schematic representation of the Renilla luciferase intronless-coding vector (pcDNAGlobinRen) showing positions of the CMV promoter and BGH polyadenylation signal. Small arrows indicate the position of the primers used for the PCR. ( B - D ) Immunoblots of HeLa cells co-transfected with pcDNAGlobinRen together with pCI-Flag EB2 as indicated in the figure. Cells were previously transfected with either a control siRNA (lanes 1 and 2 of each panel) or an siRNA specific for SRp20 (panel B, lanes 3 and 4), 9G8 (panel C, lanes 3 and 4) or SF2/ASF (panel D, lanes 3 and 4). The blots were either probed with an M2-Flag mAb to detect Flag-EB2, an anti-SRp20 mAb, an anti-9G8, an anti-SF2/ASF, or an anti-α-Tubulin as a control of total protein amounts. ( E–G ) Quantification of cytoplasmic luciferase encoding mRNAs by RT–qPCR using GAPDH as an internal control. mRNAs were extracted from cells transfected as described in B, C and D respectively. Experiments were made three times and the error bars represent standard deviations.
    Figure Legend Snippet: SRp20 is required for the EB2-dependent cytoplasmic accumulation of Renilla luciferase mRNA. ( A ) Schematic representation of the Renilla luciferase intronless-coding vector (pcDNAGlobinRen) showing positions of the CMV promoter and BGH polyadenylation signal. Small arrows indicate the position of the primers used for the PCR. ( B - D ) Immunoblots of HeLa cells co-transfected with pcDNAGlobinRen together with pCI-Flag EB2 as indicated in the figure. Cells were previously transfected with either a control siRNA (lanes 1 and 2 of each panel) or an siRNA specific for SRp20 (panel B, lanes 3 and 4), 9G8 (panel C, lanes 3 and 4) or SF2/ASF (panel D, lanes 3 and 4). The blots were either probed with an M2-Flag mAb to detect Flag-EB2, an anti-SRp20 mAb, an anti-9G8, an anti-SF2/ASF, or an anti-α-Tubulin as a control of total protein amounts. ( E–G ) Quantification of cytoplasmic luciferase encoding mRNAs by RT–qPCR using GAPDH as an internal control. mRNAs were extracted from cells transfected as described in B, C and D respectively. Experiments were made three times and the error bars represent standard deviations.

    Techniques Used: Luciferase, Plasmid Preparation, Polymerase Chain Reaction, Western Blot, Transfection, Quantitative RT-PCR

    28) Product Images from "Human Th17 Cells Lack HIV-Inhibitory RNases and Are Highly Permissive to Productive HIV Infection"

    Article Title: Human Th17 Cells Lack HIV-Inhibitory RNases and Are Highly Permissive to Productive HIV Infection

    Journal: Journal of Virology

    doi: 10.1128/JVI.02869-15

    Comparison of RNase expression and HIV inhibition in T h 17-polarized cells with those in T h 0-polarized cells. (A) Representative flow cytometry plots depicting IL-17 expression among T h 0-polarized, CCR6 − cells and T h 17-polarized, CCR6 + cells. SSC, side scatter. (B) Box plot showing differential gene expression between T h 0- and T h 17-polarized cells. Total RNA from T h 0- or T h 17-polarized cells was analyzed by using microarrays. Relative levels of gene expression in T h 17-polarized cells and T h 0-polarized cells are shown. (C) Thirty micrograms of total protein from cell lysates of CCR6 − , T h 0-polarized and CCR6 + , T h 17-polarized CD4 T cells from three donors was denatured and separated according to size by SDS-PAGE, transferred to a PVDF membrane, and then probed for the indicated RNase expression with anti-RNase antibodies. The loading control was glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (D) T h 0- and T h 17-polarized CD4 T cells were infected with HIV IIIB , washed, and then resuspended in medium containing the indicated RNases. Percent inhibition was calculated from the percentage of p24 + cells, as measured by flow cytometry.
    Figure Legend Snippet: Comparison of RNase expression and HIV inhibition in T h 17-polarized cells with those in T h 0-polarized cells. (A) Representative flow cytometry plots depicting IL-17 expression among T h 0-polarized, CCR6 − cells and T h 17-polarized, CCR6 + cells. SSC, side scatter. (B) Box plot showing differential gene expression between T h 0- and T h 17-polarized cells. Total RNA from T h 0- or T h 17-polarized cells was analyzed by using microarrays. Relative levels of gene expression in T h 17-polarized cells and T h 0-polarized cells are shown. (C) Thirty micrograms of total protein from cell lysates of CCR6 − , T h 0-polarized and CCR6 + , T h 17-polarized CD4 T cells from three donors was denatured and separated according to size by SDS-PAGE, transferred to a PVDF membrane, and then probed for the indicated RNase expression with anti-RNase antibodies. The loading control was glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (D) T h 0- and T h 17-polarized CD4 T cells were infected with HIV IIIB , washed, and then resuspended in medium containing the indicated RNases. Percent inhibition was calculated from the percentage of p24 + cells, as measured by flow cytometry.

    Techniques Used: Expressing, Inhibition, Flow Cytometry, Cytometry, SDS Page, Infection

    29) Product Images from "Rapid Changes in Connexin-43 in Response to Genotoxic Stress Stabilize Cell-Cell Communication in Corneal Endothelium"

    Article Title: Rapid Changes in Connexin-43 in Response to Genotoxic Stress Stabilize Cell-Cell Communication in Corneal Endothelium

    Journal: Investigative Ophthalmology & Visual Science

    doi: 10.1167/iovs.11-7272

    Rapid accumulation of Cx43 protein in response to genotoxic stress with mitomycin C. Cells were treated with prewarmed media containing 5 μM MMC for indicated times in minutes. ( A ) Western blot for total Cx43 using C-terminal antibody ( top ) and
    Figure Legend Snippet: Rapid accumulation of Cx43 protein in response to genotoxic stress with mitomycin C. Cells were treated with prewarmed media containing 5 μM MMC for indicated times in minutes. ( A ) Western blot for total Cx43 using C-terminal antibody ( top ) and

    Techniques Used: Western Blot

    The stability of preexisting Cx43 is altered after MMC treatment. Cells were treated with prewarmed media containing 10 μg/mL CHX with or without 5 μM MMC for indicated times in minutes. Blot shows total Cx43 using a C-terminal–specific
    Figure Legend Snippet: The stability of preexisting Cx43 is altered after MMC treatment. Cells were treated with prewarmed media containing 10 μg/mL CHX with or without 5 μM MMC for indicated times in minutes. Blot shows total Cx43 using a C-terminal–specific

    Techniques Used:

    Genotoxic stress results in reduced internalization of Cx43 plaques. Seventy percent to 80% confluent cells were transfected with a Cx43–GFP plasmid. ( A ) Twenty-four hours after transfection, cells were treated with either medium (“CTRL,”
    Figure Legend Snippet: Genotoxic stress results in reduced internalization of Cx43 plaques. Seventy percent to 80% confluent cells were transfected with a Cx43–GFP plasmid. ( A ) Twenty-four hours after transfection, cells were treated with either medium (“CTRL,”

    Techniques Used: Transfection, Plasmid Preparation

    MMC induces alteration in Cx43 association with ZO-1. Cells were treated with prewarmed media containing 5 μM MMC for 60 minutes or media alone (CTRL). ( A ) Coimmunoprecipitation with a rabbit polyclonal anti-Cx43 was performed followed by immunoblotting
    Figure Legend Snippet: MMC induces alteration in Cx43 association with ZO-1. Cells were treated with prewarmed media containing 5 μM MMC for 60 minutes or media alone (CTRL). ( A ) Coimmunoprecipitation with a rabbit polyclonal anti-Cx43 was performed followed by immunoblotting

    Techniques Used:

    Changes in Cx43 gap junction plaque size and intensity after genotoxic stress. ( A ) Cells were treated with prewarmed media containing 5 μM MMC for the indicated time in minutes then stained for Cx43 ( green ). DAPI (nuclei). Orange arrows indicate
    Figure Legend Snippet: Changes in Cx43 gap junction plaque size and intensity after genotoxic stress. ( A ) Cells were treated with prewarmed media containing 5 μM MMC for the indicated time in minutes then stained for Cx43 ( green ). DAPI (nuclei). Orange arrows indicate

    Techniques Used: Staining

    30) Product Images from "Characterization of Nuclear Localization Signals (NLSs) and Function of NLSs and Phosphorylation of Serine Residues in Subcellular and Subnuclear Localization of Transformer-2? (Tra2?) *"

    Article Title: Characterization of Nuclear Localization Signals (NLSs) and Function of NLSs and Phosphorylation of Serine Residues in Subcellular and Subnuclear Localization of Transformer-2? (Tra2?) *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.456715

    Effects of RS and RRM domains, NLSs, and phosphorylation on nuclear/cytoplasmic localization of Tra2β in COS-1 cells. Fluorescent signals of GFP (or GFP fusion proteins) and nuclear staining (DAPI, blue ) in the COS-1 cells with expression of various
    Figure Legend Snippet: Effects of RS and RRM domains, NLSs, and phosphorylation on nuclear/cytoplasmic localization of Tra2β in COS-1 cells. Fluorescent signals of GFP (or GFP fusion proteins) and nuclear staining (DAPI, blue ) in the COS-1 cells with expression of various

    Techniques Used: Staining, Expressing

    Effects of RS1 and RS2 domains on subnuclear localization of Tra2β. Confocal microscopy images showing the immunofluorescent signals of GFP or nuclear speckle marker (SC35) and the overlay (MERGE) in the SH-SY5Y cells with expression of various
    Figure Legend Snippet: Effects of RS1 and RS2 domains on subnuclear localization of Tra2β. Confocal microscopy images showing the immunofluorescent signals of GFP or nuclear speckle marker (SC35) and the overlay (MERGE) in the SH-SY5Y cells with expression of various

    Techniques Used: Confocal Microscopy, Marker, Expressing

    Effects of RS and RRM domains on nuclear/cytoplasmic localization of Tra2β in SH-SY5Y cells. A , illustrations of the DNA construct to express the recombinant fusion proteins. The GFP reporter gene was fused in-frame with the full-length Tra2β
    Figure Legend Snippet: Effects of RS and RRM domains on nuclear/cytoplasmic localization of Tra2β in SH-SY5Y cells. A , illustrations of the DNA construct to express the recombinant fusion proteins. The GFP reporter gene was fused in-frame with the full-length Tra2β

    Techniques Used: Construct, Recombinant

    Effects of NLSs and phosphorylation on nuclear/cytoplasmic localization of Tra2β. A , illustrations of the DNA construct to express the GFP-RS1 truncations and mutants. The GFP reporter gene was fused in-frame with the full-length RS1 domain (GFP-RS1)
    Figure Legend Snippet: Effects of NLSs and phosphorylation on nuclear/cytoplasmic localization of Tra2β. A , illustrations of the DNA construct to express the GFP-RS1 truncations and mutants. The GFP reporter gene was fused in-frame with the full-length RS1 domain (GFP-RS1)

    Techniques Used: Construct

    31) Product Images from "S-Adenosylmethionine regulates apoptosis and autophagy in MCF-7 breast cancer cells through the modulation of specific microRNAs"

    Article Title: S-Adenosylmethionine regulates apoptosis and autophagy in MCF-7 breast cancer cells through the modulation of specific microRNAs

    Journal: Cancer Cell International

    doi: 10.1186/s12935-018-0697-6

    Effect of AdoMet and miR-34a mimic or miR-34c mimic on the HDAC1, SIRT1 and p53 expression levels. a Alignment of miR-34a and miR-34c with HDAC1 3′UTR obtained from miRNA-mRNA integration analysis using the TargetScan microRNA target prediction software. b Cells were transfected with miR-34a and miR-34c mimic or inhibitor, in the presence or not (Control) of 500 μMAdoMet for 72 h. Then, 10 μg of cell lysates were subjected to SDS-PAGE, incubated with antibodies against the indicated proteins and analyzed by Western blotting. The housekeeping protein α-tubulin was used as loading control. Graphs show the densitometric intensity of Ac-p53/p53 band ratio. The intensities of signals were expressed as arbitrary units. The images are representative of three immunoblotting analyses obtained from at least three independent experiments. Bars, SDs
    Figure Legend Snippet: Effect of AdoMet and miR-34a mimic or miR-34c mimic on the HDAC1, SIRT1 and p53 expression levels. a Alignment of miR-34a and miR-34c with HDAC1 3′UTR obtained from miRNA-mRNA integration analysis using the TargetScan microRNA target prediction software. b Cells were transfected with miR-34a and miR-34c mimic or inhibitor, in the presence or not (Control) of 500 μMAdoMet for 72 h. Then, 10 μg of cell lysates were subjected to SDS-PAGE, incubated with antibodies against the indicated proteins and analyzed by Western blotting. The housekeeping protein α-tubulin was used as loading control. Graphs show the densitometric intensity of Ac-p53/p53 band ratio. The intensities of signals were expressed as arbitrary units. The images are representative of three immunoblotting analyses obtained from at least three independent experiments. Bars, SDs

    Techniques Used: Expressing, Software, Transfection, SDS Page, Incubation, Western Blot

    32) Product Images from "miR-122 does not impact recognition of the HCV genome by innate sensors of RNA but rather protects the 5′ end from the cellular pyrophosphatases, DOM3Z and DUSP11"

    Article Title: miR-122 does not impact recognition of the HCV genome by innate sensors of RNA but rather protects the 5′ end from the cellular pyrophosphatases, DOM3Z and DUSP11

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky273

    miR-122 binding does not shield the 5′ terminus of HCV RNA against RLR recognition. Huh-7 cells were electroporated with siRIG-I or siControl (siCon) at day –3 and at day 0 cells were electroporated again with the indicated siRNA and either ( A ) wild-type or GNN FL HCV RNA, and a firefly luciferase control mRNA, or ( B ) S1+S2p3 SGR or S1+S2p3 GND SGR, a Renilla luciferase control mRNA, and miR-122p3 (miR-122-dependent) or miCon (miR-122-independent). Replication was measured by evaluating luciferase production at the indicated timepoints. ( C ) Western blot showing knockdown efficiency with antibodies against RIG-I and β-actin. Percent knockdown ± standard deviation relative to siCon is indicated. Huh-7 cells were electroporated with siMDA5 or siControl (siCon) at day –3, at day 0 cells were electroporated again with the indicated siRNA, and either ( D ) wild-type or GNN FL HCV RNA, and a firefly luciferase control mRNA or ( E ) S1+S2p3 SGR or S1+S2p3 GND SGR, a Renilla luciferase control mRNA, and miR-122p3 (miR-122-dependent) or miCon (miR-122-independent). Replication was measured by evaluating luciferase production at the indicated timepoints. ( F ) Quantitative PCR analysis indicating knockdown efficiency using MDA5-specific and GAPDH-specific TaqMan probes. MDA5 mRNA levels were calculated relative to the siCon. ( G ) Huh-7 cells were electroporated with siMDA5 on day –3, treated with 50 IU/ml IFN-α on day –1 and harvested for western blot at day 0 using antibodies against MDA5 and β-actin. Percent knockdown ± standard deviation relative to siCon is indicated. ( H ) Northern blot analysis of FL HCV RNA accumulation during MDA5 knockdown at day 3. ( I ) Densitometry quantification of northern blot analysis in (H) normalized to siCon. Huh-7 cells were electroporated with siLGP2 or siControl (siCon) at day –2, at day 0 cells were electroporated again with the indicated siRNA, and either ( J ) wild-type or GNN FL HCV RNA, and a firefly luciferase control mRNA or ( K ) S1+S2p3 SGR or S1+S2p3 GND SGR, a Renilla luciferase control mRNA, and miR-122p3 (miR-122-dependent) or miCon (miR-122-independent). Replication was measured by evaluating luciferase production at the indicated timepoints. ( L ) Western blot showing knockdown efficiency with antibodies against LGP2 and β-actin. Percent knockdown ± standard deviation relative to siCon is indicated. All data are representative of at least three independent experiments and statistical significance was determined by paired parametric t test.
    Figure Legend Snippet: miR-122 binding does not shield the 5′ terminus of HCV RNA against RLR recognition. Huh-7 cells were electroporated with siRIG-I or siControl (siCon) at day –3 and at day 0 cells were electroporated again with the indicated siRNA and either ( A ) wild-type or GNN FL HCV RNA, and a firefly luciferase control mRNA, or ( B ) S1+S2p3 SGR or S1+S2p3 GND SGR, a Renilla luciferase control mRNA, and miR-122p3 (miR-122-dependent) or miCon (miR-122-independent). Replication was measured by evaluating luciferase production at the indicated timepoints. ( C ) Western blot showing knockdown efficiency with antibodies against RIG-I and β-actin. Percent knockdown ± standard deviation relative to siCon is indicated. Huh-7 cells were electroporated with siMDA5 or siControl (siCon) at day –3, at day 0 cells were electroporated again with the indicated siRNA, and either ( D ) wild-type or GNN FL HCV RNA, and a firefly luciferase control mRNA or ( E ) S1+S2p3 SGR or S1+S2p3 GND SGR, a Renilla luciferase control mRNA, and miR-122p3 (miR-122-dependent) or miCon (miR-122-independent). Replication was measured by evaluating luciferase production at the indicated timepoints. ( F ) Quantitative PCR analysis indicating knockdown efficiency using MDA5-specific and GAPDH-specific TaqMan probes. MDA5 mRNA levels were calculated relative to the siCon. ( G ) Huh-7 cells were electroporated with siMDA5 on day –3, treated with 50 IU/ml IFN-α on day –1 and harvested for western blot at day 0 using antibodies against MDA5 and β-actin. Percent knockdown ± standard deviation relative to siCon is indicated. ( H ) Northern blot analysis of FL HCV RNA accumulation during MDA5 knockdown at day 3. ( I ) Densitometry quantification of northern blot analysis in (H) normalized to siCon. Huh-7 cells were electroporated with siLGP2 or siControl (siCon) at day –2, at day 0 cells were electroporated again with the indicated siRNA, and either ( J ) wild-type or GNN FL HCV RNA, and a firefly luciferase control mRNA or ( K ) S1+S2p3 SGR or S1+S2p3 GND SGR, a Renilla luciferase control mRNA, and miR-122p3 (miR-122-dependent) or miCon (miR-122-independent). Replication was measured by evaluating luciferase production at the indicated timepoints. ( L ) Western blot showing knockdown efficiency with antibodies against LGP2 and β-actin. Percent knockdown ± standard deviation relative to siCon is indicated. All data are representative of at least three independent experiments and statistical significance was determined by paired parametric t test.

    Techniques Used: Binding Assay, Luciferase, Western Blot, Standard Deviation, Real-time Polymerase Chain Reaction, Northern Blot

    DOM3Z and DUSP11 partially localize to the cytoplasm in Huh-7.5 cells. ( A ) Huh-7.5 cells were plated onto 8-well chamber slides and infected with JFH-1 T (MOI = 0.1). After 3 days, cells were fixed and stained for dsRNA, DOM3Z (top panel) or DUSP11 (bottom panel) and DAPI. ( B ) Huh-7.5 cells were infected with JFH-1 T (MOI = 0.1) and h arvested 3 days post-infection. Following subcellular fractionation, cellular localization of the pyrophosphatases was determined by western blot with antibodies against DOM3Z and DUSP11. GAPDH and Lamin A–C were used as cytoplasmic and nuclear markers, respectively. HCV core was used to confirm HCV infection. Percent expression ± standard deviation relative to total expression is indicated. All data are representative of three independent experiments.
    Figure Legend Snippet: DOM3Z and DUSP11 partially localize to the cytoplasm in Huh-7.5 cells. ( A ) Huh-7.5 cells were plated onto 8-well chamber slides and infected with JFH-1 T (MOI = 0.1). After 3 days, cells were fixed and stained for dsRNA, DOM3Z (top panel) or DUSP11 (bottom panel) and DAPI. ( B ) Huh-7.5 cells were infected with JFH-1 T (MOI = 0.1) and h arvested 3 days post-infection. Following subcellular fractionation, cellular localization of the pyrophosphatases was determined by western blot with antibodies against DOM3Z and DUSP11. GAPDH and Lamin A–C were used as cytoplasmic and nuclear markers, respectively. HCV core was used to confirm HCV infection. Percent expression ± standard deviation relative to total expression is indicated. All data are representative of three independent experiments.

    Techniques Used: Infection, Staining, Fractionation, Western Blot, Expressing, Standard Deviation

    33) Product Images from "Regulation of Multiple Stages of Hepadnavirus Replication by the Carboxyl-Terminal Domain of Viral Core Protein in trans"

    Article Title: Regulation of Multiple Stages of Hepadnavirus Replication by the Carboxyl-Terminal Domain of Viral Core Protein in trans

    Journal: Journal of Virology

    doi: 10.1128/JVI.03116-14

    Interactions between CDK2 or importin α and HCTD or DCTD detected by GST pulldown assay. HEK293 cells in 100-mm dishes were transfected with 20 μg of the indicated GST-HCTD or GST-DCTD fusion constructs. Three days posttransfection, cells
    Figure Legend Snippet: Interactions between CDK2 or importin α and HCTD or DCTD detected by GST pulldown assay. HEK293 cells in 100-mm dishes were transfected with 20 μg of the indicated GST-HCTD or GST-DCTD fusion constructs. Three days posttransfection, cells

    Techniques Used: GST Pulldown Assay, Transfection, Construct

    34) Product Images from "Yersinia pestis YopK Inhibits Bacterial Adhesion to Host Cells by Binding to the Extracellular Matrix Adaptor Protein Matrilin-2"

    Article Title: Yersinia pestis YopK Inhibits Bacterial Adhesion to Host Cells by Binding to the Extracellular Matrix Adaptor Protein Matrilin-2

    Journal: Infection and Immunity

    doi: 10.1128/IAI.01069-16

    Expression of YopK Δ91–124 in the Δ yopK strain does not restore a wild-type bacterial adhesion phenotype. (A) The Y. pestis strains were cultured in TMH medium without calcium at 37°C to an OD of 1.0. Bacterial cells and culture supernatants were collected by centrifugation, and the proteins were analyzed by SDS-PAGE and immunoblotting using anti-YopK antibody. YopK Δ91–124 was secreted by Δ yopK -C yopK Δ91–124 at a level comparable to that of YopK secretion by the wild-type Y. pestis . (B) CD profiles in the far-UV range (190 to 250 nm) of YopK and YopK Δ91–124 at 10 μM. The profiles represent the mean CD profiles for 8 measurements per protein. The buffer spectrum was subtracted from the proteins' spectra, and the resulting spectrum was analyzed with the online Dichroweb server. (C) HeLa cells were infected with the indicated strains at an MOI of 100 in the presence of 1 μM cytochalasin D. The percentage of adhesion was determined as described above. (D) HeLa cells were infected as described for panel C. After 2 h of infection, cells were extensively washed in prewarmed PBS to remove unattached bacteria, and the bacteria that adhered to HeLa cells were visualized using anti-F1 antigen monoclonal antibody and donkey anti-mouse IgG secondary antibody conjugated to Alexa Fluor 555. Scale bars in the images represent 9 μm. All experiments were independently performed at least three times in triplicate. Statistical analysis was performed using one-way ANOVA with Dunnett's multiple-comparison tests to analyze the significance of differences in bacterial adhesion between the different strains (*, P
    Figure Legend Snippet: Expression of YopK Δ91–124 in the Δ yopK strain does not restore a wild-type bacterial adhesion phenotype. (A) The Y. pestis strains were cultured in TMH medium without calcium at 37°C to an OD of 1.0. Bacterial cells and culture supernatants were collected by centrifugation, and the proteins were analyzed by SDS-PAGE and immunoblotting using anti-YopK antibody. YopK Δ91–124 was secreted by Δ yopK -C yopK Δ91–124 at a level comparable to that of YopK secretion by the wild-type Y. pestis . (B) CD profiles in the far-UV range (190 to 250 nm) of YopK and YopK Δ91–124 at 10 μM. The profiles represent the mean CD profiles for 8 measurements per protein. The buffer spectrum was subtracted from the proteins' spectra, and the resulting spectrum was analyzed with the online Dichroweb server. (C) HeLa cells were infected with the indicated strains at an MOI of 100 in the presence of 1 μM cytochalasin D. The percentage of adhesion was determined as described above. (D) HeLa cells were infected as described for panel C. After 2 h of infection, cells were extensively washed in prewarmed PBS to remove unattached bacteria, and the bacteria that adhered to HeLa cells were visualized using anti-F1 antigen monoclonal antibody and donkey anti-mouse IgG secondary antibody conjugated to Alexa Fluor 555. Scale bars in the images represent 9 μm. All experiments were independently performed at least three times in triplicate. Statistical analysis was performed using one-way ANOVA with Dunnett's multiple-comparison tests to analyze the significance of differences in bacterial adhesion between the different strains (*, P

    Techniques Used: Expressing, Cell Culture, Centrifugation, SDS Page, Infection

    YopK colocalizes with the endogenous MATN2 on the HeLa cell surface. (A) Equal amounts of purified GFP-YopK, GFP-YopK Δ91–124 , or GFP were added into HeLa cells and incubated at 4°C overnight. Endogenous MATN2 on the HeLa cell surface was visualized using a rabbit anti-MATN2 antibody and a donkey anti-rabbit IgG secondary antibody conjugated to Alexa Fluor 555. All scale bars represent 9 μm. Images were acquired using an UltraVIEW Vox live-cell imaging system, and Pearson's correlation coefficient was used to quantify the degree of colocalization between the two channels (red and green) in a confocal image by using Volocity 6.1 software. (B) The bar graphs were drawn to show average Pearson's correlation coefficients (with error bars) from multiple images ( n = 7 for each treatment) of the HeLa cells incubated with different proteins. One-way ANOVA with Bonferroni's multiple-comparison test was performed to analyze the significance of difference in bacterial adhesion between the different treatments (***, P
    Figure Legend Snippet: YopK colocalizes with the endogenous MATN2 on the HeLa cell surface. (A) Equal amounts of purified GFP-YopK, GFP-YopK Δ91–124 , or GFP were added into HeLa cells and incubated at 4°C overnight. Endogenous MATN2 on the HeLa cell surface was visualized using a rabbit anti-MATN2 antibody and a donkey anti-rabbit IgG secondary antibody conjugated to Alexa Fluor 555. All scale bars represent 9 μm. Images were acquired using an UltraVIEW Vox live-cell imaging system, and Pearson's correlation coefficient was used to quantify the degree of colocalization between the two channels (red and green) in a confocal image by using Volocity 6.1 software. (B) The bar graphs were drawn to show average Pearson's correlation coefficients (with error bars) from multiple images ( n = 7 for each treatment) of the HeLa cells incubated with different proteins. One-way ANOVA with Bonferroni's multiple-comparison test was performed to analyze the significance of difference in bacterial adhesion between the different treatments (***, P

    Techniques Used: Purification, Incubation, Live Cell Imaging, Software

    35) Product Images from "Transient transgenesis of the tapeworm Taenia crassiceps"

    Article Title: Transient transgenesis of the tapeworm Taenia crassiceps

    Journal: SpringerPlus

    doi: 10.1186/s40064-015-1278-y

    Co-localization of GFP fluorescence and α-GFP immunofluorescence in tissue sections of cysticerci after 24 h of microinjection. a GFP fluorescence in a GFP-TOPO plasmid microinjected cyst (FITC filter); b Immunohistochemical localization using a 1:250 dilution of a polyclonal α -GFP rabbit IgG antibody followed by a 1:200 dilution of a goat α-rabbit IgG CY3-conjugated antibody (CY3 filter); c Merging of a and b images. d Water microinjected cysts (FITC filter); e Immunohistochemical localization as in b in a water microinjected cyst (CY3 filter); f Merging of d and e images. Arrows show subtegumentary cytons. T tegument. Photographs were obtained under Confocal Laser microscopy
    Figure Legend Snippet: Co-localization of GFP fluorescence and α-GFP immunofluorescence in tissue sections of cysticerci after 24 h of microinjection. a GFP fluorescence in a GFP-TOPO plasmid microinjected cyst (FITC filter); b Immunohistochemical localization using a 1:250 dilution of a polyclonal α -GFP rabbit IgG antibody followed by a 1:200 dilution of a goat α-rabbit IgG CY3-conjugated antibody (CY3 filter); c Merging of a and b images. d Water microinjected cysts (FITC filter); e Immunohistochemical localization as in b in a water microinjected cyst (CY3 filter); f Merging of d and e images. Arrows show subtegumentary cytons. T tegument. Photographs were obtained under Confocal Laser microscopy

    Techniques Used: Fluorescence, Immunofluorescence, Plasmid Preparation, Immunohistochemistry, Microscopy

    Time course of the GFP fluorescence after microinjection of intact Taenia crassiceps cysticerci. a GFP-TOPO plasmid ( 1 – 3 ) and a GFP-negative plasmid: pCMV-VSV-G ( 4 ) microinjected cysts, after 24 ( 1 and 4 ); 48 ( 2 ) and 72 h ( 3 ). Photographs were taken using an Olympus DSU confocal system with a FITC (450–490 nm) filter under the same conditions for all cases. b Western blots using crude extracts of GFP-TOPO ( 1 – 3 ) and pCMV-VSV-G ( 5 ) microinjected cysts; 24 ( 1 and 5 ); 48 ( 2 ) and 72 ( 3 ) h post microinjection. A crude extract of HEK 293 cells transfected with GFP-TOPO ( 4 ) is also shown as a positive control. 50 µg of each crude extract were loaded on each lane in the gel and all blots were obtained from a single gel run. For detection of GFP, a polyclonal rabbit IgG α-GFP antibody and a goat α-rabbit IgG antibody conjugated with horseradish peroxidase were used at 1:1000 and 1:10,000 dilutions, respectively. Loading controls ( 6 – 10 ) used a sheep anti-mouse albumin polyclonal antibody followed by a horseradish peroxidase conjugated anti-sheep IgG secondary antibody (Aldridge et al. 2006 ). Development of peroxidase was carried out using an enhanced chemiluminescence kit
    Figure Legend Snippet: Time course of the GFP fluorescence after microinjection of intact Taenia crassiceps cysticerci. a GFP-TOPO plasmid ( 1 – 3 ) and a GFP-negative plasmid: pCMV-VSV-G ( 4 ) microinjected cysts, after 24 ( 1 and 4 ); 48 ( 2 ) and 72 h ( 3 ). Photographs were taken using an Olympus DSU confocal system with a FITC (450–490 nm) filter under the same conditions for all cases. b Western blots using crude extracts of GFP-TOPO ( 1 – 3 ) and pCMV-VSV-G ( 5 ) microinjected cysts; 24 ( 1 and 5 ); 48 ( 2 ) and 72 ( 3 ) h post microinjection. A crude extract of HEK 293 cells transfected with GFP-TOPO ( 4 ) is also shown as a positive control. 50 µg of each crude extract were loaded on each lane in the gel and all blots were obtained from a single gel run. For detection of GFP, a polyclonal rabbit IgG α-GFP antibody and a goat α-rabbit IgG antibody conjugated with horseradish peroxidase were used at 1:1000 and 1:10,000 dilutions, respectively. Loading controls ( 6 – 10 ) used a sheep anti-mouse albumin polyclonal antibody followed by a horseradish peroxidase conjugated anti-sheep IgG secondary antibody (Aldridge et al. 2006 ). Development of peroxidase was carried out using an enhanced chemiluminescence kit

    Techniques Used: Fluorescence, Plasmid Preparation, Western Blot, Transfection, Positive Control

    36) Product Images from "SUMO1 Modification Facilitates Avibirnavirus Replication by Stabilizing Polymerase VP1"

    Article Title: SUMO1 Modification Facilitates Avibirnavirus Replication by Stabilizing Polymerase VP1

    Journal: Journal of Virology

    doi: 10.1128/JVI.02227-18

    Inhibition of IBDV polymerase VP1 degradation by SUMOylation. (A) UnSUMOylated VP1 proteins with I 404 C/T and I 406 C/F mutations were unstable. 293T cells individually transfected with Flag-VP1 or its mutations for 24 h and were treated with CHX (100 μg/ml) for 0, 4, 8, and 12 h. (B) Blocking proteasome activity inhibited degradation of unSUMOylated VP1. 293T cells were transfected with the indicated plasmids for 24 h and were then treated with MG132 (10 μg/ml) for 8 h. The resultant cell lysates were subjected to Western blotting with the indicated antibodies for analyzing the life span of WT VP1 and mutant VP1 (A) and VP1 levels (B) by ImageJ. All detection was performed by three independent experiments. (C and D) Enhanced ubiquitination of unSUMOylated VP1 with I 404 C/T and I 406 C/F mutation. 293T cells were transfected with Flag-VP1 or its four mutants and HA-Ub (C) or HA-K48 (D) for 36 h. Lysates of the cells were subjected to ubiquitination assays and Western blotting with the indicated antibodies. (E) Low stability of unSUMOylated VP1 during IBDV infection. DF-1 cells were infected with IBDV at an MOI of 1 for 18 h and treated with CHX (100 μg/ml) for 0, 4, 8, and 12 h. (F) Blocking proteasome activity (MG132) inhibited VP1 degradation of unSUMOylated VP1 during IBDV infection. DF-1 cells were infected with IBDV at an MOI of 1 for 18 h and then treated with MG132 (10 μg/ml) and CHX (100 μg/ml) for 8 h. The resultant cell lysates were subjected to Western blotting with the indicated antibodies for analyzing the life span of WT VP1 and mutant VP1 (E) and VP1 levels (F) by ImageJ. All detection was performed by three independent experiments. (G) The replication complex assembly of WT and mutant IBDV was not altered. DF-1 cells were infected with WT and mutant IBDV for 12 h. The resultant cells were fixed and incubated with rabbit anti-VP1 antibody, chicken anti-VP3 antibody, and a mouse MAb specific for dsRNA and then reacted with Alexa Fluor 546 anti-rabbit, FITC goat anti-chicken, and Alexa Fluor 647 donkey anti-mouse IgG as secondary antibodies. DAPI was used to stain the nuclei. Confocal microscope images were taken under a Nikon laser microscope. Scale bars, 10 μm.
    Figure Legend Snippet: Inhibition of IBDV polymerase VP1 degradation by SUMOylation. (A) UnSUMOylated VP1 proteins with I 404 C/T and I 406 C/F mutations were unstable. 293T cells individually transfected with Flag-VP1 or its mutations for 24 h and were treated with CHX (100 μg/ml) for 0, 4, 8, and 12 h. (B) Blocking proteasome activity inhibited degradation of unSUMOylated VP1. 293T cells were transfected with the indicated plasmids for 24 h and were then treated with MG132 (10 μg/ml) for 8 h. The resultant cell lysates were subjected to Western blotting with the indicated antibodies for analyzing the life span of WT VP1 and mutant VP1 (A) and VP1 levels (B) by ImageJ. All detection was performed by three independent experiments. (C and D) Enhanced ubiquitination of unSUMOylated VP1 with I 404 C/T and I 406 C/F mutation. 293T cells were transfected with Flag-VP1 or its four mutants and HA-Ub (C) or HA-K48 (D) for 36 h. Lysates of the cells were subjected to ubiquitination assays and Western blotting with the indicated antibodies. (E) Low stability of unSUMOylated VP1 during IBDV infection. DF-1 cells were infected with IBDV at an MOI of 1 for 18 h and treated with CHX (100 μg/ml) for 0, 4, 8, and 12 h. (F) Blocking proteasome activity (MG132) inhibited VP1 degradation of unSUMOylated VP1 during IBDV infection. DF-1 cells were infected with IBDV at an MOI of 1 for 18 h and then treated with MG132 (10 μg/ml) and CHX (100 μg/ml) for 8 h. The resultant cell lysates were subjected to Western blotting with the indicated antibodies for analyzing the life span of WT VP1 and mutant VP1 (E) and VP1 levels (F) by ImageJ. All detection was performed by three independent experiments. (G) The replication complex assembly of WT and mutant IBDV was not altered. DF-1 cells were infected with WT and mutant IBDV for 12 h. The resultant cells were fixed and incubated with rabbit anti-VP1 antibody, chicken anti-VP3 antibody, and a mouse MAb specific for dsRNA and then reacted with Alexa Fluor 546 anti-rabbit, FITC goat anti-chicken, and Alexa Fluor 647 donkey anti-mouse IgG as secondary antibodies. DAPI was used to stain the nuclei. Confocal microscope images were taken under a Nikon laser microscope. Scale bars, 10 μm.

    Techniques Used: Inhibition, Transfection, Blocking Assay, Activity Assay, Western Blot, Mutagenesis, Infection, Incubation, Staining, Microscopy

    37) Product Images from "A genome-wide CRISPR screen identifies N-acetylglucosamine-1-phosphate transferase as a potential antiviral target for Ebola virus"

    Article Title: A genome-wide CRISPR screen identifies N-acetylglucosamine-1-phosphate transferase as a potential antiviral target for Ebola virus

    Journal: Nature Communications

    doi: 10.1038/s41467-018-08135-4

    Cathepsins are reduced in GNPTAB - knockout cells, but restored upon GNPTAB reconstitution. a Immunoblotting of lysates from parental, knockout and reconstituted cells for CatB and CatL. The migration of a 30 kDa molecular mass marker is shown to the right. b CatB activity in lysates from parental HAP1, or knockout GNPTAB- or NPC1-cells. Cells were treated with cathepsin B inhibitor (Bi), cathepsin L inhibitor (Li), at 10 or 1 µM, or DMSO vehicle control for 1 h at 37 °C, before lysis and incubation with fluorescent CatB peptide substrate. After 1 h incubation at room temperature, fluorescence was measured. Data represent the mean ± s.d. of three technical replicates. A representative of three independent experiments is shown. c CatB activity in lysates from reconstituted cells. Lysates from transduced HAP1 or GNPTAB − cells were generated and tested for CatB activity, as for panel b . Data represent the mean ± s.d. of three technical replicates. A representative of two independent experiments is shown. For both panels b and c , statistical analysis was performed with a two-tailed Student’s t -test with significance shown as **** P ≤ 0.0001
    Figure Legend Snippet: Cathepsins are reduced in GNPTAB - knockout cells, but restored upon GNPTAB reconstitution. a Immunoblotting of lysates from parental, knockout and reconstituted cells for CatB and CatL. The migration of a 30 kDa molecular mass marker is shown to the right. b CatB activity in lysates from parental HAP1, or knockout GNPTAB- or NPC1-cells. Cells were treated with cathepsin B inhibitor (Bi), cathepsin L inhibitor (Li), at 10 or 1 µM, or DMSO vehicle control for 1 h at 37 °C, before lysis and incubation with fluorescent CatB peptide substrate. After 1 h incubation at room temperature, fluorescence was measured. Data represent the mean ± s.d. of three technical replicates. A representative of three independent experiments is shown. c CatB activity in lysates from reconstituted cells. Lysates from transduced HAP1 or GNPTAB − cells were generated and tested for CatB activity, as for panel b . Data represent the mean ± s.d. of three technical replicates. A representative of two independent experiments is shown. For both panels b and c , statistical analysis was performed with a two-tailed Student’s t -test with significance shown as **** P ≤ 0.0001

    Techniques Used: Knock-Out, Migration, Marker, Activity Assay, Lysis, Incubation, Fluorescence, Generated, Two Tailed Test

    Blocking GNPTAB cleavage with a SKI-1/S1P inhibitor blocks EBOV infection. a PF-429242 blocks processing of GNPTAB. Huh7 cells were transfected with a plasmid expressing GNPTAB-myc. Twenty-four hours post transfection, PF-429242, or DMSO vehicle, was added to the indicated concentrations. Cell lysates were harvested 48 h post transfection and subjected to immunoblotting. b PF-429242 does not alter NPC1 expression levels. Huh7 cells were treated with PF-429242 at the indicated concentrations for 24 h before cell lysates were harvested and subjected to immunoblotting. c PF-429242 blocks EBOV-GP-mediated entry. Varying concentrations of PF-429242 were added to Huh7 cells 1 h prior to the addition of luciferase-encoding lentiviral particles pseudotyped with the Zaire-EBOV-GP (EBOVpp), or with VSV-G protein (VSVpp). Three days later luciferase activity was determined. Data represent the mean and s.d. of values relative to the DMSO vehicle control, from 8 biological replicates. A representative of 3 independent experiments is shown. d PF-429242 inhibits infection by EBOV-ZsG reporter virus. Huh7 cells were treated with varying concentrations of PF-429242, before being mock-infected or infected with EBOV-ZsG reporter virus. Three days post-infection, cell viability or ZsG fluorescence was measured, respectively. Data represent the mean and s.d. of values relative to the DMSO vehicle control, from four biological replicates. A representative of six independent experiments is shown
    Figure Legend Snippet: Blocking GNPTAB cleavage with a SKI-1/S1P inhibitor blocks EBOV infection. a PF-429242 blocks processing of GNPTAB. Huh7 cells were transfected with a plasmid expressing GNPTAB-myc. Twenty-four hours post transfection, PF-429242, or DMSO vehicle, was added to the indicated concentrations. Cell lysates were harvested 48 h post transfection and subjected to immunoblotting. b PF-429242 does not alter NPC1 expression levels. Huh7 cells were treated with PF-429242 at the indicated concentrations for 24 h before cell lysates were harvested and subjected to immunoblotting. c PF-429242 blocks EBOV-GP-mediated entry. Varying concentrations of PF-429242 were added to Huh7 cells 1 h prior to the addition of luciferase-encoding lentiviral particles pseudotyped with the Zaire-EBOV-GP (EBOVpp), or with VSV-G protein (VSVpp). Three days later luciferase activity was determined. Data represent the mean and s.d. of values relative to the DMSO vehicle control, from 8 biological replicates. A representative of 3 independent experiments is shown. d PF-429242 inhibits infection by EBOV-ZsG reporter virus. Huh7 cells were treated with varying concentrations of PF-429242, before being mock-infected or infected with EBOV-ZsG reporter virus. Three days post-infection, cell viability or ZsG fluorescence was measured, respectively. Data represent the mean and s.d. of values relative to the DMSO vehicle control, from four biological replicates. A representative of six independent experiments is shown

    Techniques Used: Blocking Assay, Infection, Transfection, Plasmid Preparation, Expressing, Luciferase, Activity Assay, Fluorescence

    38) Product Images from "SOX9 accelerates ESC differentiation to three germ layer lineages by repressing SOX2 expression through P21 (WAF1/CIP1)"

    Article Title: SOX9 accelerates ESC differentiation to three germ layer lineages by repressing SOX2 expression through P21 (WAF1/CIP1)

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.115436

    p21 ( Waf1 / Cip1 ) induced by Sox9 inhibits Sox2 expression through direct binding to SRR2 enhancer. (A) Growth profiles of SOX9-inducible ESCs cultured in Dox+ (control) or Dox− conditions (three independent experiments; data are mean±s.e.m.;
    Figure Legend Snippet: p21 ( Waf1 / Cip1 ) induced by Sox9 inhibits Sox2 expression through direct binding to SRR2 enhancer. (A) Growth profiles of SOX9-inducible ESCs cultured in Dox+ (control) or Dox− conditions (three independent experiments; data are mean±s.e.m.;

    Techniques Used: Expressing, Binding Assay, Cell Culture

    Effects of Sox9 on early ESC differentiation requires p21 . (A) Western blots detecting P21 and β-actin (control) in SOX9-inducible ESCs cultured for 4 days in Dox+ (control) or Dox− conditions after treating with siRNA against
    Figure Legend Snippet: Effects of Sox9 on early ESC differentiation requires p21 . (A) Western blots detecting P21 and β-actin (control) in SOX9-inducible ESCs cultured for 4 days in Dox+ (control) or Dox− conditions after treating with siRNA against

    Techniques Used: Western Blot, Cell Culture

    39) Product Images from "Internalization mechanisms of brain-derived tau oligomers from patients with Alzheimer’s disease, progressive supranuclear palsy and dementia with Lewy bodies"

    Article Title: Internalization mechanisms of brain-derived tau oligomers from patients with Alzheimer’s disease, progressive supranuclear palsy and dementia with Lewy bodies

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-020-2503-3

    Exogenous tau oligomers mediated alternations of autophagy–lysosomal pathway (ALP) in neurons. Cells were pretreated with NT or Ext2 siRNA followed by the presence or absence of 0.1 μM or 0.5 μM biotin-tagged TauO from AD ( a ), PSP ( b ), or DLB ( c ). All experiments were performed with similar conditions and parameters as for Fig. 4f–h (cells pretreated with NT or Ext2 siRNA followed by the presence or absence of 0.1 μM biotin-tagged TauO), together with samples from cells preincubated with NT or Ext2 siRNA followed by 0.5 μM biotin-tagged TauO. Representative Western blot images revealed the expression of p62 (autophagy receptor), LC3B-II (autophagosome membrane formation), and LAMP-2 (lysosome). Quantification of band intensity shown below was normalized to βIII-tubulin. The same immunoblots probed with loading controls shown in Fig. 4f–h were reused in Fig. 5a–c. a AD TauO-exposed cells showed a significant increase in p62 and LAMP-2 levels, yet a drastic decrease was seen in LC3B-II/LC3B-I ratio. Reverse effects were observed in Ext2 siRNA-pretreated group. b PSP TauO-treated cells slightly changed the expression of p62, but did not alter LAMP-2 expression. LCB-II/LC3B-I ratio was significantly reduced in PSP TauO-treated, while Ext2 siRNA exposure alleviated the PSP TauO effect without reaching statistical significance. c Reductions of p62, LCB-II/LC3B-I ratio, and LAMP-2 were found in DLB TauO-exposed neurons, while the reverse effects from Ext2 siRNA-pretreated were not observed. Statistical analyses were measured by one-way ANOVA with Tukey’s test from three biological independent experiments. Results showed as the value of mean ± SEM, * p
    Figure Legend Snippet: Exogenous tau oligomers mediated alternations of autophagy–lysosomal pathway (ALP) in neurons. Cells were pretreated with NT or Ext2 siRNA followed by the presence or absence of 0.1 μM or 0.5 μM biotin-tagged TauO from AD ( a ), PSP ( b ), or DLB ( c ). All experiments were performed with similar conditions and parameters as for Fig. 4f–h (cells pretreated with NT or Ext2 siRNA followed by the presence or absence of 0.1 μM biotin-tagged TauO), together with samples from cells preincubated with NT or Ext2 siRNA followed by 0.5 μM biotin-tagged TauO. Representative Western blot images revealed the expression of p62 (autophagy receptor), LC3B-II (autophagosome membrane formation), and LAMP-2 (lysosome). Quantification of band intensity shown below was normalized to βIII-tubulin. The same immunoblots probed with loading controls shown in Fig. 4f–h were reused in Fig. 5a–c. a AD TauO-exposed cells showed a significant increase in p62 and LAMP-2 levels, yet a drastic decrease was seen in LC3B-II/LC3B-I ratio. Reverse effects were observed in Ext2 siRNA-pretreated group. b PSP TauO-treated cells slightly changed the expression of p62, but did not alter LAMP-2 expression. LCB-II/LC3B-I ratio was significantly reduced in PSP TauO-treated, while Ext2 siRNA exposure alleviated the PSP TauO effect without reaching statistical significance. c Reductions of p62, LCB-II/LC3B-I ratio, and LAMP-2 were found in DLB TauO-exposed neurons, while the reverse effects from Ext2 siRNA-pretreated were not observed. Statistical analyses were measured by one-way ANOVA with Tukey’s test from three biological independent experiments. Results showed as the value of mean ± SEM, * p

    Techniques Used: Western Blot, Expressing

    Localization of internalized tau oligomers with the endosomal– lysosomal system. a , e Neurons were exposed to AF568-tagged TauO (Red) from AD, PSP, or DLB for 1 h with (+) or without (−) Heparin pretreatment. Cells were fixed and immunostained for a mature neuronal marker (βIII-tubulin, blue), an early endosomal marker (Rab5, green) ( a ) and a lysosomal marker (LAMP-2, green) ( e ). Representative orthogonal images indicate AF568-tagged TauO co-localized to early endosomes and lysosomes, indicated by arrows. Scale bar: 2 and 10 μm. b – d Pearson’s correlation coefficient analysis of internalized AD TauO ( b ), PSP TauO ( c ), DLB TauO ( d ) with early endosome over 1 h was demonstrated with similar experimental conditions and parameters as for ( a ). Each treatment group was randomly imaged in five different regions of interest, and performed in duplicate. f – h Pearson’s correlation coefficient analysis of lysosome with internalized AD TauO ( f ), PSP TauO ( g ), and DLB TauO ( h ) was demonstrated, using the same experimental conditions as for ( e ). Image analyses were calculated by unpaired and two-tailed Student’s t test. Results showed as the value of mean ± SEM, ** p
    Figure Legend Snippet: Localization of internalized tau oligomers with the endosomal– lysosomal system. a , e Neurons were exposed to AF568-tagged TauO (Red) from AD, PSP, or DLB for 1 h with (+) or without (−) Heparin pretreatment. Cells were fixed and immunostained for a mature neuronal marker (βIII-tubulin, blue), an early endosomal marker (Rab5, green) ( a ) and a lysosomal marker (LAMP-2, green) ( e ). Representative orthogonal images indicate AF568-tagged TauO co-localized to early endosomes and lysosomes, indicated by arrows. Scale bar: 2 and 10 μm. b – d Pearson’s correlation coefficient analysis of internalized AD TauO ( b ), PSP TauO ( c ), DLB TauO ( d ) with early endosome over 1 h was demonstrated with similar experimental conditions and parameters as for ( a ). Each treatment group was randomly imaged in five different regions of interest, and performed in duplicate. f – h Pearson’s correlation coefficient analysis of lysosome with internalized AD TauO ( f ), PSP TauO ( g ), and DLB TauO ( h ) was demonstrated, using the same experimental conditions as for ( e ). Image analyses were calculated by unpaired and two-tailed Student’s t test. Results showed as the value of mean ± SEM, ** p

    Techniques Used: Marker, Two Tailed Test

    40) Product Images from "Porcine deltacoronavirus activates the Raf/MEK/ERK pathway to promote its replication"

    Article Title: Porcine deltacoronavirus activates the Raf/MEK/ERK pathway to promote its replication

    Journal: Virus Research

    doi: 10.1016/j.virusres.2020.197961

    Inhibition of ERK1/2 activation impairs PDCoV propagation. (A) ST cells were preincubated with DMSO, PD98059 (50 and 100 μM), or U0126 (50 and 100 μM) for 1 h prior to infection and were mock infected or infected with PDCoV at an MOI of 1. The virus-infected cells were further maintained for 12 h in the presence of DMSO or inhibitors. PDCoV-specific CPEs were monitored and photographed at 12 hpi under an inverted microscope at the magnification of 200× (first panels). For immunostaining, the infected cells were fixed at 12 hpi and incubated with MAb against the PDCoV N protein, followed by incubation with Alexa green-conjugated goat anti-mouse secondary antibody (second panels). The cells were then counterstained with DAPI (third panels) and examined under a fluorescent microscope at 200× magnification. (B) PDCoV production in the presence of each inhibitor was quantified by measuring the percentage of cells expressing N proteins through flow cytometry. (C) Chemical inhibition of ERK1/2 was quantitatively determined using a FACE assay. ST cells were mock infected or PDCoV infected in the presence of DMSO, PD98059, or U0126. The cells were fixed at 6 hpi with 4% formaldehyde and incubated with an anti-ERK1/2 or anti-phospho-ERK1/2 antibody followed by HRP-conjugated IgG antibodies. The absorbance of the solution was determined at 450 nm using a spectrophotometer. (D) At 6 hpi, cellular lysates were prepared and subjected to immunoblotting using an antibody against p-ERK1/2, ERK1/2, p-Elk-1, or PDCoV N. The blot was also reacted with an anti-β-actin antibody to verify equal protein leading. Each protein expression was quantitatively analyzed by densitometry, and fold changes in each p-ERK1/2:total ERK1/2, p-Elk-1:β-actin, and PDCoV N:β-actin ratio are independently plotted (right panel). Data are the representative of the means from three independent experiments, and error bars denote the mean ± SDM. * P
    Figure Legend Snippet: Inhibition of ERK1/2 activation impairs PDCoV propagation. (A) ST cells were preincubated with DMSO, PD98059 (50 and 100 μM), or U0126 (50 and 100 μM) for 1 h prior to infection and were mock infected or infected with PDCoV at an MOI of 1. The virus-infected cells were further maintained for 12 h in the presence of DMSO or inhibitors. PDCoV-specific CPEs were monitored and photographed at 12 hpi under an inverted microscope at the magnification of 200× (first panels). For immunostaining, the infected cells were fixed at 12 hpi and incubated with MAb against the PDCoV N protein, followed by incubation with Alexa green-conjugated goat anti-mouse secondary antibody (second panels). The cells were then counterstained with DAPI (third panels) and examined under a fluorescent microscope at 200× magnification. (B) PDCoV production in the presence of each inhibitor was quantified by measuring the percentage of cells expressing N proteins through flow cytometry. (C) Chemical inhibition of ERK1/2 was quantitatively determined using a FACE assay. ST cells were mock infected or PDCoV infected in the presence of DMSO, PD98059, or U0126. The cells were fixed at 6 hpi with 4% formaldehyde and incubated with an anti-ERK1/2 or anti-phospho-ERK1/2 antibody followed by HRP-conjugated IgG antibodies. The absorbance of the solution was determined at 450 nm using a spectrophotometer. (D) At 6 hpi, cellular lysates were prepared and subjected to immunoblotting using an antibody against p-ERK1/2, ERK1/2, p-Elk-1, or PDCoV N. The blot was also reacted with an anti-β-actin antibody to verify equal protein leading. Each protein expression was quantitatively analyzed by densitometry, and fold changes in each p-ERK1/2:total ERK1/2, p-Elk-1:β-actin, and PDCoV N:β-actin ratio are independently plotted (right panel). Data are the representative of the means from three independent experiments, and error bars denote the mean ± SDM. * P

    Techniques Used: Inhibition, Activation Assay, Infection, Inverted Microscopy, Immunostaining, Incubation, Microscopy, Expressing, Flow Cytometry, Spectrophotometry

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    Incubation:

    Article Title: Influences of cyclosporin A and non-immunosuppressive derivatives on cellular cyclophilins and viral nucleocapsid protein during human coronavirus 229E replication.
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    other:

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    Mass Spectrometry:

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    c-di-GMP regulates TfoY protein levels. (A) Western blot of PAGE-separated protein isolated from V. cholerae parent strains carrying either the P 1234 -TL-FULL gfp reporter fusion or the G20U variant. Blots were hybridized with anti-GFP primary antibody and HRP-conjugated secondary antibody and detected using ECL. The blot shown is representative of multiple experiments. (B) Quantification of TfoY-GFP fusion protein present in the lanes depicted in panel A ( n = 3 or 4). Relative amounts of protein were quantified for each lane and normalized to the WT Vc2 riboswitch with the P tac -qrgB * group for each blot (set equal to 1 relative unit of protein). L, low; I, intermediate; H, high. Error bars for all graphs indicate the standard deviation. *, P

    Journal: Journal of Bacteriology

    Article Title: Cyclic di-GMP Regulates TfoY in Vibrio cholerae To Control Motility by both Transcriptional and Posttranscriptional Mechanisms

    doi: 10.1128/JB.00578-17

    Figure Lengend Snippet: c-di-GMP regulates TfoY protein levels. (A) Western blot of PAGE-separated protein isolated from V. cholerae parent strains carrying either the P 1234 -TL-FULL gfp reporter fusion or the G20U variant. Blots were hybridized with anti-GFP primary antibody and HRP-conjugated secondary antibody and detected using ECL. The blot shown is representative of multiple experiments. (B) Quantification of TfoY-GFP fusion protein present in the lanes depicted in panel A ( n = 3 or 4). Relative amounts of protein were quantified for each lane and normalized to the WT Vc2 riboswitch with the P tac -qrgB * group for each blot (set equal to 1 relative unit of protein). L, low; I, intermediate; H, high. Error bars for all graphs indicate the standard deviation. *, P

    Article Snippet: Blots were blocked with 5% nonfat milk in Tris-buffered saline with 0.1% Tween 20 (TBS-T) and hybridized with a mouse anti-GFP primary antibody and a rabbit anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody (both Thermo Fisher Scientific).

    Techniques: Western Blot, Polyacrylamide Gel Electrophoresis, Isolation, Variant Assay, Standard Deviation

    Histological analysis of mice pre-treated with CPMO1v. BALB/c mice (6-day old, n = 6) were i.p injected with 15 μg/g CPMO1v or sCPMO1v. CHIKV infection and treatment regimen is carried out as aforementioned in Fig. 5 . All mice were sacrificed at day 7 p.i. and their hind limbs and liver were harvested, fixed with formalin, ethanol-dehydrated and paraffin embedded in sections for ( a–h ) H E staining or ( i–p ) IHC staining using primary rabbit anti-CHIKV E2 IgG followed by secondary goat anti-rabbit HRP IgG. ( d ) ( f ) Arrow indicates smudgy nuclei inclusions while arrowhead indicates multi-nucleated inclusions of pale necrotic hepatocytes. ( k , l ) Arrow indicates positive CHIKV antigen staining. Images were viewed and captured at 400× under Olympus microscope. Representative images at 50 μm scale are shown.

    Journal: Scientific Reports

    Article Title: Antiviral Phosphorodiamidate Morpholino Oligomers are Protective against Chikungunya Virus Infection on Cell-based and Murine Models

    doi: 10.1038/srep12727

    Figure Lengend Snippet: Histological analysis of mice pre-treated with CPMO1v. BALB/c mice (6-day old, n = 6) were i.p injected with 15 μg/g CPMO1v or sCPMO1v. CHIKV infection and treatment regimen is carried out as aforementioned in Fig. 5 . All mice were sacrificed at day 7 p.i. and their hind limbs and liver were harvested, fixed with formalin, ethanol-dehydrated and paraffin embedded in sections for ( a–h ) H E staining or ( i–p ) IHC staining using primary rabbit anti-CHIKV E2 IgG followed by secondary goat anti-rabbit HRP IgG. ( d ) ( f ) Arrow indicates smudgy nuclei inclusions while arrowhead indicates multi-nucleated inclusions of pale necrotic hepatocytes. ( k , l ) Arrow indicates positive CHIKV antigen staining. Images were viewed and captured at 400× under Olympus microscope. Representative images at 50 μm scale are shown.

    Article Snippet: For IHC, tissue samples were labeled with primary rabbit E2 antibody diluted to 1:100, followed by a secondary goat anti-rabbit HRP conjugate (Thermoscientific).

    Techniques: Mouse Assay, Injection, Infection, Staining, Immunohistochemistry, Microscopy

    Histological analysis of mice pre-treated with CPMO1v. BALB/c mice (6-day old, n = 6) were i.p injected with 15 μg/g CPMO1v or sCPMO1v. CHIKV infection and treatment regimen is carried out as aforementioned in Fig. 5 . All mice were sacrificed at day 7 p.i. and their hind limbs and liver were harvested, fixed with formalin, ethanol-dehydrated and paraffin embedded in sections for ( a–h ) H E staining or ( i–p ) IHC staining using primary rabbit anti-CHIKV E2 IgG followed by secondary goat anti-rabbit HRP IgG. ( d ) ( f ) Arrow indicates smudgy nuclei inclusions while arrowhead indicates multi-nucleated inclusions of pale necrotic hepatocytes. ( k , l ) Arrow indicates positive CHIKV antigen staining. Images were viewed and captured at 400× under Olympus microscope. Representative images at 50 μm scale are shown.

    Journal: Scientific Reports

    Article Title: Antiviral Phosphorodiamidate Morpholino Oligomers are Protective against Chikungunya Virus Infection on Cell-based and Murine Models

    doi: 10.1038/srep12727

    Figure Lengend Snippet: Histological analysis of mice pre-treated with CPMO1v. BALB/c mice (6-day old, n = 6) were i.p injected with 15 μg/g CPMO1v or sCPMO1v. CHIKV infection and treatment regimen is carried out as aforementioned in Fig. 5 . All mice were sacrificed at day 7 p.i. and their hind limbs and liver were harvested, fixed with formalin, ethanol-dehydrated and paraffin embedded in sections for ( a–h ) H E staining or ( i–p ) IHC staining using primary rabbit anti-CHIKV E2 IgG followed by secondary goat anti-rabbit HRP IgG. ( d ) ( f ) Arrow indicates smudgy nuclei inclusions while arrowhead indicates multi-nucleated inclusions of pale necrotic hepatocytes. ( k , l ) Arrow indicates positive CHIKV antigen staining. Images were viewed and captured at 400× under Olympus microscope. Representative images at 50 μm scale are shown.

    Article Snippet: CHIKV E2 protein was probed using rabbit anti-CHIKV E2 13893 B3 polyclonal antibody (in-house produced) at 1:3000 dilution and goat anti-rabbit secondary antibody conjugated to HRP (Thermoscientific) at 1:6000 dilution.

    Techniques: Mouse Assay, Injection, Infection, Staining, Immunohistochemistry, Microscopy