pml 5e10 mab  (Thermo Fisher)


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    Structured Review

    Thermo Fisher pml 5e10 mab
    Fig. 2.  PML and eIF4E co-localize in several cell types. U937 ( A – C ) and K562 ( D – F ) cells were stained for eIF4E (in green) and PML (in red) with the overlay in yellow. PML and eIF4E were detected using polyclonal antiPML and eIF4E mAb, respectively. Identical results were obtained using anti-PML 5E10 mAb and eIF4E mAb conjugated directly to FITC when U937 cells ( G – I ) were stained for eIF4E (in green) and PML (in blue) with the overlay in aqua. The objective is 100×. Scale bars = 10 µM. Confocal micrographs represent single sections through the plane of the cells.
    Pml 5e10 Mab, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 20207 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 85 stars, based on 20207 article reviews
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    pml 5e10 mab - by Bioz Stars, 2020-07
    85/100 stars

    Images

    1) Product Images from "PML RING suppresses oncogenic transformation by reducing the affinity of eIF4E for mRNA"

    Article Title: PML RING suppresses oncogenic transformation by reducing the affinity of eIF4E for mRNA

    Journal: The EMBO Journal

    doi: 10.1093/emboj/20.16.4547

    Fig. 2.  PML and eIF4E co-localize in several cell types. U937 ( A – C ) and K562 ( D – F ) cells were stained for eIF4E (in green) and PML (in red) with the overlay in yellow. PML and eIF4E were detected using polyclonal antiPML and eIF4E mAb, respectively. Identical results were obtained using anti-PML 5E10 mAb and eIF4E mAb conjugated directly to FITC when U937 cells ( G – I ) were stained for eIF4E (in green) and PML (in blue) with the overlay in aqua. The objective is 100×. Scale bars = 10 µM. Confocal micrographs represent single sections through the plane of the cells.
    Figure Legend Snippet: Fig. 2. PML and eIF4E co-localize in several cell types. U937 ( A – C ) and K562 ( D – F ) cells were stained for eIF4E (in green) and PML (in red) with the overlay in yellow. PML and eIF4E were detected using polyclonal antiPML and eIF4E mAb, respectively. Identical results were obtained using anti-PML 5E10 mAb and eIF4E mAb conjugated directly to FITC when U937 cells ( G – I ) were stained for eIF4E (in green) and PML (in blue) with the overlay in aqua. The objective is 100×. Scale bars = 10 µM. Confocal micrographs represent single sections through the plane of the cells.

    Techniques Used: Staining

    2) 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:

    3) Product Images from "Angiotensin II Type 1 Receptor Antagonist Attenuates Lacrimal Gland, Lung, and Liver Fibrosis in a Murine Model of Chronic Graft-Versus-Host Disease"

    Article Title: Angiotensin II Type 1 Receptor Antagonist Attenuates Lacrimal Gland, Lung, and Liver Fibrosis in a Murine Model of Chronic Graft-Versus-Host Disease

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0064724

    Fibrotic changes in control and cGVHD murine lacrimal glands. A: Light microscopy of cGVHD lacrimal glands showing the infiltration of inflammatory cells around medium-sized interlobular ducts, and a severely fibrotic interstitium that is more intense in the periductal areas. Inset: Infiltrated inflammatory cells (white arrowhead) and interstitial fibroblasts (red arrowhead). B: Collagen deposition was quantified as the ratio of the blue-stained area to the total stained area in Mallory staining and expressed as % fibrotic area. Collagen deposition was more severe in the cGVHD lacrimal glands compared with the controls (p = 0.000005). C: Immunostaining for CD45 and HSP47 in lacrimal glands shows intense brown staining of many cells in the interstitium and periductal areas in the cGVHD lacrimal glands. CD45-positive cells indicate infiltrating inflammatory cells. HSP47-positive cells are spindle-shaped cells with oval nuclei, which indicate fibroblasts (inset). D: The mean densities of CD45 + inflammatory cells and HSP47 + fibroblasts were significantly higher in the cGVHD compared with control lacrimal glands (CD45 + inflammatory cells; p = 0.001, HSP47 + fibroblasts; p = 0.0001). E: Quantitative real-time PCR revealed that the HSP47, collagen type I alpha 1, collagen type I alpha 2, and collagen type III alpha 1 expressions in the cGVHD group were higher than in the control group (HSP47; p = 0.004, collage type I alpha 1; p = 0.0002, collagen type I alpha 2; p = 0.03, collagen type III alpha 1; p = 0.002). Magnification, A: X200, C: X200. Scale bar, A: 50 µm, C: 50 µm. The results represent the mean ± SD. B: n = 9; D: n = 8; E: n = 9 in each group. E: The vertical axis shows the expression ratio of mRNAs. Fold change shows the expression ratio of mRNAs in the control and cGVHD group. The expression level in the control group was defined as 1. *p
    Figure Legend Snippet: Fibrotic changes in control and cGVHD murine lacrimal glands. A: Light microscopy of cGVHD lacrimal glands showing the infiltration of inflammatory cells around medium-sized interlobular ducts, and a severely fibrotic interstitium that is more intense in the periductal areas. Inset: Infiltrated inflammatory cells (white arrowhead) and interstitial fibroblasts (red arrowhead). B: Collagen deposition was quantified as the ratio of the blue-stained area to the total stained area in Mallory staining and expressed as % fibrotic area. Collagen deposition was more severe in the cGVHD lacrimal glands compared with the controls (p = 0.000005). C: Immunostaining for CD45 and HSP47 in lacrimal glands shows intense brown staining of many cells in the interstitium and periductal areas in the cGVHD lacrimal glands. CD45-positive cells indicate infiltrating inflammatory cells. HSP47-positive cells are spindle-shaped cells with oval nuclei, which indicate fibroblasts (inset). D: The mean densities of CD45 + inflammatory cells and HSP47 + fibroblasts were significantly higher in the cGVHD compared with control lacrimal glands (CD45 + inflammatory cells; p = 0.001, HSP47 + fibroblasts; p = 0.0001). E: Quantitative real-time PCR revealed that the HSP47, collagen type I alpha 1, collagen type I alpha 2, and collagen type III alpha 1 expressions in the cGVHD group were higher than in the control group (HSP47; p = 0.004, collage type I alpha 1; p = 0.0002, collagen type I alpha 2; p = 0.03, collagen type III alpha 1; p = 0.002). Magnification, A: X200, C: X200. Scale bar, A: 50 µm, C: 50 µm. The results represent the mean ± SD. B: n = 9; D: n = 8; E: n = 9 in each group. E: The vertical axis shows the expression ratio of mRNAs. Fold change shows the expression ratio of mRNAs in the control and cGVHD group. The expression level in the control group was defined as 1. *p

    Techniques Used: Light Microscopy, Staining, Immunostaining, Real-time Polymerase Chain Reaction, Expressing

    4) Product Images from "Peroxisome proliferator-activated receptor-gamma dependent pathway reduces the phosphorylation of dynamin-related protein 1 and ameliorates hippocampal injury induced by global ischemia in rats"

    Article Title: Peroxisome proliferator-activated receptor-gamma dependent pathway reduces the phosphorylation of dynamin-related protein 1 and ameliorates hippocampal injury induced by global ischemia in rats

    Journal: Journal of Biomedical Science

    doi: 10.1186/s12929-016-0262-3

    Drp1-siRNA downregulates p-Drp1(Ser616) expression in the hippocampal CA1 subfield after TGI. Fluorescent double staining of p-Drp1 (green) and NeuN (red) in the hippocampal CA1 subfield in a sham control group, b ischemia/reperfusion 24 h with negative control siRNA and c siRNA for Drp1 and ischemia/reperfusion 24 h. NeuN showed the nuclear distribution while p-Drp1 were dispersed in the cytoplasm. Scale bars, 50 μm Merged images with higher magnification demonstrate that p-Drp1(Ser616) and NeuN-positive cells localized separately in the nucleus and non-nuclear cytoplasm in neurons in ( d ). Scale bars, 2 μm. A semi-quantitative data about the change of p-Drp1(Ser616) expression after Drp1-siRNA for Fig. 5 a-c was shown in ( f ). Fluorescent double staining of p-Drp1(Ser616) (green) and COXIV (blue) in the neuron of the hippocampal CA1 subfield; merged image shows the co-localization in mitochondria in neurons under the condition of ischemia/reperfusion for 24 h ( e ). Scale bars, 2 μm. I/R: ischemia/reperfusion, NC: negative control siRNA. COXIV: cytochrome c oxidase subunit 4
    Figure Legend Snippet: Drp1-siRNA downregulates p-Drp1(Ser616) expression in the hippocampal CA1 subfield after TGI. Fluorescent double staining of p-Drp1 (green) and NeuN (red) in the hippocampal CA1 subfield in a sham control group, b ischemia/reperfusion 24 h with negative control siRNA and c siRNA for Drp1 and ischemia/reperfusion 24 h. NeuN showed the nuclear distribution while p-Drp1 were dispersed in the cytoplasm. Scale bars, 50 μm Merged images with higher magnification demonstrate that p-Drp1(Ser616) and NeuN-positive cells localized separately in the nucleus and non-nuclear cytoplasm in neurons in ( d ). Scale bars, 2 μm. A semi-quantitative data about the change of p-Drp1(Ser616) expression after Drp1-siRNA for Fig. 5 a-c was shown in ( f ). Fluorescent double staining of p-Drp1(Ser616) (green) and COXIV (blue) in the neuron of the hippocampal CA1 subfield; merged image shows the co-localization in mitochondria in neurons under the condition of ischemia/reperfusion for 24 h ( e ). Scale bars, 2 μm. I/R: ischemia/reperfusion, NC: negative control siRNA. COXIV: cytochrome c oxidase subunit 4

    Techniques Used: Expressing, Double Staining, Negative Control

    5) Product Images from "Chemerin-ChemR23 Signaling in Tooth Development"

    Article Title: Chemerin-ChemR23 Signaling in Tooth Development

    Journal: Journal of Dental Research

    doi: 10.1177/0022034512464777

    Immunofluorescent histochemical analyses of cultured porcine DE and DM cells. Cultured porcine DE (top panels) and DM (bottom panels) cells were double-labeled with anti-CK14 and anti-Chemerin, anti-Vimentin, and anti-ChemR23 antibodies, or isotype control
    Figure Legend Snippet: Immunofluorescent histochemical analyses of cultured porcine DE and DM cells. Cultured porcine DE (top panels) and DM (bottom panels) cells were double-labeled with anti-CK14 and anti-Chemerin, anti-Vimentin, and anti-ChemR23 antibodies, or isotype control

    Techniques Used: Cell Culture, Labeling

    Chemerin/ChemR23-mediated ribosomal protein S6 (rS6) phosphorylation and Runx2 expression in cultured porcine DM cells. ( A) Chemerin-induced rS6 phosphorylation in DM cells. Serum-depleted cultured DM cells were treated with recombinant Chemerin, followed
    Figure Legend Snippet: Chemerin/ChemR23-mediated ribosomal protein S6 (rS6) phosphorylation and Runx2 expression in cultured porcine DM cells. ( A) Chemerin-induced rS6 phosphorylation in DM cells. Serum-depleted cultured DM cells were treated with recombinant Chemerin, followed

    Techniques Used: Expressing, Cell Culture, Recombinant

    Chemerin and ChemR23 expression in mouse molar tooth development. DAPI-stained nuclei of developmental-stage mouse tooth frozen sections (Panels A1, B1, C1, D1 ). IF analysis detected Chemerin (red) in E12.5 DE (Panel A2 ), E14.5 DE-derived enamel knot
    Figure Legend Snippet: Chemerin and ChemR23 expression in mouse molar tooth development. DAPI-stained nuclei of developmental-stage mouse tooth frozen sections (Panels A1, B1, C1, D1 ). IF analysis detected Chemerin (red) in E12.5 DE (Panel A2 ), E14.5 DE-derived enamel knot

    Techniques Used: Expressing, Staining, Derivative Assay

    6) Product Images from "Transmission of Chronic Wasting Disease Identifies a Prion Strain Causing Cachexia and Heart Infection in Hamsters"

    Article Title: Transmission of Chronic Wasting Disease Identifies a Prion Strain Causing Cachexia and Heart Infection in Hamsters

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0028026

    Immunohistochemistry for PrP Sc in heart in Syrian golden hamsters infected with chronic wasting disease. Skeletal muscle of heart from mock (D) and WST CWD-infected (A through C) hamsters. Panels A through C are the same field of view that are separated into three panels according to the immunofluorescence staining. Heart was analyzed by dual immunofluorescence for desmin (A, C, D) and PrP Sc (B, C, D). ToPro®-3 staining of nuclei is indicated by blue fluorescence. Scale bar in A is 10 µm.
    Figure Legend Snippet: Immunohistochemistry for PrP Sc in heart in Syrian golden hamsters infected with chronic wasting disease. Skeletal muscle of heart from mock (D) and WST CWD-infected (A through C) hamsters. Panels A through C are the same field of view that are separated into three panels according to the immunofluorescence staining. Heart was analyzed by dual immunofluorescence for desmin (A, C, D) and PrP Sc (B, C, D). ToPro®-3 staining of nuclei is indicated by blue fluorescence. Scale bar in A is 10 µm.

    Techniques Used: Immunohistochemistry, Infection, Immunofluorescence, Staining, Fluorescence

    Distribution of PrP Sc in olfactory sensory epithelium in Syrian golden hamsters infected with chronic wasting disease. Laser scanning confocal microscopy of olfactory marker protein (OMP)( A, G ), PrP Sc ( B, H ), and for both OMP and PrP Sc (Merge)( C, I ) in Syrian golden hamsters infected with TgMo-sghPrP CWD (A, B, C) and mock-infected hamsters (G, H, I). Laser scanning confocal microscopy of adenylyl cyclase III (ACIII)( D ), PrP Sc ( E ), and for both ACIII and PrP Sc (Merge)( F ) in CWD infected SGH. Panels A through C , D through F and G through I are the same field of view that are separated into three panels according to the immunofluorescence staining. ToPro®-3 staining of nuclei is indicated by blue fluorescence. Olfactory receptor neurons in the olfactory sensory epithelium (OSE width indicated by white line), and nerve fibers in the subepithelial layer (SE), both express high levels of OMP (A, C and G and I). ACIII is located on the sensory cilia that project from the terminal dendrites of ORNs and its distribution was prominent at the border between the OSE and airway lumen (D and F). The white arrow points to the distal edge of the OSE where it borders the lumen of the nasal airway. Scale bar in A, D and G is 50 µm.
    Figure Legend Snippet: Distribution of PrP Sc in olfactory sensory epithelium in Syrian golden hamsters infected with chronic wasting disease. Laser scanning confocal microscopy of olfactory marker protein (OMP)( A, G ), PrP Sc ( B, H ), and for both OMP and PrP Sc (Merge)( C, I ) in Syrian golden hamsters infected with TgMo-sghPrP CWD (A, B, C) and mock-infected hamsters (G, H, I). Laser scanning confocal microscopy of adenylyl cyclase III (ACIII)( D ), PrP Sc ( E ), and for both ACIII and PrP Sc (Merge)( F ) in CWD infected SGH. Panels A through C , D through F and G through I are the same field of view that are separated into three panels according to the immunofluorescence staining. ToPro®-3 staining of nuclei is indicated by blue fluorescence. Olfactory receptor neurons in the olfactory sensory epithelium (OSE width indicated by white line), and nerve fibers in the subepithelial layer (SE), both express high levels of OMP (A, C and G and I). ACIII is located on the sensory cilia that project from the terminal dendrites of ORNs and its distribution was prominent at the border between the OSE and airway lumen (D and F). The white arrow points to the distal edge of the OSE where it borders the lumen of the nasal airway. Scale bar in A, D and G is 50 µm.

    Techniques Used: Infection, Confocal Microscopy, Marker, Immunofluorescence, Staining, Fluorescence

    7) Product Images from "Timely Synthesis of the Adenovirus Type 5 E1B 55-Kilodalton Protein Is Required for Efficient Genome Replication in Normal Human Cells"

    Article Title: Timely Synthesis of the Adenovirus Type 5 E1B 55-Kilodalton Protein Is Required for Efficient Genome Replication in Normal Human Cells

    Journal: Journal of Virology

    doi: 10.1128/JVI.06764-11

    Localization of Mre11, E2 DBP, and the E4 Orf3 proteins in infected HFFs. HFFs at ∼70% confluence were infected with 50 PFU/cell AdEasyE1 (WT), the AdEasyE1Δ2347 mutant (Δ2347), or mock-infected cells (M) for 24 h. They were then processed for immunofluorescence, and Mre11, E2 DBP, and E4 Orf3 were visualized as described in Materials and Methods. The E4 Orf3 protein signal is false-colored in blue. Nuclei stained with DAPI are shown false-colored in cyan. The merged images do not include the nuclear stain.
    Figure Legend Snippet: Localization of Mre11, E2 DBP, and the E4 Orf3 proteins in infected HFFs. HFFs at ∼70% confluence were infected with 50 PFU/cell AdEasyE1 (WT), the AdEasyE1Δ2347 mutant (Δ2347), or mock-infected cells (M) for 24 h. They were then processed for immunofluorescence, and Mre11, E2 DBP, and E4 Orf3 were visualized as described in Materials and Methods. The E4 Orf3 protein signal is false-colored in blue. Nuclei stained with DAPI are shown false-colored in cyan. The merged images do not include the nuclear stain.

    Techniques Used: Infection, Mutagenesis, Immunofluorescence, Staining

    8) Product Images from "Anti-serpin Antibody-mediated Regulation of Proteases in Autoimmune Diabetes *"

    Article Title: Anti-serpin Antibody-mediated Regulation of Proteases in Autoimmune Diabetes *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.409664

    Effect of anti-serpin B13 mAb on CD4 and CD19 in pancreas-associated lymphocytes. A , left panels , FACS analysis of CD4 expression in the islets and inguinal lymph nodes of NOD mice treated with control IgG ( n = 4), anti-serpin B13 mAb ( n = 4), the E64
    Figure Legend Snippet: Effect of anti-serpin B13 mAb on CD4 and CD19 in pancreas-associated lymphocytes. A , left panels , FACS analysis of CD4 expression in the islets and inguinal lymph nodes of NOD mice treated with control IgG ( n = 4), anti-serpin B13 mAb ( n = 4), the E64

    Techniques Used: FACS, Expressing, Mouse Assay

    Effect of anti-serpin B13 natural autoantibodies on CD4 in the pancreas-associated lymphocytes. Left panels , analysis of CD4 expression in 4-week-old female NOD mice that had been prescreened for low (SBA low ) or high (SBA high ) secretion of anti-serpin
    Figure Legend Snippet: Effect of anti-serpin B13 natural autoantibodies on CD4 in the pancreas-associated lymphocytes. Left panels , analysis of CD4 expression in 4-week-old female NOD mice that had been prescreened for low (SBA low ) or high (SBA high ) secretion of anti-serpin

    Techniques Used: Expressing, Mouse Assay

    Effect of anti-serpin B13 mAb on protease targets. A , serum-binding activity of serpin B13 in NOD mice isolated from different sources. Left panel , serum samples that were positive ( S1–S3 ; n = 3) or negative ( S4–S6 ; n = 3) for binding
    Figure Legend Snippet: Effect of anti-serpin B13 mAb on protease targets. A , serum-binding activity of serpin B13 in NOD mice isolated from different sources. Left panel , serum samples that were positive ( S1–S3 ; n = 3) or negative ( S4–S6 ; n = 3) for binding

    Techniques Used: Binding Assay, Activity Assay, Mouse Assay, Isolation

    Expression of serpin B13 in the pancreas. Shown is the costaining of frozen pancreatic sections obtained from 6-week-old NOD mouse with anti-serpin B13 mAb and antibodies directed against glucagon ( A ), CD31 ( B ), and keratin-19 ( C ). Staining with the isotype
    Figure Legend Snippet: Expression of serpin B13 in the pancreas. Shown is the costaining of frozen pancreatic sections obtained from 6-week-old NOD mouse with anti-serpin B13 mAb and antibodies directed against glucagon ( A ), CD31 ( B ), and keratin-19 ( C ). Staining with the isotype

    Techniques Used: Expressing, Staining

    9) Product Images from "Eya2, a Target Activated by Plzf, Is Critical for PLZF-RARA-Induced Leukemogenesis"

    Article Title: Eya2, a Target Activated by Plzf, Is Critical for PLZF-RARA-Induced Leukemogenesis

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00585-16

    Myeloid immortalization of KSL and MP cells by Eya2 . (A) Experimental strategy for myeloid immortalization assays with retroviral transduction using pMYs-IN. (B) Structure of Eya2 and missense mutants. Arrows show primers used in RT-qPCR. (C) Expression of Eya2 and its mutants by Western blotting analyses using anti-Eya2 and anti-α-tubulin (αTub [an internal control]) antibodies (αEya2 and αTub). (D) Expression levels of Eya2 and its mutants by RT-qPCR of colony-forming cells at the end of the first plating in panel A. (E) Myeloid immortalization assays of KSL and MP cells after retroviral transduction. (F to H) Typical morphology of colonies of Eya2 -transduced KSL cells at the third round of plating (F) and typical morphology (G) and immunophenotype (H) of the cells constituting the colonies. Cells were stained with Wright-Giemsa stain. (I) Localization of Eya2 in the FLAG-tagged Eya2 -immortalized KSL cells analyzed by immunofluorescent confocal microscopy. Alexa Fluor 568-conjugated secondary antibody reacting with anti-DDDDK-tag antibody (αDDDDK) in the immortalized cells visualized their cellular localization (top). Nuclei were visualized with TO-PRO-3 iodide (middle), and a merged image is displayed (bottom). Magnifications (bar lengths): F, ×40 (200 μm); G, ×400 (20 μm); I, ×400 (30 μm). Bar graphs show means ± SD from three independent experiments.
    Figure Legend Snippet: Myeloid immortalization of KSL and MP cells by Eya2 . (A) Experimental strategy for myeloid immortalization assays with retroviral transduction using pMYs-IN. (B) Structure of Eya2 and missense mutants. Arrows show primers used in RT-qPCR. (C) Expression of Eya2 and its mutants by Western blotting analyses using anti-Eya2 and anti-α-tubulin (αTub [an internal control]) antibodies (αEya2 and αTub). (D) Expression levels of Eya2 and its mutants by RT-qPCR of colony-forming cells at the end of the first plating in panel A. (E) Myeloid immortalization assays of KSL and MP cells after retroviral transduction. (F to H) Typical morphology of colonies of Eya2 -transduced KSL cells at the third round of plating (F) and typical morphology (G) and immunophenotype (H) of the cells constituting the colonies. Cells were stained with Wright-Giemsa stain. (I) Localization of Eya2 in the FLAG-tagged Eya2 -immortalized KSL cells analyzed by immunofluorescent confocal microscopy. Alexa Fluor 568-conjugated secondary antibody reacting with anti-DDDDK-tag antibody (αDDDDK) in the immortalized cells visualized their cellular localization (top). Nuclei were visualized with TO-PRO-3 iodide (middle), and a merged image is displayed (bottom). Magnifications (bar lengths): F, ×40 (200 μm); G, ×400 (20 μm); I, ×400 (30 μm). Bar graphs show means ± SD from three independent experiments.

    Techniques Used: Transduction, Quantitative RT-PCR, Expressing, Western Blot, Staining, Giemsa Stain, Confocal Microscopy

    Inducible immortalization of KSL and MP cells by ER-Eya2 . (A) Experimental strategy for myeloid immortalization assays of KSL and MP cells with retroviral transduction of the ER-Eya2 fusion gene using pMYs-IN. ER , estrogen receptor gene; 4OHT, 4-hydroxytamoxifen. (B) Structure of Eya2 and ER-Eya2. LBD, mutant ligand-binding domain of the mouse ER. (C) Expression of ER-Eya2 by Western blotting using anti-Eya2 (αEya2 [top]), anti-ER antibody (αER [middle]), and anti-α-tubulin (αTub [an internal control; bottom]). (D) Myeloid immortalization assays of ER-Eya2 -transduced cells in the presence or absence of 4OHT. (E) 4OHT-dependent clonogenicity of cells inducibly immortalized by ER-Eya2 . (F) Localization of ER-Eya2 in the inducibly immortalized KSL cells analyzed by immunofluorescent confocal microscopy, following a 3-day culture in the presence (left panels) and absence (right panels) of 4OHT. Alexa Fluor 568-conjugated secondary antibody reacting with anti-ER in the immortalized cells visualized its cellular localization (top). Nuclei were visualized with TO-PRO-3 iodide (middle), and merged images are displayed (bottom). Magnification (bar length), ×400 (30 μm). Bar graphs show the means ± SD from three independent experiments.
    Figure Legend Snippet: Inducible immortalization of KSL and MP cells by ER-Eya2 . (A) Experimental strategy for myeloid immortalization assays of KSL and MP cells with retroviral transduction of the ER-Eya2 fusion gene using pMYs-IN. ER , estrogen receptor gene; 4OHT, 4-hydroxytamoxifen. (B) Structure of Eya2 and ER-Eya2. LBD, mutant ligand-binding domain of the mouse ER. (C) Expression of ER-Eya2 by Western blotting using anti-Eya2 (αEya2 [top]), anti-ER antibody (αER [middle]), and anti-α-tubulin (αTub [an internal control; bottom]). (D) Myeloid immortalization assays of ER-Eya2 -transduced cells in the presence or absence of 4OHT. (E) 4OHT-dependent clonogenicity of cells inducibly immortalized by ER-Eya2 . (F) Localization of ER-Eya2 in the inducibly immortalized KSL cells analyzed by immunofluorescent confocal microscopy, following a 3-day culture in the presence (left panels) and absence (right panels) of 4OHT. Alexa Fluor 568-conjugated secondary antibody reacting with anti-ER in the immortalized cells visualized its cellular localization (top). Nuclei were visualized with TO-PRO-3 iodide (middle), and merged images are displayed (bottom). Magnification (bar length), ×400 (30 μm). Bar graphs show the means ± SD from three independent experiments.

    Techniques Used: Transduction, Mutagenesis, Ligand Binding Assay, Expressing, Western Blot, Confocal Microscopy

    10) 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

    11) Product Images from "Sec16A, a key protein in COPII vesicle formation, regulates the stability and localization of the novel ubiquitin ligase RNF183"

    Article Title: Sec16A, a key protein in COPII vesicle formation, regulates the stability and localization of the novel ubiquitin ligase RNF183

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0190407

    Characterization of the novel ubiquitin ligase RNF183. ]. Blue box, transmembrane domain; red triangle, RING-finger domain; purple box, low complexity sequence. The number at right indicates the peptide length. (C) In vitro auto-ubiquitination of wild type and mutant RNF183. In vitro transcribed/translated V5-tagged RNF183 tagged was mixed and incubated with recombinant E1, E2, and HA-ubiquitin. The reaction mixture was immunoprecipitated with an anti-V5 antibody and subjected to Western blotting with anti-polyubiquitin ( upper panel) and anti-V5 antibodies ( lower panel). WT, wild type; ΔR, RING-finger domain deletion mutant; CS, Cys13-, and Cys16-to-Ser point mutations in the RING domain; IP, immunoprecipitation; Ub, ubiquitin. Asterisk indicates the immunoglobulin heavy chain. (D) In vitro auto-ubiquitination of RNF183 in the absence of each component. (E) Subcellular localization of RNF183. HeLa cells stably transfected with RNF183-V5 ( red ) were subjected to immunofluorescence staining with various antibodies for organelle makers (Calnexin, GM130, EEA1, Rab7 and LAMP1; green ) and DAPI for nuclear staining ( blue ).
    Figure Legend Snippet: Characterization of the novel ubiquitin ligase RNF183. ]. Blue box, transmembrane domain; red triangle, RING-finger domain; purple box, low complexity sequence. The number at right indicates the peptide length. (C) In vitro auto-ubiquitination of wild type and mutant RNF183. In vitro transcribed/translated V5-tagged RNF183 tagged was mixed and incubated with recombinant E1, E2, and HA-ubiquitin. The reaction mixture was immunoprecipitated with an anti-V5 antibody and subjected to Western blotting with anti-polyubiquitin ( upper panel) and anti-V5 antibodies ( lower panel). WT, wild type; ΔR, RING-finger domain deletion mutant; CS, Cys13-, and Cys16-to-Ser point mutations in the RING domain; IP, immunoprecipitation; Ub, ubiquitin. Asterisk indicates the immunoglobulin heavy chain. (D) In vitro auto-ubiquitination of RNF183 in the absence of each component. (E) Subcellular localization of RNF183. HeLa cells stably transfected with RNF183-V5 ( red ) were subjected to immunofluorescence staining with various antibodies for organelle makers (Calnexin, GM130, EEA1, Rab7 and LAMP1; green ) and DAPI for nuclear staining ( blue ).

    Techniques Used: Sequencing, In Vitro, Mutagenesis, Incubation, Recombinant, Immunoprecipitation, Western Blot, Stable Transfection, Transfection, Immunofluorescence, Staining

    Effects of Sec16 on RNF183 subcellular localization. (A) Effect of Sec16A downregulation on RNF183 subcellular localization. HeLa cells stably expressing RNF183-V5 were transfected with NC (1st, 3rd, 5th panels) or Sec16A (2nd, 4th, 6th panels) siRNAs. At 48 h after transfection, cells were subjected to immunofluorescence staining with anti-V5 ( green ) and anti-calnexin, GM130, or LAMP1 ( red ) antibodies, and DAPI ( blue ). (B) Effect of proteasome inhibition on RNF183 subcellular localization. HeLa cells stably expressing RNF183-V5 were transfected with NC ( top panels) or Sec16A ( middle and bottom panels) siRNAs. At 36 h after transfection, cells were incubated with ( bottom panels) or without ( top and middle panels) 10 μM MG132 for 12 h.
    Figure Legend Snippet: Effects of Sec16 on RNF183 subcellular localization. (A) Effect of Sec16A downregulation on RNF183 subcellular localization. HeLa cells stably expressing RNF183-V5 were transfected with NC (1st, 3rd, 5th panels) or Sec16A (2nd, 4th, 6th panels) siRNAs. At 48 h after transfection, cells were subjected to immunofluorescence staining with anti-V5 ( green ) and anti-calnexin, GM130, or LAMP1 ( red ) antibodies, and DAPI ( blue ). (B) Effect of proteasome inhibition on RNF183 subcellular localization. HeLa cells stably expressing RNF183-V5 were transfected with NC ( top panels) or Sec16A ( middle and bottom panels) siRNAs. At 36 h after transfection, cells were incubated with ( bottom panels) or without ( top and middle panels) 10 μM MG132 for 12 h.

    Techniques Used: Stable Transfection, Expressing, Transfection, Immunofluorescence, Staining, Inhibition, Incubation

    Effects of Sec16 on other ubiquitin ligases. (A) Interactions of RNF152 and HRD1 with Sec16A. Coimmunoprecipitation was performed in HEK293 cells engineered to stably express V5-tagged RNF183, V5-tagged RNF152, or myc-tagged HRD1. Cell lysates were immunoprecipitated with anti-V5 or anti-myc antibodies or normal mouse immunoglobulin G (IgG; negative control). Immune complexes were analyzed by Western blotting with an anti-Sec16A antibody ( top panel) and anti-V5 ( second panel) or anti-myc antibodies ( third panel). (B, D) Effect of Sec16A downregulation on RNF152 and HRD1 protein stability. Stable RNF152-V5- or HRD1-myc-expressing HEK293 cells were transfected with NC or Sec16A siRNA. At 48 h after transfection, cells were subjected to a CHX assay. (C, E) Asterisks represent significant differences (n = 3; Student’s t test with Bonferroni correction, *p
    Figure Legend Snippet: Effects of Sec16 on other ubiquitin ligases. (A) Interactions of RNF152 and HRD1 with Sec16A. Coimmunoprecipitation was performed in HEK293 cells engineered to stably express V5-tagged RNF183, V5-tagged RNF152, or myc-tagged HRD1. Cell lysates were immunoprecipitated with anti-V5 or anti-myc antibodies or normal mouse immunoglobulin G (IgG; negative control). Immune complexes were analyzed by Western blotting with an anti-Sec16A antibody ( top panel) and anti-V5 ( second panel) or anti-myc antibodies ( third panel). (B, D) Effect of Sec16A downregulation on RNF152 and HRD1 protein stability. Stable RNF152-V5- or HRD1-myc-expressing HEK293 cells were transfected with NC or Sec16A siRNA. At 48 h after transfection, cells were subjected to a CHX assay. (C, E) Asterisks represent significant differences (n = 3; Student’s t test with Bonferroni correction, *p

    Techniques Used: Stable Transfection, Immunoprecipitation, Negative Control, Western Blot, Expressing, Transfection

    Effects of Sec16 on RNF183 protein stability. (A) Effect of Sec16A downregulation on RNF183 protein stability. HeLa cells stably expressing RNF183-V5 were transfected with NC (1st, 3rd, and 5th panels) or Sec16A (2nd, 4th and 6th panels) siRNA. At 44 h after transfection, cells were treated with 30 μg/ml cycloheximide (CHX) and 10 μM MG132 for the indicated periods. Total cell lysates were analyzed by Western blotting with an anti-V5 (1st and 2nd panels), Sec16A (3rd and 4th panels), and β-actin (5th and 6th panels) antibodies. (B) Quantitative curves of data from (A). RNF183 levels at each time point were plotted relative to the level at time 0 (n = 3). Asterisks represent significant differences (Student’s t test with Bonferroni correction, *p
    Figure Legend Snippet: Effects of Sec16 on RNF183 protein stability. (A) Effect of Sec16A downregulation on RNF183 protein stability. HeLa cells stably expressing RNF183-V5 were transfected with NC (1st, 3rd, and 5th panels) or Sec16A (2nd, 4th and 6th panels) siRNA. At 44 h after transfection, cells were treated with 30 μg/ml cycloheximide (CHX) and 10 μM MG132 for the indicated periods. Total cell lysates were analyzed by Western blotting with an anti-V5 (1st and 2nd panels), Sec16A (3rd and 4th panels), and β-actin (5th and 6th panels) antibodies. (B) Quantitative curves of data from (A). RNF183 levels at each time point were plotted relative to the level at time 0 (n = 3). Asterisks represent significant differences (Student’s t test with Bonferroni correction, *p

    Techniques Used: Stable Transfection, Expressing, Transfection, Western Blot

    12) Product Images from "Endosomal sorting of VAMP3 is regulated by PI4K2A"

    Article Title: Endosomal sorting of VAMP3 is regulated by PI4K2A

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.148809

    TfR delivery to the ERC and rate of recycling are reduced upon PI4K2A depletion. After treatment of COS-7 cells with control siRNA or siRNA directed against PI4K2A, cells were serum-starved for 30 min, then incubated with Alexa-Fluor-488-conjugated transferrin for 10 min and either fixed (A,B) or chased in complete medium for 30 min prior to fixation in 4% paraformaldehyde (C,D). PI4K2A knockdown (A, right panel) impedes delivery of transferrin to the ERC in comparison to the control siRNA-treated cells (A, left panels). (B) Appearance of transferrin in the perinuclear ERC in cells treated with siRNA oligonucleotides, shown in A, or transfected with TeNT. The ratio of transferrin fluorescence in the ERC (F ERC ) and total fluorescence (F total ) was determined as described in the Materials and Methods. Shown are ratios of mean+s.e.m. fluorescence values ( n = 50). Statistical significance was determined by one-way ANOVA analysis. * P
    Figure Legend Snippet: TfR delivery to the ERC and rate of recycling are reduced upon PI4K2A depletion. After treatment of COS-7 cells with control siRNA or siRNA directed against PI4K2A, cells were serum-starved for 30 min, then incubated with Alexa-Fluor-488-conjugated transferrin for 10 min and either fixed (A,B) or chased in complete medium for 30 min prior to fixation in 4% paraformaldehyde (C,D). PI4K2A knockdown (A, right panel) impedes delivery of transferrin to the ERC in comparison to the control siRNA-treated cells (A, left panels). (B) Appearance of transferrin in the perinuclear ERC in cells treated with siRNA oligonucleotides, shown in A, or transfected with TeNT. The ratio of transferrin fluorescence in the ERC (F ERC ) and total fluorescence (F total ) was determined as described in the Materials and Methods. Shown are ratios of mean+s.e.m. fluorescence values ( n = 50). Statistical significance was determined by one-way ANOVA analysis. * P

    Techniques Used: Incubation, Transfection, Fluorescence

    13) Product Images from "Immunohistochemical Study of the Laminin α5 Chain and Its Specific Receptor, Basal Cell Adhesion Molecule (BCAM), in both Fetal and Adult Rat Pituitary Glands"

    Article Title: Immunohistochemical Study of the Laminin α5 Chain and Its Specific Receptor, Basal Cell Adhesion Molecule (BCAM), in both Fetal and Adult Rat Pituitary Glands

    Journal: Acta Histochemica et Cytochemica

    doi: 10.1267/ahc.18014

    BCAM-expressing cells in the rat anterior pituitary gland at P60. a–c : Double immunostaining of S100 protein ( a , a marker of folliculo-stellate cells, green) and BCAM ( b , red) in the anterior pituitary gland. d–f : Double immunostaining of isolectin B4 ( d , a marker of endothelial cells, green) and BCAM ( e , red) in the anterior pituitary gland. Bar = 10 μm ( a–f ).
    Figure Legend Snippet: BCAM-expressing cells in the rat anterior pituitary gland at P60. a–c : Double immunostaining of S100 protein ( a , a marker of folliculo-stellate cells, green) and BCAM ( b , red) in the anterior pituitary gland. d–f : Double immunostaining of isolectin B4 ( d , a marker of endothelial cells, green) and BCAM ( e , red) in the anterior pituitary gland. Bar = 10 μm ( a–f ).

    Techniques Used: Expressing, Double Immunostaining, Marker

    14) Product Images from "Identification and Characterization of the V(D)J Recombination Activating Gene 1 in Long-Term Memory of Context Fear Conditioning"

    Article Title: Identification and Characterization of the V(D)J Recombination Activating Gene 1 in Long-Term Memory of Context Fear Conditioning

    Journal: Neural Plasticity

    doi: 10.1155/2016/1752176

    RAG1 protein expression in amygdalar neuronal cells. Amygdalar coronal sections of context fear conditioning-trained mice, perfused 1 h after conditioning, were used for immunofluorescence and analyzed by confocal microscopy. Antibodies from immunofluorescence were validated by Western blot analysis. (a) Amygdalar area representative images of a double immunostaining using RAG1 antibody labeled with Alexa Fluor 488, green channel signal, and NeuN antibody labeled with Alexa Fluor 568, red channel signal. The left panel shows the NeuN positive neuronal nuclei, while the middle panel depicts RAG1 immunopositive cells. The right panel is the merge image showing colocalization of the NeuN neuronal nuclei marker and RAG1. Arrows point to some of the RAG1 immunopositive neurons. These immunofluorescent images revealed colocalization of RAG1 protein expressing cells with those expressing NeuN, suggesting the presence of RAG1 in neurons, although not all neurons expressed RAG1. (b) Tissue punches from amygdala (Amy) were obtained 1 h after context fear conditioning and analyzed in Western blot by comparative comigration with a standard molecular weight (MW) marker and protein extracts from bone marrow (BM) ((b)-1) and thymus (Thy) ((b)-2). Both sets of experiments consistently showed comigration between the tissues with a band corresponding to ~120 KD of RAG1 protein (green channel corresponding to RAG1 and red channel corresponding to beta-actin, ~42 KD); prestained molecular weight (MW) marker (ladder) was included in all the Western blots. ((b)-3) Additionally, tissue protein extracts from leg muscle (Mus) (negative control) were analyzed compared to amygdalar extracts with respect to RAG1 expression. As expected, RAG1 was not expressed in muscle compared to amygdala ((b)-3), bone marrow ((b)-1), and thymus ((b)-2). ((b)-4) RAG1 antibody preabsorption assays, either with muscle or with bone marrow extracts, showed that only bone marrow extracts, which express RAG1 as opposed to muscle, were able to block the ~120 KD band from amygdalar protein extracts in the Western blots, indicating that RAG1 antibody was preabsorbed (blocked) only by RAG1 protein expressing tissue (bone marrow).
    Figure Legend Snippet: RAG1 protein expression in amygdalar neuronal cells. Amygdalar coronal sections of context fear conditioning-trained mice, perfused 1 h after conditioning, were used for immunofluorescence and analyzed by confocal microscopy. Antibodies from immunofluorescence were validated by Western blot analysis. (a) Amygdalar area representative images of a double immunostaining using RAG1 antibody labeled with Alexa Fluor 488, green channel signal, and NeuN antibody labeled with Alexa Fluor 568, red channel signal. The left panel shows the NeuN positive neuronal nuclei, while the middle panel depicts RAG1 immunopositive cells. The right panel is the merge image showing colocalization of the NeuN neuronal nuclei marker and RAG1. Arrows point to some of the RAG1 immunopositive neurons. These immunofluorescent images revealed colocalization of RAG1 protein expressing cells with those expressing NeuN, suggesting the presence of RAG1 in neurons, although not all neurons expressed RAG1. (b) Tissue punches from amygdala (Amy) were obtained 1 h after context fear conditioning and analyzed in Western blot by comparative comigration with a standard molecular weight (MW) marker and protein extracts from bone marrow (BM) ((b)-1) and thymus (Thy) ((b)-2). Both sets of experiments consistently showed comigration between the tissues with a band corresponding to ~120 KD of RAG1 protein (green channel corresponding to RAG1 and red channel corresponding to beta-actin, ~42 KD); prestained molecular weight (MW) marker (ladder) was included in all the Western blots. ((b)-3) Additionally, tissue protein extracts from leg muscle (Mus) (negative control) were analyzed compared to amygdalar extracts with respect to RAG1 expression. As expected, RAG1 was not expressed in muscle compared to amygdala ((b)-3), bone marrow ((b)-1), and thymus ((b)-2). ((b)-4) RAG1 antibody preabsorption assays, either with muscle or with bone marrow extracts, showed that only bone marrow extracts, which express RAG1 as opposed to muscle, were able to block the ~120 KD band from amygdalar protein extracts in the Western blots, indicating that RAG1 antibody was preabsorbed (blocked) only by RAG1 protein expressing tissue (bone marrow).

    Techniques Used: Expressing, Mouse Assay, Immunofluorescence, Confocal Microscopy, Western Blot, Double Immunostaining, Labeling, Marker, Molecular Weight, Negative Control, Blocking Assay

    15) Product Images from "Identification of Endothelial Cell Junctional Proteins and Lymphocyte Receptors Involved in Transendothelial Migration of Human Effector Memory CD4+ T Cells"

    Article Title: Identification of Endothelial Cell Junctional Proteins and Lymphocyte Receptors Involved in Transendothelial Migration of Human Effector Memory CD4+ T Cells

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1002835

    Knockdown of EC JAM-1 inhibits chemokine-dependent TEM of EM CD4 + T cells. CIITA HDMECs were transfected with control siRNA or two different siRNAs targeting JAM-1 (JAM-1 siRNA-5 and JAM-1 siRNA-6), treated with TNF, either harvested for FACS analysis ( A ) or overlaid with TSST-1 superantigen, and used in flow TEM assays with EM CD4 + T cells ( B ). A , Histograms show FACS analysis of cells stained with isotype control IgG (thin line) or anti–JAM-1 (thick lines) demonstrating effective knockdown. B , Left lower panel (VB2 − ) shows TEM of Vβ2TCR − cells at 15 min. Right lower panel on (VB2 + ) shows TEM of Vβ2TCR + cells at 60 min. Graphs display data from one representative experiment of three (VB2 − ) and two (VB2 + ) separate experiments using T cells from different donors. * p
    Figure Legend Snippet: Knockdown of EC JAM-1 inhibits chemokine-dependent TEM of EM CD4 + T cells. CIITA HDMECs were transfected with control siRNA or two different siRNAs targeting JAM-1 (JAM-1 siRNA-5 and JAM-1 siRNA-6), treated with TNF, either harvested for FACS analysis ( A ) or overlaid with TSST-1 superantigen, and used in flow TEM assays with EM CD4 + T cells ( B ). A , Histograms show FACS analysis of cells stained with isotype control IgG (thin line) or anti–JAM-1 (thick lines) demonstrating effective knockdown. B , Left lower panel (VB2 − ) shows TEM of Vβ2TCR − cells at 15 min. Right lower panel on (VB2 + ) shows TEM of Vβ2TCR + cells at 60 min. Graphs display data from one representative experiment of three (VB2 − ) and two (VB2 + ) separate experiments using T cells from different donors. * p

    Techniques Used: Transmission Electron Microscopy, Transfection, FACS, Flow Cytometry, Staining

    Blocking of T cell LFA-1 but not Mac-1 inhibits TCR-dependent TEM of EM CD4 + T cells. A , Mac-1 is expressed on EM CD4 + T cells. Contour plots of FACS analysis of CD45RA − CD4 + T cells stained with isotype-matched control PE- and FITC-conjugated IgG ( left plot ) or PE-conjugated anti-CD11b and FITC-conjugated anti-CCR7 ( right plot ). Note that there is a significant proportion of CD11b + cells in the CCR7 low (i.e., EM) population. B , TEM assays. EM CD4 + T cells were preincubated with isotype control IgG (control), anti–LFA-1 (LFA-1), or anti–Mac-1 (Mac-1) blocking mAb 30 min prior to flow TEM on TNF-treated CIITA-transduced HDMECs overlaid with TSST-1. Panel on the left (VB2 − ) shows TEM of Vβ2TCR − cells at 15 min. Panel on the right (VB2 + ) shows TEM of Vβ2TCR + cells at 60 min. Graphs display data pooled from three separate experiments using T cells isolated from three different donors. *** p
    Figure Legend Snippet: Blocking of T cell LFA-1 but not Mac-1 inhibits TCR-dependent TEM of EM CD4 + T cells. A , Mac-1 is expressed on EM CD4 + T cells. Contour plots of FACS analysis of CD45RA − CD4 + T cells stained with isotype-matched control PE- and FITC-conjugated IgG ( left plot ) or PE-conjugated anti-CD11b and FITC-conjugated anti-CCR7 ( right plot ). Note that there is a significant proportion of CD11b + cells in the CCR7 low (i.e., EM) population. B , TEM assays. EM CD4 + T cells were preincubated with isotype control IgG (control), anti–LFA-1 (LFA-1), or anti–Mac-1 (Mac-1) blocking mAb 30 min prior to flow TEM on TNF-treated CIITA-transduced HDMECs overlaid with TSST-1. Panel on the left (VB2 − ) shows TEM of Vβ2TCR − cells at 15 min. Panel on the right (VB2 + ) shows TEM of Vβ2TCR + cells at 60 min. Graphs display data pooled from three separate experiments using T cells isolated from three different donors. *** p

    Techniques Used: Blocking Assay, Transmission Electron Microscopy, FACS, Staining, Flow Cytometry, Isolation

    Blocking of EC nectin-2 and/or PVR inhibits TCR-dependent TEM of EM CD4 + T cells. TNF-treated CIITA-transduced HDMECs overlaid with TSST-1 were preincubated with isotype control (control), anti–nectin-2 (nectin-2), anti-PVR (PVR), and both blocking Abs (nectin-2+PVR) prior to flow TEM. Panel on the left shows TEM of Vβ2TCR − cells at 15 min. Panel on the right shows TEM of Vβ2TCR + cells at 60 min. Graphs display data from one representative of eight different experiments, testing each condition with T cells from at least three different donors. *** p
    Figure Legend Snippet: Blocking of EC nectin-2 and/or PVR inhibits TCR-dependent TEM of EM CD4 + T cells. TNF-treated CIITA-transduced HDMECs overlaid with TSST-1 were preincubated with isotype control (control), anti–nectin-2 (nectin-2), anti-PVR (PVR), and both blocking Abs (nectin-2+PVR) prior to flow TEM. Panel on the left shows TEM of Vβ2TCR − cells at 15 min. Panel on the right shows TEM of Vβ2TCR + cells at 60 min. Graphs display data from one representative of eight different experiments, testing each condition with T cells from at least three different donors. *** p

    Techniques Used: Blocking Assay, Transmission Electron Microscopy, Flow Cytometry

    Blocking of T cell DNAM-1 and/or Tactile inhibits TCR-dependent TEM of EM CD4 + T cells. A , Contour plots of FACS analysis of total CD4 + T cells stained with FITC-conjugated IgG (IgG–FITC) and PE-conjugated IgG (IgG–PE, upper left ) or FITC-conjugated anti-CCR7 (CCR7–FITC) and PE-conjugated anti–DNAM-1 (DNAM-1–PE, lower left ) or FITC-conjugated anti-CCR7 and Alexa Fluor 647-complexed IgG (IgG-647, upper right ) or FITC-conjugated anti-CCR7 and Alexa Fluor 647-complexed anti-Tactile (Tactile-647, lower right ). Note the distinct population of cells that are DNAM-1 high, CCR7 low or Tactile high, CCR7 low in the middle , left , and right plots , respectively. Lower histograms show overlays of the isotype-matched control IgG and anti–DNAM-1 ( left ) or anti-Tactile ( right ) plots. B , EM CD4 + T cells were preincubated with isotype-matched control IgG (control), anti–DNAM-1 (DNAM-1), anti-Tactile (Tactile), and both anti–DNAM-1 and anti-Tactile blocking mAbs (DNAM-1+Tactile) prior to flow TEM. Left panel shows TEM of Vβ2TCR − cells at 15 min. Right panel shows TEM of Vβ2TCR + cells at 60 min. Graphs display data from one representative experiment of two (VB2 − ) and four (VB2 + ) separate experiments using T cells isolated from different donors. ** p
    Figure Legend Snippet: Blocking of T cell DNAM-1 and/or Tactile inhibits TCR-dependent TEM of EM CD4 + T cells. A , Contour plots of FACS analysis of total CD4 + T cells stained with FITC-conjugated IgG (IgG–FITC) and PE-conjugated IgG (IgG–PE, upper left ) or FITC-conjugated anti-CCR7 (CCR7–FITC) and PE-conjugated anti–DNAM-1 (DNAM-1–PE, lower left ) or FITC-conjugated anti-CCR7 and Alexa Fluor 647-complexed IgG (IgG-647, upper right ) or FITC-conjugated anti-CCR7 and Alexa Fluor 647-complexed anti-Tactile (Tactile-647, lower right ). Note the distinct population of cells that are DNAM-1 high, CCR7 low or Tactile high, CCR7 low in the middle , left , and right plots , respectively. Lower histograms show overlays of the isotype-matched control IgG and anti–DNAM-1 ( left ) or anti-Tactile ( right ) plots. B , EM CD4 + T cells were preincubated with isotype-matched control IgG (control), anti–DNAM-1 (DNAM-1), anti-Tactile (Tactile), and both anti–DNAM-1 and anti-Tactile blocking mAbs (DNAM-1+Tactile) prior to flow TEM. Left panel shows TEM of Vβ2TCR − cells at 15 min. Right panel shows TEM of Vβ2TCR + cells at 60 min. Graphs display data from one representative experiment of two (VB2 − ) and four (VB2 + ) separate experiments using T cells isolated from different donors. ** p

    Techniques Used: Blocking Assay, Transmission Electron Microscopy, FACS, Staining, Flow Cytometry, Isolation

    Knockdown of EC CD99 inhibits TCR-dependent TEM of EM CD4 + T cells. CIITA HDMEC were transfected with control siRNA or two different siRNAs targeting CD99 (CD99 siRNA-2 and CD99 siRNA-5), treated with TNF, analyzed by FACS ( A ) or overlaid with TSST-1 superantigen (recognized by those T cells with the germline-encoded Vβ2 segment in the TCR), and used in flow TEM assays with EM CD4 + T cells ( B ). A , FACS plots of HDMECs stained with isotype-matched control IgG (thin lines) or anti-CD99 or -CD31 (thick lines in left panels and right panels , respectively) demonstrating knockdown of CD99 without reduction of CD31 expression. B , TEM assays. Graph on the left (VB2 − ) shows TEM of Vβ2TCR − cells (i.e., those T cells with TCR that are not activated by TSST-1) at 15 min. Graph on the right (VB2 + ) shows TEM of Vβ2TCR + cells at 60 min. Graphs display data from one representative experiment of two (VB2 − ) or three (VB2 + ) separate experiments using T cells from different donors. *** p
    Figure Legend Snippet: Knockdown of EC CD99 inhibits TCR-dependent TEM of EM CD4 + T cells. CIITA HDMEC were transfected with control siRNA or two different siRNAs targeting CD99 (CD99 siRNA-2 and CD99 siRNA-5), treated with TNF, analyzed by FACS ( A ) or overlaid with TSST-1 superantigen (recognized by those T cells with the germline-encoded Vβ2 segment in the TCR), and used in flow TEM assays with EM CD4 + T cells ( B ). A , FACS plots of HDMECs stained with isotype-matched control IgG (thin lines) or anti-CD99 or -CD31 (thick lines in left panels and right panels , respectively) demonstrating knockdown of CD99 without reduction of CD31 expression. B , TEM assays. Graph on the left (VB2 − ) shows TEM of Vβ2TCR − cells (i.e., those T cells with TCR that are not activated by TSST-1) at 15 min. Graph on the right (VB2 + ) shows TEM of Vβ2TCR + cells at 60 min. Graphs display data from one representative experiment of two (VB2 − ) or three (VB2 + ) separate experiments using T cells from different donors. *** p

    Techniques Used: Transmission Electron Microscopy, Transfection, FACS, Flow Cytometry, Staining, Expressing

    16) Product Images from "Reiterative Use of the Notch Signal During Zebrafish Intrahepatic Biliary Development"

    Article Title: Reiterative Use of the Notch Signal During Zebrafish Intrahepatic Biliary Development

    Journal: Developmental dynamics : an official publication of the American Association of Anatomists

    doi: 10.1002/dvdy.22220

    Notch signaling is required for canalicular development Confocal projections (5 μm) through the liver of a 70 hpf larva (A) and two 92 hpf (C, D) larvae treated with DAPT from 48 – 70 hpf (A); DAPT from 48 – 92 hpf (C) and DAPT from 48 – 70 hpf (D). A 70 hpf (B) and 92 hpf wild type larva (E) are also shown. All larvae were immunostained with Mdr (red) and 2F11 (green) antibodies. DAPT treatment arrests canalicular development (red) and biliary development (green). Canalicular development reinitiates with DAPT withdrawal (D). Note that the depth of these confocal projections is too small to detect reinitiation of duct development upon DAPT withdrawal.
    Figure Legend Snippet: Notch signaling is required for canalicular development Confocal projections (5 μm) through the liver of a 70 hpf larva (A) and two 92 hpf (C, D) larvae treated with DAPT from 48 – 70 hpf (A); DAPT from 48 – 92 hpf (C) and DAPT from 48 – 70 hpf (D). A 70 hpf (B) and 92 hpf wild type larva (E) are also shown. All larvae were immunostained with Mdr (red) and 2F11 (green) antibodies. DAPT treatment arrests canalicular development (red) and biliary development (green). Canalicular development reinitiates with DAPT withdrawal (D). Note that the depth of these confocal projections is too small to detect reinitiation of duct development upon DAPT withdrawal.

    Techniques Used:

    Notch reporter expression overlaps with the 2F11 epitope in developing intrahepatic biliary cells Whole mount confocal images through the liver of developing Notch reporter larvae stained with the 2F11 antibody (A–D) and a GFP antibody (A′–D′) with overlap of the two markers (A″–D″). GFP positive biliary epithelia are first detected at 45 hpf (A′). At this stage there is strong 2F11 expression in the gallbladder (g), extrahepatic duct (ed) and in a few liver parenchymal cells (l). Only partial overlap between GFP and 2F11 is seen in the liver at this stage. GFP positive cells are detected in the pancreas. From 50 hpf – 70 hpf there is progressive increase in the number of GFP positive cells. At 50 hpf there is significant overlap between the GFP and 2F11 epitopes in the biliary cells. At 60 hpf and 70 hpf there is nearly complete overlap between these markers in the biliary cells. The gallbladder and extrahepatic duct remain GPF negative at all stages. The pancreatic ductal network is also GFP positive (p).
    Figure Legend Snippet: Notch reporter expression overlaps with the 2F11 epitope in developing intrahepatic biliary cells Whole mount confocal images through the liver of developing Notch reporter larvae stained with the 2F11 antibody (A–D) and a GFP antibody (A′–D′) with overlap of the two markers (A″–D″). GFP positive biliary epithelia are first detected at 45 hpf (A′). At this stage there is strong 2F11 expression in the gallbladder (g), extrahepatic duct (ed) and in a few liver parenchymal cells (l). Only partial overlap between GFP and 2F11 is seen in the liver at this stage. GFP positive cells are detected in the pancreas. From 50 hpf – 70 hpf there is progressive increase in the number of GFP positive cells. At 50 hpf there is significant overlap between the GFP and 2F11 epitopes in the biliary cells. At 60 hpf and 70 hpf there is nearly complete overlap between these markers in the biliary cells. The gallbladder and extrahepatic duct remain GPF negative at all stages. The pancreatic ductal network is also GFP positive (p).

    Techniques Used: Expressing, Staining

    Development of hepatocyte canaliculi and intrahepatic biliary network Developmental pattern of the 2F11 epitope (A–E) and Mdr epitope, a canalicular transporter (A′–E′) with overlap of the two markers (A″–E″). The length and number of the canaliculi increases between 60 hpf and 120 hpf. The overlap of the two markers shows that each canaliculus develops in close association with the 2F11 positive biliary epithelia. Each canaliculus drains into a single intrahepatic bile duct. The ducts associated with many of the canaliculi in the 120 hpf sample are out of the plane of focus. (F, F′ and F″) Comparable confocal projections through the liver of a larva immunostained with the keratin-18 and Mdr antibodies. Note overlap (yellow) of the keratin-18 epitope in the terminal ductules with the Mdr protein in the canalicular membrane.
    Figure Legend Snippet: Development of hepatocyte canaliculi and intrahepatic biliary network Developmental pattern of the 2F11 epitope (A–E) and Mdr epitope, a canalicular transporter (A′–E′) with overlap of the two markers (A″–E″). The length and number of the canaliculi increases between 60 hpf and 120 hpf. The overlap of the two markers shows that each canaliculus develops in close association with the 2F11 positive biliary epithelia. Each canaliculus drains into a single intrahepatic bile duct. The ducts associated with many of the canaliculi in the 120 hpf sample are out of the plane of focus. (F, F′ and F″) Comparable confocal projections through the liver of a larva immunostained with the keratin-18 and Mdr antibodies. Note overlap (yellow) of the keratin-18 epitope in the terminal ductules with the Mdr protein in the canalicular membrane.

    Techniques Used:

    Notch reporter expression in developing intrahepatic biliary cells Confocal projections through the liver of 45 hpf – 120 hpf Notch reporter larvae following anti-GFP immunostains. The GFP expression pattern closely resembles the distribution of the 2F11 epitope within intrahepatic biliary cells.
    Figure Legend Snippet: Notch reporter expression in developing intrahepatic biliary cells Confocal projections through the liver of 45 hpf – 120 hpf Notch reporter larvae following anti-GFP immunostains. The GFP expression pattern closely resembles the distribution of the 2F11 epitope within intrahepatic biliary cells.

    Techniques Used: Expressing

    Zebrafish intrahepatic biliary development (A, B) Confocal projections through the liver of a 120 hpf larvae stained with the anti-Keratin 18 antibody. The low power image (A) shows the intrahepatic biliary network (l), extraehepatic ducts (ed) and gallbladder (g). The high power image (B) shows that the keratin-18 protein detects 3 classes of intrahepatic ducts: long ducts (arrow), interconnecting ducts (open arrowhead) and terminal ductules (arrowhead). (C, D) Confocal projections through the liver of a 120 hpf larvae stained with the 2F11 antibody. The low power image (C) shows that the 2F11 epitope is presented within the intrahepatic (l) and extrahepatic (ed) ductal systems as well as the gallbladder (g). The high power image (D) shows that the 2F11 epitope is present in the nucleus of the biliary epithelial cell (arrowhead). This epitope is also detected on the long ducts (arrow) and interconnecting ducts (open arrowhead) but not the terminal ductules. (E–H) Developmental pattern of the 2F11 epitope in the intrahepatic biliary system between 36 hpf and 96 hpf.
    Figure Legend Snippet: Zebrafish intrahepatic biliary development (A, B) Confocal projections through the liver of a 120 hpf larvae stained with the anti-Keratin 18 antibody. The low power image (A) shows the intrahepatic biliary network (l), extraehepatic ducts (ed) and gallbladder (g). The high power image (B) shows that the keratin-18 protein detects 3 classes of intrahepatic ducts: long ducts (arrow), interconnecting ducts (open arrowhead) and terminal ductules (arrowhead). (C, D) Confocal projections through the liver of a 120 hpf larvae stained with the 2F11 antibody. The low power image (C) shows that the 2F11 epitope is presented within the intrahepatic (l) and extrahepatic (ed) ductal systems as well as the gallbladder (g). The high power image (D) shows that the 2F11 epitope is present in the nucleus of the biliary epithelial cell (arrowhead). This epitope is also detected on the long ducts (arrow) and interconnecting ducts (open arrowhead) but not the terminal ductules. (E–H) Developmental pattern of the 2F11 epitope in the intrahepatic biliary system between 36 hpf and 96 hpf.

    Techniques Used: Staining

    17) Product Images from "NS5A Inhibitors Impair NS5A–Phosphatidylinositol 4-Kinase IIIα Complex Formation and Cause a Decrease of Phosphatidylinositol 4-Phosphate and Cholesterol Levels in Hepatitis C Virus-Associated Membranes"

    Article Title: NS5A Inhibitors Impair NS5A–Phosphatidylinositol 4-Kinase IIIα Complex Formation and Cause a Decrease of Phosphatidylinositol 4-Phosphate and Cholesterol Levels in Hepatitis C Virus-Associated Membranes

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.03293-14

    Chemically diverse NS5A inhibitors block NS5A hyperphosphorylation. 10A-IFN cells were transfected with the construct pCD-BlaRep-wt (NS3-5B wt) (A) or pCD-BlaRep-K@67 (NS3-5B 5A-K@67) or pCD-BlaRep-K@67-Y93H (NS3-5B 5A-K@67, Y93H) (B) and Con1 HCV polyprotein
    Figure Legend Snippet: Chemically diverse NS5A inhibitors block NS5A hyperphosphorylation. 10A-IFN cells were transfected with the construct pCD-BlaRep-wt (NS3-5B wt) (A) or pCD-BlaRep-K@67 (NS3-5B 5A-K@67) or pCD-BlaRep-K@67-Y93H (NS3-5B 5A-K@67, Y93H) (B) and Con1 HCV polyprotein

    Techniques Used: Blocking Assay, Transfection, Construct

    NS5A inhibitors promote formation of the large-cluster NS5A phenotype. (A) IF staining of NS5A of Huh7.5-10A and Huh7.5-10A-Y93H cells treated for 8 h with 0.2% DMSO as a control or with the indicated compounds at 20× the EC 50 : DCV, 250 pM; I-7,
    Figure Legend Snippet: NS5A inhibitors promote formation of the large-cluster NS5A phenotype. (A) IF staining of NS5A of Huh7.5-10A and Huh7.5-10A-Y93H cells treated for 8 h with 0.2% DMSO as a control or with the indicated compounds at 20× the EC 50 : DCV, 250 pM; I-7,

    Techniques Used: Staining

    NS5A inhibitors impair NS5A-PI4KIIIα protein complex formation. (A) 10A-IFN cells were cotransfected with the constructs pEF1A-PIK4CA (PI4KIIIα) and pCD-BlaRep-K@67 (NS3-5B 5A-K@67) or pCD-BlaRep-K@67-Y93H (NS3-5B 5A-K@67, Y93H), and the
    Figure Legend Snippet: NS5A inhibitors impair NS5A-PI4KIIIα protein complex formation. (A) 10A-IFN cells were cotransfected with the constructs pEF1A-PIK4CA (PI4KIIIα) and pCD-BlaRep-K@67 (NS3-5B 5A-K@67) or pCD-BlaRep-K@67-Y93H (NS3-5B 5A-K@67, Y93H), and the

    Techniques Used: Construct

    NS5A inhibitors interfere with the enrichment of PI4P in HCV replication membranes. (A) IF staining of PI4P and NS5A in Huh7.5-10A and Huh7.5-10A-Y93H cells treated for 8 h with 0.2% DMSO as a control or with the indicated compounds at 20× the
    Figure Legend Snippet: NS5A inhibitors interfere with the enrichment of PI4P in HCV replication membranes. (A) IF staining of PI4P and NS5A in Huh7.5-10A and Huh7.5-10A-Y93H cells treated for 8 h with 0.2% DMSO as a control or with the indicated compounds at 20× the

    Techniques Used: Staining

    Chemically diverse NS5A inhibitors block NS5A hyperphosphorylation.
    Figure Legend Snippet: Chemically diverse NS5A inhibitors block NS5A hyperphosphorylation.

    Techniques Used: Blocking Assay

    Both PI4KIIIα and NS5A inhibitors reduce cholesterol concentration in the HCV-induced membranous web. Fluorescence staining of intracellular PI4P and unesterified cholesterol. The detection of PI4P (green) and Filipin (red) was performed as detailed
    Figure Legend Snippet: Both PI4KIIIα and NS5A inhibitors reduce cholesterol concentration in the HCV-induced membranous web. Fluorescence staining of intracellular PI4P and unesterified cholesterol. The detection of PI4P (green) and Filipin (red) was performed as detailed

    Techniques Used: Concentration Assay, Fluorescence, Staining

    18) Product Images from "Hepatitis E Virus Genotype 1 Infection of Swine Kidney Cells In Vitro Is Inhibited at Multiple Levels"

    Article Title: Hepatitis E Virus Genotype 1 Infection of Swine Kidney Cells In Vitro Is Inhibited at Multiple Levels

    Journal: Journal of Virology

    doi: 10.1128/JVI.02205-13

    Flow cytometry of transfected cells stained separately for the ORF2 or ORF3 protein. HepG2/C3A and LLC-PK cells electroporated with transcripts from P6 (A), Sar55 (B), or Sar/S17 (C, D) were plated in 6 wells of a 6-well culture plate, incubated at 34.5°C, and harvested 7 (A, B) or 5 (C, D) days later. Cells in triplicate wells were stained for the ORF2 protein, followed by goat anti-human IgG labeled with Alexa Fluor 488 (open bars); cells in the other 3 wells were stained for the ORF3 protein, followed by goat anti-rabbit IgG also labeled with Alexa Fluor 488 (hatched bars). Flow cytometry was performed with the same settings for all samples. (A to C) Mean percentage of positive cells, in triplicate; (D) mean of the geometric mean fluorescence intensity in the same triplicate samples assayed for panel C. Shaded bars, ORF2; dotted bars, ORF3. Error bars indicate standard deviations. P values were determined by Student's t test; P values of less than 0.05 were statistically significant.
    Figure Legend Snippet: Flow cytometry of transfected cells stained separately for the ORF2 or ORF3 protein. HepG2/C3A and LLC-PK cells electroporated with transcripts from P6 (A), Sar55 (B), or Sar/S17 (C, D) were plated in 6 wells of a 6-well culture plate, incubated at 34.5°C, and harvested 7 (A, B) or 5 (C, D) days later. Cells in triplicate wells were stained for the ORF2 protein, followed by goat anti-human IgG labeled with Alexa Fluor 488 (open bars); cells in the other 3 wells were stained for the ORF3 protein, followed by goat anti-rabbit IgG also labeled with Alexa Fluor 488 (hatched bars). Flow cytometry was performed with the same settings for all samples. (A to C) Mean percentage of positive cells, in triplicate; (D) mean of the geometric mean fluorescence intensity in the same triplicate samples assayed for panel C. Shaded bars, ORF2; dotted bars, ORF3. Error bars indicate standard deviations. P values were determined by Student's t test; P values of less than 0.05 were statistically significant.

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

    19) Product Images from "Acinetobacter baumannii invades epithelial cells and outer membrane protein A mediates interactions with epithelial cells"

    Article Title: Acinetobacter baumannii invades epithelial cells and outer membrane protein A mediates interactions with epithelial cells

    Journal: BMC Microbiology

    doi: 10.1186/1471-2180-8-216

    Cell surface binding of rAbOmpA . The grey shaded region represents the control fluorescent level seen in the cells treated with polyclonal anti-rabbit AbOmpA antibody and Alexa Fluor ® 488-conjugated secondary antibody without the addition of rAbOmpA. The solid line represents the fluorescent level seen in the cells treated with 6 μg/ml of rAbOmpA.
    Figure Legend Snippet: Cell surface binding of rAbOmpA . The grey shaded region represents the control fluorescent level seen in the cells treated with polyclonal anti-rabbit AbOmpA antibody and Alexa Fluor ® 488-conjugated secondary antibody without the addition of rAbOmpA. The solid line represents the fluorescent level seen in the cells treated with 6 μg/ml of rAbOmpA.

    Techniques Used: Binding Assay

    Adherence and invasion of A. baumannii ATCC19606 T and isogenic AbOmpA - mutant in epithelial cells . (A). Adherence of A. baumannii to epithelial cells. NCI-H292 cells were infected with A. baumannii strains at an MOI of 100 for 1 h. Actin was stained with Alexa Fluor ® 488 phalloidin (green). Bacteria were stained with polyclonal anti-rabbit AbOmpA antibody, followed by a secondary antibody Alexa Fluor ® 568 (red). (B). NCI-H292 and HEp-2 cells were infected with A. baumannii at an MOI of 100 for 5 h. Invasion efficiency was expressed as the number of bacteria per well using the gentamicin protection assay. Results represent the mean and standard deviation of three separate experiments on separately days. * P
    Figure Legend Snippet: Adherence and invasion of A. baumannii ATCC19606 T and isogenic AbOmpA - mutant in epithelial cells . (A). Adherence of A. baumannii to epithelial cells. NCI-H292 cells were infected with A. baumannii strains at an MOI of 100 for 1 h. Actin was stained with Alexa Fluor ® 488 phalloidin (green). Bacteria were stained with polyclonal anti-rabbit AbOmpA antibody, followed by a secondary antibody Alexa Fluor ® 568 (red). (B). NCI-H292 and HEp-2 cells were infected with A. baumannii at an MOI of 100 for 5 h. Invasion efficiency was expressed as the number of bacteria per well using the gentamicin protection assay. Results represent the mean and standard deviation of three separate experiments on separately days. * P

    Techniques Used: Mutagenesis, Infection, Staining, Standard Deviation

    Invasion of A. baumannii in epithelial cells . (A) NCI-H292 cells were infected with A. baumannii ATCC 19606 T at an MOI of 100 up to 7 h. The colony-forming units were enumerated to measure the time-course of invasion. The result represents the mean ± standard deviation in duplicate wells and repeated a minimum of three separate times on separate days. (B) NCI-H292, HEp-2, and HeLa cells were infected with A. baumannii strains at an MOI of 100 for 5 h. (C) NCI-H292 cells were infected with A. baumannii 05KA103 at an MOI of 100 for 5 h. Actin was stained with Alexa Fluor ® 488 phalloidin (green). Bacteria were stained with polyclonal anti-rabbit AbOmpA antibody, followed by a secondary antibody Alexa Fluor ® 568 (red). The analytical sectioning was performed from the top to the bottom of the cells. The figure represents a single section of the cells. (D). NCI-H292 cells were infected with A. baumannii ATCC 19606 T at an MOI of 100 for 5 h. Actin filaments have wrap-around-bacteria (white arrow). Red arrow indicates the extracellular bacteria. The figure represents all projection of sections in one picture.
    Figure Legend Snippet: Invasion of A. baumannii in epithelial cells . (A) NCI-H292 cells were infected with A. baumannii ATCC 19606 T at an MOI of 100 up to 7 h. The colony-forming units were enumerated to measure the time-course of invasion. The result represents the mean ± standard deviation in duplicate wells and repeated a minimum of three separate times on separate days. (B) NCI-H292, HEp-2, and HeLa cells were infected with A. baumannii strains at an MOI of 100 for 5 h. (C) NCI-H292 cells were infected with A. baumannii 05KA103 at an MOI of 100 for 5 h. Actin was stained with Alexa Fluor ® 488 phalloidin (green). Bacteria were stained with polyclonal anti-rabbit AbOmpA antibody, followed by a secondary antibody Alexa Fluor ® 568 (red). The analytical sectioning was performed from the top to the bottom of the cells. The figure represents a single section of the cells. (D). NCI-H292 cells were infected with A. baumannii ATCC 19606 T at an MOI of 100 for 5 h. Actin filaments have wrap-around-bacteria (white arrow). Red arrow indicates the extracellular bacteria. The figure represents all projection of sections in one picture.

    Techniques Used: Infection, Standard Deviation, Staining

    20) Product Images from "NTPDase3 and ecto-5′-nucleotidase/CD73 are differentially expressed during mouse bladder cancer progression"

    Article Title: NTPDase3 and ecto-5′-nucleotidase/CD73 are differentially expressed during mouse bladder cancer progression

    Journal: Purinergic Signalling

    doi: 10.1007/s11302-014-9405-8

    a The images correspond to immunofluorescent staining of ecto-5′-NT/CD73 in different times of bladder cancer induction. Cryosections of mouse bladders were labeled with antibody to ecto-5′-NT/CD73 ( red ), Alexa 488 phalloidin to label
    Figure Legend Snippet: a The images correspond to immunofluorescent staining of ecto-5′-NT/CD73 in different times of bladder cancer induction. Cryosections of mouse bladders were labeled with antibody to ecto-5′-NT/CD73 ( red ), Alexa 488 phalloidin to label

    Techniques Used: Staining, Labeling

    a The images correspond to immunofluorescence of NTPDase3 in bladder urothelium at different times of bladder cancer induction. Cryosections of mouse bladders were labeled with antibody to NTPDase3 ( red ), Alexa 488 phalloidin to label actin cytoskeleton
    Figure Legend Snippet: a The images correspond to immunofluorescence of NTPDase3 in bladder urothelium at different times of bladder cancer induction. Cryosections of mouse bladders were labeled with antibody to NTPDase3 ( red ), Alexa 488 phalloidin to label actin cytoskeleton

    Techniques Used: Immunofluorescence, Labeling

    21) Product Images from "Persistent fibroblast growth factor 23 signalling in the parathyroid glands for secondary hyperparathyroidism in mice with chronic kidney disease"

    Article Title: Persistent fibroblast growth factor 23 signalling in the parathyroid glands for secondary hyperparathyroidism in mice with chronic kidney disease

    Journal: Scientific Reports

    doi: 10.1038/srep40534

    Expression of FGFRs (FGFR1, FGFR2, FGFR3, and FGFR4) and αKlotho in normal and genetically engineered parathyroid glands. A specific gene or genes were conditionally manipulated in the parathyroid glands by mating Fgfr1–3 flox/flox , αKlotho flox/flox , or Fgfr1–4 flox/flox mice with PTH-Cre mice (cKO) or without mating (non-cKO). The mice were treated with heminephrectomy plus a high-phosphate diet (CKD) or not treated (non-CKD) using the protocol described in the Methods section. Paraffin-embedded thyro-parathyroid glands were sectioned and immunostained (red) using an indirect immunofluorescence technique as described in the Methods section. Nuclei were stained with DAPI (blue). Scale bars: 10 μm.
    Figure Legend Snippet: Expression of FGFRs (FGFR1, FGFR2, FGFR3, and FGFR4) and αKlotho in normal and genetically engineered parathyroid glands. A specific gene or genes were conditionally manipulated in the parathyroid glands by mating Fgfr1–3 flox/flox , αKlotho flox/flox , or Fgfr1–4 flox/flox mice with PTH-Cre mice (cKO) or without mating (non-cKO). The mice were treated with heminephrectomy plus a high-phosphate diet (CKD) or not treated (non-CKD) using the protocol described in the Methods section. Paraffin-embedded thyro-parathyroid glands were sectioned and immunostained (red) using an indirect immunofluorescence technique as described in the Methods section. Nuclei were stained with DAPI (blue). Scale bars: 10 μm.

    Techniques Used: Expressing, Mouse Assay, Immunofluorescence, Staining

    22) Product Images from "Control of mitochondrial homeostasis by endocytic regulatory proteins"

    Article Title: Control of mitochondrial homeostasis by endocytic regulatory proteins

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.204537

    Depletion of EHD1, rabankyrin-5 or VPS35 does not induce Mfn2 accumulation. HeLa cells were either mock treated, or treated with EHD1, rabankyrin-5 or Vps35 siRNA for 72 h. Depletion efficacy was validated by immunoblotting with antibodies against EHD1, rabankyrin-5 and VPS35 (A; top three panels), and the effect of the siRNA was assessed with antibodies against Mfn2 (A; second panel from the top), Drp1 (A; third panel from the top) and actin (A; bottom panel). (B–G) Densitometric quantification from three separate experiments. * P
    Figure Legend Snippet: Depletion of EHD1, rabankyrin-5 or VPS35 does not induce Mfn2 accumulation. HeLa cells were either mock treated, or treated with EHD1, rabankyrin-5 or Vps35 siRNA for 72 h. Depletion efficacy was validated by immunoblotting with antibodies against EHD1, rabankyrin-5 and VPS35 (A; top three panels), and the effect of the siRNA was assessed with antibodies against Mfn2 (A; second panel from the top), Drp1 (A; third panel from the top) and actin (A; bottom panel). (B–G) Densitometric quantification from three separate experiments. * P

    Techniques Used:

    EHD1 interacts with Mul1. (A) Model for the potential role of EHD1 in regulating mitochondrial dynamics via Mul1. Under normal conditions, the ubiquitin ligase Mul1 is released from an interaction with VPS35 and the retromer components (including EHD1), and relocates to the mitochondrial membrane, where it ubiquitylates Mfn2, inducing its proteasomal degradation and promoting normal mitochondrial fission. Upon EHD1 depletion, Mul1 would be retained in association with VPS35 and the retromer, preventing Mfn2 degradation and thus enhancing mitochondrial membrane fusion. (B) GST pulldown from bovine brain cytosol was performed with GST only, a GST-tagged EH domain of EHD1 (GST–EH1) and GST–EHD1. Eluates were immunoblotted with antibodies against MICAL-L1 (top panel), as a positive interactor with EHD1, and Mul1 (middle panel). GST fusion protein samples were immunoblotted with anti-GST (bottom panel). (C) Co-immunoprecipitation (IP) of proteins from a HeLa cell lysate using anti-Mul1 (αMul1), and immunoblotted with anti-Vps26 and anti-rabankyrin-5 antibodies. 25 kDa immunoglobulin light chains detected by the secondary anti-light chain antibody are indicated in the bottom panel.
    Figure Legend Snippet: EHD1 interacts with Mul1. (A) Model for the potential role of EHD1 in regulating mitochondrial dynamics via Mul1. Under normal conditions, the ubiquitin ligase Mul1 is released from an interaction with VPS35 and the retromer components (including EHD1), and relocates to the mitochondrial membrane, where it ubiquitylates Mfn2, inducing its proteasomal degradation and promoting normal mitochondrial fission. Upon EHD1 depletion, Mul1 would be retained in association with VPS35 and the retromer, preventing Mfn2 degradation and thus enhancing mitochondrial membrane fusion. (B) GST pulldown from bovine brain cytosol was performed with GST only, a GST-tagged EH domain of EHD1 (GST–EH1) and GST–EHD1. Eluates were immunoblotted with antibodies against MICAL-L1 (top panel), as a positive interactor with EHD1, and Mul1 (middle panel). GST fusion protein samples were immunoblotted with anti-GST (bottom panel). (C) Co-immunoprecipitation (IP) of proteins from a HeLa cell lysate using anti-Mul1 (αMul1), and immunoblotted with anti-Vps26 and anti-rabankyrin-5 antibodies. 25 kDa immunoglobulin light chains detected by the secondary anti-light chain antibody are indicated in the bottom panel.

    Techniques Used: Immunoprecipitation

    Depletion of EHD1 and rabankyrin-5 results in reduced and sequestered VPS35, respectively. (A–D) HeLa cells were either mock treated, treated with EHD1 siRNA (A) or treated with rabankyrin-5 (Rank-5) siRNA (C) for 72 h and immunoblotted for VPS35, EHD1, Rank-5 and actin. The asterisk (in A) indicates reduced VPS35 protein levels. (B,D) Quantification of protein levels from three independent experiments. * P
    Figure Legend Snippet: Depletion of EHD1 and rabankyrin-5 results in reduced and sequestered VPS35, respectively. (A–D) HeLa cells were either mock treated, treated with EHD1 siRNA (A) or treated with rabankyrin-5 (Rank-5) siRNA (C) for 72 h and immunoblotted for VPS35, EHD1, Rank-5 and actin. The asterisk (in A) indicates reduced VPS35 protein levels. (B,D) Quantification of protein levels from three independent experiments. * P

    Techniques Used:

    Rabankyrin-5 mediates the interaction between EHD1 and Mul1, and its depletion induces an elongated mitochondrial network similar to that observed upon EHD1 depletion. (A,B) RPE cells were either mock treated (A) or treated with rabankyrin-5 siRNA for 72 h (B) and immunostained for the mitochondrial membrane marker Tom20. (C) The Mito Morphology Macro plugin in ImageJ was used for quantifying mean±s.d. for mitochondrial size, perimeter and circularity in three independent experiments each using 10 cells per treatment. * P
    Figure Legend Snippet: Rabankyrin-5 mediates the interaction between EHD1 and Mul1, and its depletion induces an elongated mitochondrial network similar to that observed upon EHD1 depletion. (A,B) RPE cells were either mock treated (A) or treated with rabankyrin-5 siRNA for 72 h (B) and immunostained for the mitochondrial membrane marker Tom20. (C) The Mito Morphology Macro plugin in ImageJ was used for quantifying mean±s.d. for mitochondrial size, perimeter and circularity in three independent experiments each using 10 cells per treatment. * P

    Techniques Used: Marker

    23) Product Images from "Profiling human breast epithelial cells using single cell RNA sequencing identifies cell diversity"

    Article Title: Profiling human breast epithelial cells using single cell RNA sequencing identifies cell diversity

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04334-1

    Validation and spatial integration of two distinct luminal cell types. a Immunofluorescence analysis of NY-BR-1 protein expression (green) in combination with basal marker SLPI (red) and DNA stain using DAPI (blue) within tissue sections from primary human reduction mammoplasty samples revealed that NY-BR-1 and SLPI are markers for distinct luminal subpopulations. b – e Immunofluorescence analysis of NY-BR-1 and SLPI (red) protein expression with: hormone receptors for estrogen receptor ( b ), progesterone ( c ), and androgen ( d ) and proliferation marker Ki67 e in green. f Summary of hormone receptor and proliferation marker expression in L1 and L2 cells. g Violin plot showing expression of KRT8 in the luminal subpopulations, higher expression is seen in the luminal L1.1 and L1.2 subpopulation. h Sample frame for detection of KRT8 protein content from individual cells using single cell Western blot following detection using microarray scanner. i for violin plots displaying expression of relevant hormone receptors as well as proliferation and luminal progenitor markers. All scale bars = 25 µm
    Figure Legend Snippet: Validation and spatial integration of two distinct luminal cell types. a Immunofluorescence analysis of NY-BR-1 protein expression (green) in combination with basal marker SLPI (red) and DNA stain using DAPI (blue) within tissue sections from primary human reduction mammoplasty samples revealed that NY-BR-1 and SLPI are markers for distinct luminal subpopulations. b – e Immunofluorescence analysis of NY-BR-1 and SLPI (red) protein expression with: hormone receptors for estrogen receptor ( b ), progesterone ( c ), and androgen ( d ) and proliferation marker Ki67 e in green. f Summary of hormone receptor and proliferation marker expression in L1 and L2 cells. g Violin plot showing expression of KRT8 in the luminal subpopulations, higher expression is seen in the luminal L1.1 and L1.2 subpopulation. h Sample frame for detection of KRT8 protein content from individual cells using single cell Western blot following detection using microarray scanner. i for violin plots displaying expression of relevant hormone receptors as well as proliferation and luminal progenitor markers. All scale bars = 25 µm

    Techniques Used: Immunofluorescence, Expressing, Marker, Staining, Western Blot, Microarray

    24) Product Images from "LRRK2 interactions with ?-synuclein in Parkinson's disease brains and in cell models"

    Article Title: LRRK2 interactions with ?-synuclein in Parkinson's disease brains and in cell models

    Journal: Journal of Molecular Medicine (Berlin, Germany)

    doi: 10.1007/s00109-012-0984-y

    Co-localization of LRRK2 and α-synuclein in PD brain and cell models. In PD brains ( a – d , f – g ), merged images clearly outline single neurons in the substantia nigra ( a , b ) and Lewy bodies ( b – d , f – g ) using double-labelling immunofluorescence. There is an increase of LRRK2 and α-synuclein immunoreactivity in brainstem neurons without Lewy body formation ( a ), with LRRK2 co-localizing with α-synuclein in Lewy bodies ( donut inclusion in b ) in these neurons. The co-localisation of LRRK2 and α-synuclein was also observed in cortical Lewy bodies ( c ). Cortical Lewy bodies without LRRK2 immunoreactivity were also observed ( d ). S129 phosphorylated α-synuclein antibody also confirmed co-localisation of LRRK2 with phosphorylated α-synuclein, with LRRK2 often centralized to a radiating pattern of phosphorylated α-synuclein fibrils ( f – h ). In the H4 cell model, double-labelling immunofluorescence for α-synuclein inclusion formation shows that endogenous LRRK2 co-localizes with α-synuclein inclusions ( e ). Scales in all panels are equivalent to 10 μm
    Figure Legend Snippet: Co-localization of LRRK2 and α-synuclein in PD brain and cell models. In PD brains ( a – d , f – g ), merged images clearly outline single neurons in the substantia nigra ( a , b ) and Lewy bodies ( b – d , f – g ) using double-labelling immunofluorescence. There is an increase of LRRK2 and α-synuclein immunoreactivity in brainstem neurons without Lewy body formation ( a ), with LRRK2 co-localizing with α-synuclein in Lewy bodies ( donut inclusion in b ) in these neurons. The co-localisation of LRRK2 and α-synuclein was also observed in cortical Lewy bodies ( c ). Cortical Lewy bodies without LRRK2 immunoreactivity were also observed ( d ). S129 phosphorylated α-synuclein antibody also confirmed co-localisation of LRRK2 with phosphorylated α-synuclein, with LRRK2 often centralized to a radiating pattern of phosphorylated α-synuclein fibrils ( f – h ). In the H4 cell model, double-labelling immunofluorescence for α-synuclein inclusion formation shows that endogenous LRRK2 co-localizes with α-synuclein inclusions ( e ). Scales in all panels are equivalent to 10 μm

    Techniques Used: Immunofluorescence

    Increased levels of total and S129 phosphorylated α-synuclein in PD brain. a – c Peroxidase immunohistochemistry of brain sections from the same PD case showing the regional density of Lewy pathology as revealed by immunohistochemistry using phosphorylated α-synuclein antibody and counterstained with cresyl violet. Scale in c = 100 μm and is equivalent for a and b . Severe pathology is observed in the amygdala ( a ) with moderate pathology in the anterior cingulate cortex ( b ). Neuronal inclusions are not observed in the visual cortex ( c ). d , e Quantitation ( d ) of Western blots ( e ) in the same three brain regions in the PD cases (represented as an increase over control levels) confirmed the regional changes noted histologically in PD and showed considerably more phosphorylated α-synuclein compared with total α-synuclein in each regions (note the percentage at left versus fold change at right in d ). Error bars = SEM
    Figure Legend Snippet: Increased levels of total and S129 phosphorylated α-synuclein in PD brain. a – c Peroxidase immunohistochemistry of brain sections from the same PD case showing the regional density of Lewy pathology as revealed by immunohistochemistry using phosphorylated α-synuclein antibody and counterstained with cresyl violet. Scale in c = 100 μm and is equivalent for a and b . Severe pathology is observed in the amygdala ( a ) with moderate pathology in the anterior cingulate cortex ( b ). Neuronal inclusions are not observed in the visual cortex ( c ). d , e Quantitation ( d ) of Western blots ( e ) in the same three brain regions in the PD cases (represented as an increase over control levels) confirmed the regional changes noted histologically in PD and showed considerably more phosphorylated α-synuclein compared with total α-synuclein in each regions (note the percentage at left versus fold change at right in d ). Error bars = SEM

    Techniques Used: Immunohistochemistry, Quantitation Assay, Western Blot

    Knockdown of LRRK2 expression alters the size and number of α-synuclein inclusions. a Western blots showing that H4 cells infected with LRRK2-shRNA have the expected knockdown of LRRK2 protein (LRRK2-KD) compared with the scramble shRNA control, but have no significant change on the level of endogenous α-synuclein or phosphorylated α-synuclein at S129. b The model for α-synuclein inclusions was reproduced in a LRRK2 knockdown cell line and in parental control cells. Cells were classified into two groups according to the number of α-synuclein-immunoreactive inclusions observed: cells with five or more inclusions and cells with less than five inclusions. Scale bar = 10 μm. c Data from three independent experiments shows a greater proportion of cells containing five or more inclusions in the LRRK2 knockdown cells compared with controls. d LRRK2 silencing (LRRK2-KD) promotes a significant reduction in the average size of the inclusions, resulting in a more punctate aggregation pattern in the cells. Student’s test ( n = 3; ** p
    Figure Legend Snippet: Knockdown of LRRK2 expression alters the size and number of α-synuclein inclusions. a Western blots showing that H4 cells infected with LRRK2-shRNA have the expected knockdown of LRRK2 protein (LRRK2-KD) compared with the scramble shRNA control, but have no significant change on the level of endogenous α-synuclein or phosphorylated α-synuclein at S129. b The model for α-synuclein inclusions was reproduced in a LRRK2 knockdown cell line and in parental control cells. Cells were classified into two groups according to the number of α-synuclein-immunoreactive inclusions observed: cells with five or more inclusions and cells with less than five inclusions. Scale bar = 10 μm. c Data from three independent experiments shows a greater proportion of cells containing five or more inclusions in the LRRK2 knockdown cells compared with controls. d LRRK2 silencing (LRRK2-KD) promotes a significant reduction in the average size of the inclusions, resulting in a more punctate aggregation pattern in the cells. Student’s test ( n = 3; ** p

    Techniques Used: Expressing, Western Blot, Infection, shRNA

    Co-immunoprecipitation of LRRK2 and α-synuclein. a Western blots showing the immunoprecipitation of endogenous α-synuclein in lysates from WT and LRRK2 knockout mouse brains. The co-immunoprecipitation with endogenous LRRK2 occurs in WT but not in the LRRK2 knockout brain sample. b , c Over-expression of Myc-LRRK2 (WT or G2019S) together with α-synuclein in HEK-293 cells showed the co-immunoprecipitation of LRRK2 (WT or G2019S) with α-synuclein using anti-Myc as the capture antibody and anti-α-synuclein and anti-LRRK2 antibodies for Western blotting ( b ) or using anti-α-synuclein as the capture antibody and anti-α-synuclein and anti-Myc antibodies for Western blotting ( c )
    Figure Legend Snippet: Co-immunoprecipitation of LRRK2 and α-synuclein. a Western blots showing the immunoprecipitation of endogenous α-synuclein in lysates from WT and LRRK2 knockout mouse brains. The co-immunoprecipitation with endogenous LRRK2 occurs in WT but not in the LRRK2 knockout brain sample. b , c Over-expression of Myc-LRRK2 (WT or G2019S) together with α-synuclein in HEK-293 cells showed the co-immunoprecipitation of LRRK2 (WT or G2019S) with α-synuclein using anti-Myc as the capture antibody and anti-α-synuclein and anti-LRRK2 antibodies for Western blotting ( b ) or using anti-α-synuclein as the capture antibody and anti-α-synuclein and anti-Myc antibodies for Western blotting ( c )

    Techniques Used: Immunoprecipitation, Western Blot, Knock-Out, Over Expression

    LRRK2 levels correlated with α-synuclein levels in PD brain. Quantitation ( a ) of LRRK2 Western blots ( b ) in the same brain regions in the PD cases (represented as an increase over control levels) and correlations with α-synuclein levels ( c ). The protein levels of LRRK2 were increased in the disease-affected areas (amygdala and cingulate) compared to the non-affected area (visual cortex) ( a ). Error bars = SEM. Multivariate analysis revealed a significant correlation between the increasing levels of α-synuclein and LRRK2 only in PD but not controls ( c )
    Figure Legend Snippet: LRRK2 levels correlated with α-synuclein levels in PD brain. Quantitation ( a ) of LRRK2 Western blots ( b ) in the same brain regions in the PD cases (represented as an increase over control levels) and correlations with α-synuclein levels ( c ). The protein levels of LRRK2 were increased in the disease-affected areas (amygdala and cingulate) compared to the non-affected area (visual cortex) ( a ). Error bars = SEM. Multivariate analysis revealed a significant correlation between the increasing levels of α-synuclein and LRRK2 only in PD but not controls ( c )

    Techniques Used: Quantitation Assay, Western Blot

    25) Product Images from "Modulation of Glucose Takeup by Glucose Transport on the Isolated OHCs"

    Article Title: Modulation of Glucose Takeup by Glucose Transport on the Isolated OHCs

    Journal: Neural Plasticity

    doi: 10.1155/2018/7513217

    Basal staining for GLUT-4 may result from intracellular penetration of the antibody. OHCs were costained using anti-GLUT-4 antibodies and di-8-ANEPPS: (a) GLUT-4 staining; (b) di-8-ANEPPS staining; (c) merged image; (d) high magnification; (e) Nomarski image. Scale bar, 10 μ m in all images except the inset (2 μ m).
    Figure Legend Snippet: Basal staining for GLUT-4 may result from intracellular penetration of the antibody. OHCs were costained using anti-GLUT-4 antibodies and di-8-ANEPPS: (a) GLUT-4 staining; (b) di-8-ANEPPS staining; (c) merged image; (d) high magnification; (e) Nomarski image. Scale bar, 10 μ m in all images except the inset (2 μ m).

    Techniques Used: Staining

    26) Product Images from "Intra-articular injection of synthetic microRNA-210 accelerates avascular meniscal healing in rat medial meniscal injured model"

    Article Title: Intra-articular injection of synthetic microRNA-210 accelerates avascular meniscal healing in rat medial meniscal injured model

    Journal: Arthritis Research & Therapy

    doi: 10.1186/s13075-014-0488-y

    Immunohistochemistry of meniscus at 4 weeks after intra-articular injection. (A) Immunohistochemistry of vascular endothelial growth factor (VEGF; upper) and fibroblast growth factor-2 (FGF2; lower). VEGF and FGF2 expressed intensely on the surface of the meniscus, around the injured site and in the red zone in the miR-210 group compared with the control group. Arrow, injured site. Bar = 100 μm. (B) Immunohistochemistry of type 2 collagen. Type 2 collagen expression was observed around the injured site of the meniscus in the miR-210 group compared with the control group. Arrow, injured site. Bar = 100 μm. (C) Isolectin B4 staining and the number of blood vessels. Newly formed vessels were observed around the injured site in the miR-210 group, while little blood vessels were observed in the control. White arrow, injured site; yellow arrow, blood vessels. Bar = 100 μm. Number of blood vessels in the miR-210 group was significantly higher than that in the control group (* P
    Figure Legend Snippet: Immunohistochemistry of meniscus at 4 weeks after intra-articular injection. (A) Immunohistochemistry of vascular endothelial growth factor (VEGF; upper) and fibroblast growth factor-2 (FGF2; lower). VEGF and FGF2 expressed intensely on the surface of the meniscus, around the injured site and in the red zone in the miR-210 group compared with the control group. Arrow, injured site. Bar = 100 μm. (B) Immunohistochemistry of type 2 collagen. Type 2 collagen expression was observed around the injured site of the meniscus in the miR-210 group compared with the control group. Arrow, injured site. Bar = 100 μm. (C) Isolectin B4 staining and the number of blood vessels. Newly formed vessels were observed around the injured site in the miR-210 group, while little blood vessels were observed in the control. White arrow, injured site; yellow arrow, blood vessels. Bar = 100 μm. Number of blood vessels in the miR-210 group was significantly higher than that in the control group (* P

    Techniques Used: Immunohistochemistry, Injection, Expressing, Staining

    Gene expression analysis by real-time PCR in the meniscus at 4 weeks following intraarticular injection. Expression of mature miR-210, collagen type 1 alpha 1 (Col1a1), collagen type 2 alpha 1 (Col2a1), vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF2) was examined using real-time PCR. Expression of miR-210, Col2a1, VEGF and FGF2 in the miR-210 group was significantly higher than that in the control and normal groups (* P
    Figure Legend Snippet: Gene expression analysis by real-time PCR in the meniscus at 4 weeks following intraarticular injection. Expression of mature miR-210, collagen type 1 alpha 1 (Col1a1), collagen type 2 alpha 1 (Col2a1), vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF2) was examined using real-time PCR. Expression of miR-210, Col2a1, VEGF and FGF2 in the miR-210 group was significantly higher than that in the control and normal groups (* P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Injection

    Gene expression analyses in inner meniscus cells after overexpression of miR-210. (A) Real-time PCR analysis of collagen type 1 alpha 1 (Col1a1), collagen type 2 alpha 1 (Col2a1), vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF2) at 7 days after in vitro transfection of inner meniscus cells. Expression of only Col2a1 was significantly higher than that in the control group (* P
    Figure Legend Snippet: Gene expression analyses in inner meniscus cells after overexpression of miR-210. (A) Real-time PCR analysis of collagen type 1 alpha 1 (Col1a1), collagen type 2 alpha 1 (Col2a1), vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF2) at 7 days after in vitro transfection of inner meniscus cells. Expression of only Col2a1 was significantly higher than that in the control group (* P

    Techniques Used: Expressing, Over Expression, Real-time Polymerase Chain Reaction, In Vitro, Transfection

    Gene expression analyses in synovial cells after overexpression of miR-210. (A) Real-time PCR analysis of collagen type 1 alpha 1 (Col1a1), collagen type 2 alpha 1 (Col2a1), vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF2) at 7 days after in vitro transfection of synovial cells. Expression of Col2a1 was not detected in both groups. Expression of VEGF and FGF2 was significantly higher than in the control group (* P
    Figure Legend Snippet: Gene expression analyses in synovial cells after overexpression of miR-210. (A) Real-time PCR analysis of collagen type 1 alpha 1 (Col1a1), collagen type 2 alpha 1 (Col2a1), vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF2) at 7 days after in vitro transfection of synovial cells. Expression of Col2a1 was not detected in both groups. Expression of VEGF and FGF2 was significantly higher than in the control group (* P

    Techniques Used: Expressing, Over Expression, Real-time Polymerase Chain Reaction, In Vitro, Transfection

    27) Product Images from "Ubiquilin1 Represses Migration and Epithelial to Mesenchymal Transition of Human Non-small Cell Lung Cancer Cells"

    Article Title: Ubiquilin1 Represses Migration and Epithelial to Mesenchymal Transition of Human Non-small Cell Lung Cancer Cells

    Journal: Oncogene

    doi: 10.1038/onc.2014.97

    Coordinate regulation of EMT by UBQLN1 and ZEB1 (a) Loss of Zeb1 increases expression of UBQLN1 and increases epithelial markers in A549 and H358 cells. Western blot analysis of ZEB1, UBQLN1 and EMT markers in A549 and H358 cells. Cells were transfected with either non-targeting siRNA (siNT) or with siRNA targeting ZEB1 (siZEB1). After 72 hrs of transfection, cells were harvested and subjected to western blot for protein expression analysis for UBQLN1, ZEB1 along with other EMT markers (b) UBQLN1 loss requires ZEB1 to induce EMT in A549 and H358 cells. Cells were transfected with non-targeting siRNA, with siUBQLN1, siZEB1 or the combination of siUBQLN1 and siZEB1. After 72 hrs of transfection, cells were harvested. Western blot analysis confirming knockdown of UBQLN1 and ZEB1 along with different EMT markers. (c) Fluorescence staining for E-cadherin in A549. After 24 hrs of transfection either with non-targeting siRNA (siNT) or with siRNAs targeting UBQLN1 (siU1), siZeb1 or combination of siU1 and siZeb1, cells were trypsinized and plated on chamber slides and stained for E-cadherin. i, iii, v and vii: E-cadherin was detected using Alexa Fluor 488 goat anti-rabbit IgG (green). ii, iv, vi and viii: overlay of respective E-cadherin and F-actin (Alexa Fluor 568 Phalloidin; red) staining with DAPI counter stain. (d) A549 cells were prepared as described in (c) and F-actin was detected with Alexa Fluor 568 Phalloidin (red) with 60x objective. Re-organization of actin cytoskeleton through destruction and cellular protrusion formation is indicated by arrows.
    Figure Legend Snippet: Coordinate regulation of EMT by UBQLN1 and ZEB1 (a) Loss of Zeb1 increases expression of UBQLN1 and increases epithelial markers in A549 and H358 cells. Western blot analysis of ZEB1, UBQLN1 and EMT markers in A549 and H358 cells. Cells were transfected with either non-targeting siRNA (siNT) or with siRNA targeting ZEB1 (siZEB1). After 72 hrs of transfection, cells were harvested and subjected to western blot for protein expression analysis for UBQLN1, ZEB1 along with other EMT markers (b) UBQLN1 loss requires ZEB1 to induce EMT in A549 and H358 cells. Cells were transfected with non-targeting siRNA, with siUBQLN1, siZEB1 or the combination of siUBQLN1 and siZEB1. After 72 hrs of transfection, cells were harvested. Western blot analysis confirming knockdown of UBQLN1 and ZEB1 along with different EMT markers. (c) Fluorescence staining for E-cadherin in A549. After 24 hrs of transfection either with non-targeting siRNA (siNT) or with siRNAs targeting UBQLN1 (siU1), siZeb1 or combination of siU1 and siZeb1, cells were trypsinized and plated on chamber slides and stained for E-cadherin. i, iii, v and vii: E-cadherin was detected using Alexa Fluor 488 goat anti-rabbit IgG (green). ii, iv, vi and viii: overlay of respective E-cadherin and F-actin (Alexa Fluor 568 Phalloidin; red) staining with DAPI counter stain. (d) A549 cells were prepared as described in (c) and F-actin was detected with Alexa Fluor 568 Phalloidin (red) with 60x objective. Re-organization of actin cytoskeleton through destruction and cellular protrusion formation is indicated by arrows.

    Techniques Used: Expressing, Western Blot, Transfection, Fluorescence, Staining

    Loss of UBQLN1 induces EMT (a) UBQLN1 loss induces EMT in A549 and H358 cells. A549 and H358 cells were transfected with either with non-targeting siRNA (siNT) or siRNAs targeting UBQLN1 (siU1, siU1-2). After 72 hrs of transfection cells were harvested and analyzed for protein expression using the indicated antibodies. (b) Fluorescence staining for E-cadherin and Vimentin in A549. After 24 hrs of transfection either with non-targeting siRNA (siNT) or with siRNAs targeting UBQLN1 (siU1 and siU1-2) cells were trypsinized and plated on chamber slides and stained for EMT markers. i, iii and v: E-cadherin was detected using Alexa Fluor 488 goat anti-rabbit IgG (green). ii, iv and vi: overlay of respective E-cadherin and F-actin (Alexa Fluor 568 Phalloidin; red) staining with DAPI counter stain. a, c and e: Vimentin was detected using Alexa Fluor 488 goat anti-rabbit IgG (green). b, d and f: overlay of respective E-Vimentin and F-actin (Alexa Fluor 568 Phalloidin; red) staining with DAPI counter stain. (c) Cells were prepared as described in B and F-actin was detected with Alexa Fluor 568 Phalloidin (red). Re-organization of actin cytoskeleton through destruction and cellular protrusion formation is indicated by arrows. (d) Table indicating the fold change of mRNA following siRNA mediated knockdown of UBQLN1 for EMT-associated genes, as compared to non-targeting siRNA transfected cells. Values are in fold change and each value is the average of the triplicate samples for each siRNA.
    Figure Legend Snippet: Loss of UBQLN1 induces EMT (a) UBQLN1 loss induces EMT in A549 and H358 cells. A549 and H358 cells were transfected with either with non-targeting siRNA (siNT) or siRNAs targeting UBQLN1 (siU1, siU1-2). After 72 hrs of transfection cells were harvested and analyzed for protein expression using the indicated antibodies. (b) Fluorescence staining for E-cadherin and Vimentin in A549. After 24 hrs of transfection either with non-targeting siRNA (siNT) or with siRNAs targeting UBQLN1 (siU1 and siU1-2) cells were trypsinized and plated on chamber slides and stained for EMT markers. i, iii and v: E-cadherin was detected using Alexa Fluor 488 goat anti-rabbit IgG (green). ii, iv and vi: overlay of respective E-cadherin and F-actin (Alexa Fluor 568 Phalloidin; red) staining with DAPI counter stain. a, c and e: Vimentin was detected using Alexa Fluor 488 goat anti-rabbit IgG (green). b, d and f: overlay of respective E-Vimentin and F-actin (Alexa Fluor 568 Phalloidin; red) staining with DAPI counter stain. (c) Cells were prepared as described in B and F-actin was detected with Alexa Fluor 568 Phalloidin (red). Re-organization of actin cytoskeleton through destruction and cellular protrusion formation is indicated by arrows. (d) Table indicating the fold change of mRNA following siRNA mediated knockdown of UBQLN1 for EMT-associated genes, as compared to non-targeting siRNA transfected cells. Values are in fold change and each value is the average of the triplicate samples for each siRNA.

    Techniques Used: Transfection, Expressing, Fluorescence, Staining

    28) Product Images from "Enhanced viral-mediated cochlear gene delivery in adult mice by combining canal fenestration with round window membrane inoculation"

    Article Title: Enhanced viral-mediated cochlear gene delivery in adult mice by combining canal fenestration with round window membrane inoculation

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-21233-z

    Vestibular organs are transduced following the RWM + CF injection. ( a – c ) Representative images of whole mounts of the CA of the PSCC, saccule, utricle and CAs of the LSCC and ASCC as indicated. Tissue was harvested 2 weeks after the RWM + CF injection (at P15–16), stained with Alexa Fluor 568-phalloidin (red) for labelling filamentous actin, and imaged for native eGFP (green). High magnification views of the regions marked with white dotted squares in the PSCC are shown separately and stained with Myo7a (gray) for labelling hair cells and imaged for native eGFP (green).
    Figure Legend Snippet: Vestibular organs are transduced following the RWM + CF injection. ( a – c ) Representative images of whole mounts of the CA of the PSCC, saccule, utricle and CAs of the LSCC and ASCC as indicated. Tissue was harvested 2 weeks after the RWM + CF injection (at P15–16), stained with Alexa Fluor 568-phalloidin (red) for labelling filamentous actin, and imaged for native eGFP (green). High magnification views of the regions marked with white dotted squares in the PSCC are shown separately and stained with Myo7a (gray) for labelling hair cells and imaged for native eGFP (green).

    Techniques Used: Injection, Staining

    RWM + CF approach comparing canalostomy of the LSCC and PSCC. ( a ) Inner ear schematic showing the RWM + CF approach with a LSCC canalostomy. ( b ) Representative low magnification images of whole-mount apical turns and high magnification images of the apex, middle and base 2 weeks after injection of AAV2/9 (3.90 × 10 13 vg/ml) delivered at P15–16 using a RWM + CF approach with a canalostomy in LSCC. Cochleae were stained with Myo7a (red) for labelling hair cells and imaged for native eGFP (green). ( c ) Quantification of eGFP-positive IHCs in the apex, middle and base, and cochlear apex 2 weeks following a RWM + CF approach with a canalostomy in LSCC (n = 3). Data are means. ( d ) Representative images of whole mounts of the CA of the PSCC, saccule, utricle and the CA of the LSCC and ASCC are shown. Tissue was harvested 2 weeks after injection of AAV2/9 (3.90 × 10 13 vg/ml) delivered at P15–16 using a RWM + CF approach with a canalostomy in LSCC, and stained with Alexa Flour 568-phalloidin (red) for labelling filamentous actin and imaged for native eGFP (green). High magnification views of the regions marked with white dotted squares in the PSCC are shown separately, and stained with Myo7a (gray) for labelling hair cells and imaged for native eGFP (green).
    Figure Legend Snippet: RWM + CF approach comparing canalostomy of the LSCC and PSCC. ( a ) Inner ear schematic showing the RWM + CF approach with a LSCC canalostomy. ( b ) Representative low magnification images of whole-mount apical turns and high magnification images of the apex, middle and base 2 weeks after injection of AAV2/9 (3.90 × 10 13 vg/ml) delivered at P15–16 using a RWM + CF approach with a canalostomy in LSCC. Cochleae were stained with Myo7a (red) for labelling hair cells and imaged for native eGFP (green). ( c ) Quantification of eGFP-positive IHCs in the apex, middle and base, and cochlear apex 2 weeks following a RWM + CF approach with a canalostomy in LSCC (n = 3). Data are means. ( d ) Representative images of whole mounts of the CA of the PSCC, saccule, utricle and the CA of the LSCC and ASCC are shown. Tissue was harvested 2 weeks after injection of AAV2/9 (3.90 × 10 13 vg/ml) delivered at P15–16 using a RWM + CF approach with a canalostomy in LSCC, and stained with Alexa Flour 568-phalloidin (red) for labelling filamentous actin and imaged for native eGFP (green). High magnification views of the regions marked with white dotted squares in the PSCC are shown separately, and stained with Myo7a (gray) for labelling hair cells and imaged for native eGFP (green).

    Techniques Used: Injection, Staining

    29) Product Images from "Kidney Proximal Tubule Lipoapoptosis Is Regulated by Fatty Acid Transporter-2 (FATP2)"

    Article Title: Kidney Proximal Tubule Lipoapoptosis Is Regulated by Fatty Acid Transporter-2 (FATP2)

    Journal: Journal of the American Society of Nephrology : JASN

    doi: 10.1681/ASN.2017030314

    FATP2 is expressed in proximal tubule apical membranes. Formalin-fixed paraffin sections from human kidneys were probed with (A) anti-FATP2 and Alexa Fluor 488 secondary antibodies or (B) anti– γ -glutamyl transferase-1 (GGT1) and Alexa Fluor 568–labeled secondary antibodies. (C) Nuclei were labeled with DAPI in the mounting medium. (D) Colocalization from merged images is depicted in yellow.
    Figure Legend Snippet: FATP2 is expressed in proximal tubule apical membranes. Formalin-fixed paraffin sections from human kidneys were probed with (A) anti-FATP2 and Alexa Fluor 488 secondary antibodies or (B) anti– γ -glutamyl transferase-1 (GGT1) and Alexa Fluor 568–labeled secondary antibodies. (C) Nuclei were labeled with DAPI in the mounting medium. (D) Colocalization from merged images is depicted in yellow.

    Techniques Used: Labeling

    30) Product Images from "Microglial phagocytosis of living photoreceptors contributes to inherited retinal degeneration"

    Article Title: Microglial phagocytosis of living photoreceptors contributes to inherited retinal degeneration

    Journal: EMBO Molecular Medicine

    doi: 10.15252/emmm.201505298

    Activation status of microglia infiltrating the outer nuclear layer (ONL) and the exposure of phosphatidylserine (PS) on ONL photoreceptors Microglia infiltrating the outer nuclear layer (ONL) of the rd10 retina during rod photoreceptor degeneration demonstrate markers of activation. At P18, Iba1 + microglia (green, arrow) in the outer plexiform layer were negative for TSPO (red), an activation marker. At P22, Iba1 + microglia (arrow) infiltrated the ONL and acquired TSPO immunopositivity, indicating their activated status. Scale bars, 25 μm. Phosphatidylserine (PS) exposure in ONL nuclei of unfixed cryosections of rd10 retina during rod degeneration. PS exposure in the ONL was monitored in unfixed frozen sections using fluorescently conjugated annexin V which binds cell-surface PS. While minimal annexin V staining was evident in the ONL in P18 wild-type (top row) and P18 rd10 (middle row) retinas in which rod degeneration is absent, staining was prominent in P21 rd10 retina during rod degeneration (bottom row). Scale bar, 40 μm.
    Figure Legend Snippet: Activation status of microglia infiltrating the outer nuclear layer (ONL) and the exposure of phosphatidylserine (PS) on ONL photoreceptors Microglia infiltrating the outer nuclear layer (ONL) of the rd10 retina during rod photoreceptor degeneration demonstrate markers of activation. At P18, Iba1 + microglia (green, arrow) in the outer plexiform layer were negative for TSPO (red), an activation marker. At P22, Iba1 + microglia (arrow) infiltrated the ONL and acquired TSPO immunopositivity, indicating their activated status. Scale bars, 25 μm. Phosphatidylserine (PS) exposure in ONL nuclei of unfixed cryosections of rd10 retina during rod degeneration. PS exposure in the ONL was monitored in unfixed frozen sections using fluorescently conjugated annexin V which binds cell-surface PS. While minimal annexin V staining was evident in the ONL in P18 wild-type (top row) and P18 rd10 (middle row) retinas in which rod degeneration is absent, staining was prominent in P21 rd10 retina during rod degeneration (bottom row). Scale bar, 40 μm.

    Techniques Used: Activation Assay, Marker, Staining

    31) Product Images from "Manipulation of a quasi-natural cell block for high-efficiency transplantation of adherent somatic cells"

    Article Title: Manipulation of a quasi-natural cell block for high-efficiency transplantation of adherent somatic cells

    Journal: Brazilian Journal of Medical and Biological Research

    doi: 10.1590/1414-431X20144322

    Confirmation of the allografted quasi-natural cell block in the recipient body. To confirm allogenicity of the transplanted quasi-natural cell block, gender-specific probes were used to distinguish the male-derived allografted tissue from the recipient female mouse by FISH along with DAPI staining of the nucleus ( A, B ). Green spots indicate male Y chromosome probe in the corresponding female tissue. For intercellular communication capability in the allografted tissue, specimens were stained with specific probes for signaling marker connexin43, in the quasi-natural tissue ( C ) along with native heart tissue ( D ). Immunostaining of the allografted quasi-natural tissue with epithelial linage marker CD31 revealed vascular network assembly as indicated ( E ). Immunostaining of the allografted quasi-natural tissue further showed that most of cells were stained with anti-vimentin, mesodermal lineage marker, but not with anti-cytokeratin ( F ). Images for individual channels (connexin43 with alexa 488 is green, cd31 with alexa 568 is red, vimentin with alexa 488 is green, cytokeratin with alexa 568 is red) are shown on the left, and main panels show the merged image containing all channels plus DIC. The cells nuclei were visualized with DAPI (blue). Scale bar: ( A, C, D, E and F ) 20 μm; ( B ) 100 μm.
    Figure Legend Snippet: Confirmation of the allografted quasi-natural cell block in the recipient body. To confirm allogenicity of the transplanted quasi-natural cell block, gender-specific probes were used to distinguish the male-derived allografted tissue from the recipient female mouse by FISH along with DAPI staining of the nucleus ( A, B ). Green spots indicate male Y chromosome probe in the corresponding female tissue. For intercellular communication capability in the allografted tissue, specimens were stained with specific probes for signaling marker connexin43, in the quasi-natural tissue ( C ) along with native heart tissue ( D ). Immunostaining of the allografted quasi-natural tissue with epithelial linage marker CD31 revealed vascular network assembly as indicated ( E ). Immunostaining of the allografted quasi-natural tissue further showed that most of cells were stained with anti-vimentin, mesodermal lineage marker, but not with anti-cytokeratin ( F ). Images for individual channels (connexin43 with alexa 488 is green, cd31 with alexa 568 is red, vimentin with alexa 488 is green, cytokeratin with alexa 568 is red) are shown on the left, and main panels show the merged image containing all channels plus DIC. The cells nuclei were visualized with DAPI (blue). Scale bar: ( A, C, D, E and F ) 20 μm; ( B ) 100 μm.

    Techniques Used: Blocking Assay, Derivative Assay, Fluorescence In Situ Hybridization, Staining, Marker, Immunostaining

    32) Product Images from "Miniature- and Multiple-Eyespot Loci in Chlamydomonas reinhardtii Define New Modulators of Eyespot Photoreception and Assembly"

    Article Title: Miniature- and Multiple-Eyespot Loci in Chlamydomonas reinhardtii Define New Modulators of Eyespot Photoreception and Assembly

    Journal: G3: Genes|Genomes|Genetics

    doi: 10.1534/g3.111.000679

    ChR1 photoreceptor localization and eyespot layers are altered in min1 but not min2 . (A–D) Combined immunofluorescence micrographs of fixed cells stained for channelrhodopsin-1 (ChR1, magenta) and acetylated α-tubulin (AcTub, green). (A) Wild-type cell with a ChR1 patch associated with the D4 microtubule rootlet. (B) ChR1 staining in min1 cells appears as multiple, distinct spots or stripes along the D4 rootlet, occasionally appearing in off-rootlet spots. (C) The shape and position of the ChR1 patch on the D4 rootlet are maintained in min2 mutant cells. (D) min1 min2 cells, showing ChR1 staining in multiple spots along the D4 rootlet. (E–F) Combined immunofluorescence micrographs of fixed cells stained for the pigment granule marker EYE3 (red), ChR1 (blue), and AcTub (green). (E) Pigment granules (arrow) are not apposed to the plasma membrane-localized photoreceptor spots in photoautotrophically grown min1 cells. (F) Organization of eyespot layers is unaffected in min2 cells, with ChR1 directly overlaying EYE3 staining (inset). Scale bars, 5 μm.
    Figure Legend Snippet: ChR1 photoreceptor localization and eyespot layers are altered in min1 but not min2 . (A–D) Combined immunofluorescence micrographs of fixed cells stained for channelrhodopsin-1 (ChR1, magenta) and acetylated α-tubulin (AcTub, green). (A) Wild-type cell with a ChR1 patch associated with the D4 microtubule rootlet. (B) ChR1 staining in min1 cells appears as multiple, distinct spots or stripes along the D4 rootlet, occasionally appearing in off-rootlet spots. (C) The shape and position of the ChR1 patch on the D4 rootlet are maintained in min2 mutant cells. (D) min1 min2 cells, showing ChR1 staining in multiple spots along the D4 rootlet. (E–F) Combined immunofluorescence micrographs of fixed cells stained for the pigment granule marker EYE3 (red), ChR1 (blue), and AcTub (green). (E) Pigment granules (arrow) are not apposed to the plasma membrane-localized photoreceptor spots in photoautotrophically grown min1 cells. (F) Organization of eyespot layers is unaffected in min2 cells, with ChR1 directly overlaying EYE3 staining (inset). Scale bars, 5 μm.

    Techniques Used: Immunofluorescence, Staining, Mutagenesis, Marker

    Quantification of the extent of EYE2, EYE3, and ChR1 copositioning in mlt1 and mlt2 cells. Percentage of single or copositioned spots per total spots scored is shown. Copositioned spots of combinations of EYE2, EYE3, and ChR1 are markedly more prevalent in mlt1 cells compared with mlt2 cells. Conversely, mlt2 cells have a greater proportion of single EYE2, EYE3, or ChR1 spots not copositioned with other eyespot proteins. Copositioning of EYE3 with ChR1 was never observed without EYE2.
    Figure Legend Snippet: Quantification of the extent of EYE2, EYE3, and ChR1 copositioning in mlt1 and mlt2 cells. Percentage of single or copositioned spots per total spots scored is shown. Copositioned spots of combinations of EYE2, EYE3, and ChR1 are markedly more prevalent in mlt1 cells compared with mlt2 cells. Conversely, mlt2 cells have a greater proportion of single EYE2, EYE3, or ChR1 spots not copositioned with other eyespot proteins. Copositioning of EYE3 with ChR1 was never observed without EYE2.

    Techniques Used:

    Eyespots are disorganized in mlt1 and mlt2 mutant cells. (A, B) Combined immunofluorescence micrographs of individual cells stained for ChR1 (magenta) and AcTub (green). (A) mlt1 cells have ChR1 patches in either hemisphere of the cell that are often clustered around the anterior pole and associated with acetylated rootlets. Arrow indicates ChR1 patch not associated with a rootlet. (B) mlt2 cell with multiple ChR1 patches associated with microtubule rootlets. (C) Wild-type cell showing layered arrangement of EYE2 (green), EYE3 (blue), and ChR1 (red) in the eyespot (inset). (D–F) EYE2, EYE3, and ChR1 positioning is dramatically disrupted in asynchronous stationary-phase populations of mlt1 and mlt2 cells. Combined immunofluorescence micrographs of fixed cells stained for EYE2 (green), EYE3 (blue), and ChR1 (red). a, single EYE2 spot; b:, single ChR1 spot; c, single EYE3 spot; d, EYE2/EYE3 copositioned spot; e, EYE2/ChR1 copositioned spot; f, EYE2/EYE3/ChR1 copositioned spot. (D) Z-projections of combined immunofluorescence micrographs of an individual mlt2 cell illustrating positioning of EYE2, EYE3, and ChR1 spots. (E) Z-projection of combined immunofluorescence micrographs of a mlt1 field stained for EYE2 (green), EYE3 (blue), and ChR1 (red). Various combinations of single and copositioned spots are observed (arrows). (F) Z-projection of combined immunofluorescence micrographs of a mlt2 field. Various spot-positioning combinations are again observed, with single spots predominating. Scale bars, 5 µm.
    Figure Legend Snippet: Eyespots are disorganized in mlt1 and mlt2 mutant cells. (A, B) Combined immunofluorescence micrographs of individual cells stained for ChR1 (magenta) and AcTub (green). (A) mlt1 cells have ChR1 patches in either hemisphere of the cell that are often clustered around the anterior pole and associated with acetylated rootlets. Arrow indicates ChR1 patch not associated with a rootlet. (B) mlt2 cell with multiple ChR1 patches associated with microtubule rootlets. (C) Wild-type cell showing layered arrangement of EYE2 (green), EYE3 (blue), and ChR1 (red) in the eyespot (inset). (D–F) EYE2, EYE3, and ChR1 positioning is dramatically disrupted in asynchronous stationary-phase populations of mlt1 and mlt2 cells. Combined immunofluorescence micrographs of fixed cells stained for EYE2 (green), EYE3 (blue), and ChR1 (red). a, single EYE2 spot; b:, single ChR1 spot; c, single EYE3 spot; d, EYE2/EYE3 copositioned spot; e, EYE2/ChR1 copositioned spot; f, EYE2/EYE3/ChR1 copositioned spot. (D) Z-projections of combined immunofluorescence micrographs of an individual mlt2 cell illustrating positioning of EYE2, EYE3, and ChR1 spots. (E) Z-projection of combined immunofluorescence micrographs of a mlt1 field stained for EYE2 (green), EYE3 (blue), and ChR1 (red). Various combinations of single and copositioned spots are observed (arrows). (F) Z-projection of combined immunofluorescence micrographs of a mlt2 field. Various spot-positioning combinations are again observed, with single spots predominating. Scale bars, 5 µm.

    Techniques Used: Mutagenesis, Immunofluorescence, Staining

    33) 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

    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:

    34) Product Images from "Targeting Swine Leukocyte Antigen Class I Molecules for Proteasomal Degradation by the nsp1α Replicase Protein of the Chinese Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Strain JXwn06"

    Article Title: Targeting Swine Leukocyte Antigen Class I Molecules for Proteasomal Degradation by the nsp1α Replicase Protein of the Chinese Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Strain JXwn06

    Journal: Journal of Virology

    doi: 10.1128/JVI.02307-15

    Downregulation of SLA-I cell surface accumulation of PAMs by HP-PRRSV. (A) Effect of PRRSV on SLA-I cell surface accumulation. PAMs were mock infected or infected with HP-PRRSV JXwn06 or UV-irradiated JXwn06 or HB-1/3.9 at an MOI of 1 or 0.1. At the indicated time points, the cells were harvested and subjected to FACS analysis with mouse monoclonal antibody against SLA-I (JM1E3). (B) PRRSV- or mock-inoculated PAMs were incubated with saturating amounts of JM1E3 at 4°C for 1 h before being washed three times with ice-cold PBS to remove unbound MAbs. The cells were then incubated at 37°C to promote endocytosis. At different times postinfection, the cells were collected and subjected to FACS analysis with FITC-conjugated goat anti-mouse IgG. (C) PRRSV- or mock-infected PAMs were incubated with saturating amounts of unlabeled MAb JM1E3 to SLA-I at 4°C for 1 h and then at 37°C for various times before being subjected to FACS analysis with FITC-conjugated JM1E3. The trend curves were fitted based on the MFIs, and the data shown are means and standard deviations of results from three independent experiments (*, P
    Figure Legend Snippet: Downregulation of SLA-I cell surface accumulation of PAMs by HP-PRRSV. (A) Effect of PRRSV on SLA-I cell surface accumulation. PAMs were mock infected or infected with HP-PRRSV JXwn06 or UV-irradiated JXwn06 or HB-1/3.9 at an MOI of 1 or 0.1. At the indicated time points, the cells were harvested and subjected to FACS analysis with mouse monoclonal antibody against SLA-I (JM1E3). (B) PRRSV- or mock-inoculated PAMs were incubated with saturating amounts of JM1E3 at 4°C for 1 h before being washed three times with ice-cold PBS to remove unbound MAbs. The cells were then incubated at 37°C to promote endocytosis. At different times postinfection, the cells were collected and subjected to FACS analysis with FITC-conjugated goat anti-mouse IgG. (C) PRRSV- or mock-infected PAMs were incubated with saturating amounts of unlabeled MAb JM1E3 to SLA-I at 4°C for 1 h and then at 37°C for various times before being subjected to FACS analysis with FITC-conjugated JM1E3. The trend curves were fitted based on the MFIs, and the data shown are means and standard deviations of results from three independent experiments (*, P

    Techniques Used: Infection, Irradiation, FACS, Incubation

    35) Product Images from "The Assembly of GM1 Glycolipid- and Cholesterol-Enriched Raft-Like Membrane Microdomains Is Important for Giardial Encystation"

    Article Title: The Assembly of GM1 Glycolipid- and Cholesterol-Enriched Raft-Like Membrane Microdomains Is Important for Giardial Encystation

    Journal: Infection and Immunity

    doi: 10.1128/IAI.03118-14

    Raft-like microdomains in Giardia trophozoites. Trophozoites were labeled with Alexa Fluor 488-conjugated CTXB (image a, green) and GM1 antibody (image b, red). Labeling of plasma membranes, the ventral disc, and the flagella of trophozoites is visible. FAST Dil oil labels the cytoplasmic and nuclear lipids of trophozoites (image c). DAPI-stained nuclei are also noticeable. N, nucleus; PM, plasma membrane; F, flagella; VD, ventral disc; ab, antibody. Bars, 5 μm. (B) 3D representation. The image of CTXB-labeled Giardia trophozoites was captured using Zen 2009 software. z-stacks were acquired, and a 3D model was reconstructed from the 12 optical sections of the z-stacks with a slice thickness of 0.37 μm each.
    Figure Legend Snippet: Raft-like microdomains in Giardia trophozoites. Trophozoites were labeled with Alexa Fluor 488-conjugated CTXB (image a, green) and GM1 antibody (image b, red). Labeling of plasma membranes, the ventral disc, and the flagella of trophozoites is visible. FAST Dil oil labels the cytoplasmic and nuclear lipids of trophozoites (image c). DAPI-stained nuclei are also noticeable. N, nucleus; PM, plasma membrane; F, flagella; VD, ventral disc; ab, antibody. Bars, 5 μm. (B) 3D representation. The image of CTXB-labeled Giardia trophozoites was captured using Zen 2009 software. z-stacks were acquired, and a 3D model was reconstructed from the 12 optical sections of the z-stacks with a slice thickness of 0.37 μm each.

    Techniques Used: Labeling, Staining, Software

    (A) Expression of raft-like microdomains by encysting Giardia cultured in 5% DFBS-containing medium. The cells were labeled with Alexa Fluor 488-conjugated CTXB and DAPI and viewed under a confocal microscope. (Image a) Nonencysting trophozoites; (image b) encysting cells at 10 h p.i. of encystation; (image c) encysting cells at 18 h p.i. of encystation; (image d) water-resistant cysts. (B) ESV biogenesis and expression of trophozoite proteins. Encysting cells at 10 h p.i. of encystation (control and inhibitor treated) were labeled with trophozoite (green) and cyst (red) antibodies as described in Materials and Methods. (Image a) Labeling of encysting cells at 10 h p.i. of encystation in medium supplemented with ABS; (image b) encysting trophozoites at 10 h p.i. of encystation differentiated in medium containing DFBS; (image c) nystatin (27 μM) treatment; (image d) filipin III (7.6 μM) treatment; (image e) oseltamivir (20 μM) treatment. (C) Induction of encystation in DFBS-supplemented medium affects cyst production. Control and inhibitor-treated trophozoites were allowed to encyst for 18 h before labeling with cyst (red) and trophozoite (green) antibodies. (Image a) Cyst antibody-labeled water-resistant cysts produced in ABS-supplemented medium; (image b) cysts generated in ABS-supplemented medium; (image c) nystatin (27 μM) treatment; image d, filipin III (7.6 μM) treatment; image e, oseltamivir (20 μM) treatment. N, nucleus; ESV, encystation-specific vesicle; PM, plasma membrane; N, nucleus; VD, ventral disc; F, flagella; CW, cyst wall; NCW, nascent cyst wall; CL, cyst-like structure; TL, trophozoite-like structure. Bars, 5 μm. (D) Quantitative estimates of water-resistant cells expressing trophozoite proteins or cyst proteins. For quantification, 125 cells from 6 randomly selected fields from 2 separate experiments were counted. Data were analyzed by a one-way ANOVA followed by the Holm-Šídák method. **, P
    Figure Legend Snippet: (A) Expression of raft-like microdomains by encysting Giardia cultured in 5% DFBS-containing medium. The cells were labeled with Alexa Fluor 488-conjugated CTXB and DAPI and viewed under a confocal microscope. (Image a) Nonencysting trophozoites; (image b) encysting cells at 10 h p.i. of encystation; (image c) encysting cells at 18 h p.i. of encystation; (image d) water-resistant cysts. (B) ESV biogenesis and expression of trophozoite proteins. Encysting cells at 10 h p.i. of encystation (control and inhibitor treated) were labeled with trophozoite (green) and cyst (red) antibodies as described in Materials and Methods. (Image a) Labeling of encysting cells at 10 h p.i. of encystation in medium supplemented with ABS; (image b) encysting trophozoites at 10 h p.i. of encystation differentiated in medium containing DFBS; (image c) nystatin (27 μM) treatment; (image d) filipin III (7.6 μM) treatment; (image e) oseltamivir (20 μM) treatment. (C) Induction of encystation in DFBS-supplemented medium affects cyst production. Control and inhibitor-treated trophozoites were allowed to encyst for 18 h before labeling with cyst (red) and trophozoite (green) antibodies. (Image a) Cyst antibody-labeled water-resistant cysts produced in ABS-supplemented medium; (image b) cysts generated in ABS-supplemented medium; (image c) nystatin (27 μM) treatment; image d, filipin III (7.6 μM) treatment; image e, oseltamivir (20 μM) treatment. N, nucleus; ESV, encystation-specific vesicle; PM, plasma membrane; N, nucleus; VD, ventral disc; F, flagella; CW, cyst wall; NCW, nascent cyst wall; CL, cyst-like structure; TL, trophozoite-like structure. Bars, 5 μm. (D) Quantitative estimates of water-resistant cells expressing trophozoite proteins or cyst proteins. For quantification, 125 cells from 6 randomly selected fields from 2 separate experiments were counted. Data were analyzed by a one-way ANOVA followed by the Holm-Šídák method. **, P

    Techniques Used: Expressing, Cell Culture, Labeling, Microscopy, Produced, Generated

    36) Product Images from "Long-Term Stability and Safety of Transgenic Cultured Epidermal Stem Cells in Gene Therapy of Junctional Epidermolysis Bullosa"

    Article Title: Long-Term Stability and Safety of Transgenic Cultured Epidermal Stem Cells in Gene Therapy of Junctional Epidermolysis Bullosa

    Journal: Stem Cell Reports

    doi: 10.1016/j.stemcr.2013.11.001

    Expression of LAM332 and α6β4 Integrins (A–J) IF analysis of laminin 332-β3 (A and B), 332-γ2 (C and D) 332-α3 (E and F), α6 integrin (G and H), and β4 integrin (I and J) in control (WT) and transgenic (Claudio) skin sections. The transgenic epidermis expresses normal amounts of laminin 332 and α6β4 integrins properly located at the epidermal-dermal junction. Scale bars, 40 μm.
    Figure Legend Snippet: Expression of LAM332 and α6β4 Integrins (A–J) IF analysis of laminin 332-β3 (A and B), 332-γ2 (C and D) 332-α3 (E and F), α6 integrin (G and H), and β4 integrin (I and J) in control (WT) and transgenic (Claudio) skin sections. The transgenic epidermis expresses normal amounts of laminin 332 and α6β4 integrins properly located at the epidermal-dermal junction. Scale bars, 40 μm.

    Techniques Used: Expressing, Transgenic Assay

    37) Product Images from "The Golgin GCC88 Is Required for Efficient Retrograde Transport of Cargo from the Early Endosomes to the Trans"

    Article Title: The Golgin GCC88 Is Required for Efficient Retrograde Transport of Cargo from the Early Endosomes to the Trans

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E07-06-0622

    Depletion of endogenous GCC88 by an inducible shRNA. HeLa cells (clone A8) stably expressing a tetracycline on inducible shRNA to GCC88 (tet R GCC88KD A8) were either untreated (control) or treated with 10 or 100 ng/ml doxycycline (Dox) for 96 h, and monolayers were fixed with 4% paraformaldehyde. (A) Endogenous GCC88 was detected with rabbit anti-GCC88 antibodies followed by Alexa 488-conjugated anti-rabbit IgG. (B) HeLa A8 cells were incubated with 10 ng/ml doxycycline for 96 h, lysed in SDS-PAGE reducing buffer, and then extracts were subjected to SDS-PAGE on a 7.5% polyacrylamide gel. Proteins were transfer to a polyvinylidene difluoride membrane and probed with rabbit anti-GCC88 antibodies using a chemiluminescence detection system. The membrane were then stripped and reprobed with anti-α-tubulin, followed by anti-GCC185 and anti-golgin-97 antibodies. Bar, 10 μm.
    Figure Legend Snippet: Depletion of endogenous GCC88 by an inducible shRNA. HeLa cells (clone A8) stably expressing a tetracycline on inducible shRNA to GCC88 (tet R GCC88KD A8) were either untreated (control) or treated with 10 or 100 ng/ml doxycycline (Dox) for 96 h, and monolayers were fixed with 4% paraformaldehyde. (A) Endogenous GCC88 was detected with rabbit anti-GCC88 antibodies followed by Alexa 488-conjugated anti-rabbit IgG. (B) HeLa A8 cells were incubated with 10 ng/ml doxycycline for 96 h, lysed in SDS-PAGE reducing buffer, and then extracts were subjected to SDS-PAGE on a 7.5% polyacrylamide gel. Proteins were transfer to a polyvinylidene difluoride membrane and probed with rabbit anti-GCC88 antibodies using a chemiluminescence detection system. The membrane were then stripped and reprobed with anti-α-tubulin, followed by anti-GCC185 and anti-golgin-97 antibodies. Bar, 10 μm.

    Techniques Used: shRNA, Stable Transfection, Expressing, Incubation, SDS Page

    Defect in TGN38 recycling is rescued by expression of wild-type syntaxin 6. HeLa A8 were either untreated (control) or incubated in 10 ng/ml doxycycline for 96 h (GCC88 depleted) and cotransfected with TGN38 and either GFP-syntaxin6 FL (GFP-Syn6 FL ) (A), GFP-syntaxin6 cyto (GFP-Syn6 cyto ) (B), or cherry-syntaxin 16 FL (Cherry-Syn16 FL ) (C) for 24 h before the internalization assay. Monolayers were incubated with monoclonal mouse anti-TGN38 antibodies for 30 min on ice, washed in PBS, and then incubated at 37°C in serum-free media 120 min to internalize the antibody–TGN38 complex. Monolayers were fixed in 4% paraformaldehyde, permeabilized, and stained with Alexa 568-conjugated anti-mouse IgG for 60 min. Endogenous GCC88 was stained with rabbit anti-GCC88 antibodies, followed by Alexa 647-conjugated anti-rabbit IgG. Cells with no GCC88 staining and perinuclear level of syntaxin 6 or syntaxin 16 expression were analyzed (n = 15, in duplicate). Bars, 10 μm.
    Figure Legend Snippet: Defect in TGN38 recycling is rescued by expression of wild-type syntaxin 6. HeLa A8 were either untreated (control) or incubated in 10 ng/ml doxycycline for 96 h (GCC88 depleted) and cotransfected with TGN38 and either GFP-syntaxin6 FL (GFP-Syn6 FL ) (A), GFP-syntaxin6 cyto (GFP-Syn6 cyto ) (B), or cherry-syntaxin 16 FL (Cherry-Syn16 FL ) (C) for 24 h before the internalization assay. Monolayers were incubated with monoclonal mouse anti-TGN38 antibodies for 30 min on ice, washed in PBS, and then incubated at 37°C in serum-free media 120 min to internalize the antibody–TGN38 complex. Monolayers were fixed in 4% paraformaldehyde, permeabilized, and stained with Alexa 568-conjugated anti-mouse IgG for 60 min. Endogenous GCC88 was stained with rabbit anti-GCC88 antibodies, followed by Alexa 647-conjugated anti-rabbit IgG. Cells with no GCC88 staining and perinuclear level of syntaxin 6 or syntaxin 16 expression were analyzed (n = 15, in duplicate). Bars, 10 μm.

    Techniques Used: Expressing, Incubation, Staining

    Shiga toxin is transported efficiently to the Golgi in GCC88-depleted cells. HeLa A8 cells were either untreated (control) (A) or incubated with 10 ng/ml doxycycline for 96 h (GCC88 depleted) (B). Monolayers were incubated with Cy3-conjugated STx-B for 45 min on ice, and then they were either fixed immediately (0 min) or incubated at 37°C for either 20 or 60 min, followed by fixation in 4% paraformaldehyde. Cells were stained with monoclonal antibodies to GM130 followed by Alexa-conjugated anti-mouse IgG. Bars, 10 μm.
    Figure Legend Snippet: Shiga toxin is transported efficiently to the Golgi in GCC88-depleted cells. HeLa A8 cells were either untreated (control) (A) or incubated with 10 ng/ml doxycycline for 96 h (GCC88 depleted) (B). Monolayers were incubated with Cy3-conjugated STx-B for 45 min on ice, and then they were either fixed immediately (0 min) or incubated at 37°C for either 20 or 60 min, followed by fixation in 4% paraformaldehyde. Cells were stained with monoclonal antibodies to GM130 followed by Alexa-conjugated anti-mouse IgG. Bars, 10 μm.

    Techniques Used: Incubation, Staining

    Anterograde transport of E-cadherin in GCC88-depleted cells is unaffected. HeLa A8 cells were either untreated (control) or incubated with 10 ng/ml Dox for 72 h and then transfected with Ecad-GFP for 24 h before staining. (A) Monolayers were fixed with 4% paraformaldehyde, permeabilized, and endogenous GCC88 was detected with rabbit anti-GCC88 antibodies followed by Alexa 568-conjugated anti-rabbit IgG and Ecad-GFP by GFP fluorescence. (B) Monolayers were fixed and stained with monoclonal anti-Ecad antibodies followed by Alexa 568-conjugated goat ant-mouse IgG. Fixed monolayers were then permeabilized and stained with rabbit anti-GCC88 antibodies followed by Alexa 647-conjugated goat anti-rabbit IgG. Bars, 10 μm.
    Figure Legend Snippet: Anterograde transport of E-cadherin in GCC88-depleted cells is unaffected. HeLa A8 cells were either untreated (control) or incubated with 10 ng/ml Dox for 72 h and then transfected with Ecad-GFP for 24 h before staining. (A) Monolayers were fixed with 4% paraformaldehyde, permeabilized, and endogenous GCC88 was detected with rabbit anti-GCC88 antibodies followed by Alexa 568-conjugated anti-rabbit IgG and Ecad-GFP by GFP fluorescence. (B) Monolayers were fixed and stained with monoclonal anti-Ecad antibodies followed by Alexa 568-conjugated goat ant-mouse IgG. Fixed monolayers were then permeabilized and stained with rabbit anti-GCC88 antibodies followed by Alexa 647-conjugated goat anti-rabbit IgG. Bars, 10 μm.

    Techniques Used: Incubation, Transfection, Staining, Fluorescence

    Syntaxin 6 depletion impairs TGN38, but not Shiga toxin, trafficking to the Golgi apparatus. (A) HeLa cells were transfected with syntaxin 6 siRNA for 72 h, fixed in 4% paraformaldehyde, saponin permeablized, and stained with monoclonal anti-syntaxin 6 antibodies followed by Alexa-conjugated mouse IgG. (B) HeLa cells transfected with siRNA as described above for 48 h, and then they were transfected a second time with CFP-TGN38 for a further 24 h. Monolayers were then incubated with monoclonal mouse anti-TGN38 antibodies on ice for 30 min, washed in PBS, and incubated in serum-free media at 37°C for 120 min. Monolayers were fixed in 4% paraformaldehyde, permeabilized, and stained with Alexa-conjugated anti-mouse IgG. Endogenous GMAP-210 was stained with rabbit anti-GMAP-210, followed by Alexa-conjugated anti-rabbit IgG. C) HeLa cells were transfected with syntaxin 6 siRNA for 72 h and incubated with Cy3-conjugated STx-B for 45 min on ice and then either fixed immediately (0 min) or incubated at 37°C for 60 min followed by fixation. Cells were stained with monoclonal antibodies to GM130 followed by Alexa 647-conjugated mouse IgG. Bars, 10 μm.
    Figure Legend Snippet: Syntaxin 6 depletion impairs TGN38, but not Shiga toxin, trafficking to the Golgi apparatus. (A) HeLa cells were transfected with syntaxin 6 siRNA for 72 h, fixed in 4% paraformaldehyde, saponin permeablized, and stained with monoclonal anti-syntaxin 6 antibodies followed by Alexa-conjugated mouse IgG. (B) HeLa cells transfected with siRNA as described above for 48 h, and then they were transfected a second time with CFP-TGN38 for a further 24 h. Monolayers were then incubated with monoclonal mouse anti-TGN38 antibodies on ice for 30 min, washed in PBS, and incubated in serum-free media at 37°C for 120 min. Monolayers were fixed in 4% paraformaldehyde, permeabilized, and stained with Alexa-conjugated anti-mouse IgG. Endogenous GMAP-210 was stained with rabbit anti-GMAP-210, followed by Alexa-conjugated anti-rabbit IgG. C) HeLa cells were transfected with syntaxin 6 siRNA for 72 h and incubated with Cy3-conjugated STx-B for 45 min on ice and then either fixed immediately (0 min) or incubated at 37°C for 60 min followed by fixation. Cells were stained with monoclonal antibodies to GM130 followed by Alexa 647-conjugated mouse IgG. Bars, 10 μm.

    Techniques Used: Transfection, Staining, Incubation

    Depletion of GCC88 results in mislocalization of syntaxin 6. (A–E) HeLa A8 were either untreated (control) or incubated in 10 ng/ml doxycycline for 96 h (GCC88 depleted) and then fixed in 4% paraformaldehyde and permeablized. Fixed monolayers were stained for endogenous VAMP4 (A), VAMP3 (B), Vti1a (C), syntaxin 6 (D), and syntaxin 16 (E), with rabbit polyclonal antibodies to VAMP4, VAMP3, and syntaxin 16, respectively, followed by Alexa 568-conjugated rabbit IgG and mouse monoclonal antibodies to vti1a and syntaxin 6, respectively, followed by Alexa 568-conjugated anti-mouse IgG. (F) Untransfected HeLa cells (control) or HeLa cells transfected with p230 siRNA (p230 depleted) for 72 h were fixed in 4% paraformaldehyde and saponin permeabilized. Endogenous p230 was stained with human anti-p230 antibodies followed by FITC-conjugated anti-human IgG and syntaxin 6 stained with monoclonal mouse anti-syntaxin 6, followed by Alexa 568-conjugated anti-mouse IgG. Bars, 10 μm.
    Figure Legend Snippet: Depletion of GCC88 results in mislocalization of syntaxin 6. (A–E) HeLa A8 were either untreated (control) or incubated in 10 ng/ml doxycycline for 96 h (GCC88 depleted) and then fixed in 4% paraformaldehyde and permeablized. Fixed monolayers were stained for endogenous VAMP4 (A), VAMP3 (B), Vti1a (C), syntaxin 6 (D), and syntaxin 16 (E), with rabbit polyclonal antibodies to VAMP4, VAMP3, and syntaxin 16, respectively, followed by Alexa 568-conjugated rabbit IgG and mouse monoclonal antibodies to vti1a and syntaxin 6, respectively, followed by Alexa 568-conjugated anti-mouse IgG. (F) Untransfected HeLa cells (control) or HeLa cells transfected with p230 siRNA (p230 depleted) for 72 h were fixed in 4% paraformaldehyde and saponin permeabilized. Endogenous p230 was stained with human anti-p230 antibodies followed by FITC-conjugated anti-human IgG and syntaxin 6 stained with monoclonal mouse anti-syntaxin 6, followed by Alexa 568-conjugated anti-mouse IgG. Bars, 10 μm.

    Techniques Used: Incubation, Staining, Transfection

    38) Product Images from "Identification of Endothelial Cell Junctional Proteins and Lymphocyte Receptors Involved in Transendothelial Migration of Human Effector Memory CD4+ T Cells"

    Article Title: Identification of Endothelial Cell Junctional Proteins and Lymphocyte Receptors Involved in Transendothelial Migration of Human Effector Memory CD4+ T Cells

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1002835

    Blocking of EC nectin-2 and/or PVR inhibits TCR-dependent TEM of EM CD4 + T cells. TNF-treated CIITA-transduced HDMECs overlaid with TSST-1 were preincubated with isotype control (control), anti–nectin-2 (nectin-2), anti-PVR (PVR), and both blocking Abs (nectin-2+PVR) prior to flow TEM. Panel on the left shows TEM of Vβ2TCR − cells at 15 min. Panel on the right shows TEM of Vβ2TCR + cells at 60 min. Graphs display data from one representative of eight different experiments, testing each condition with T cells from at least three different donors. *** p
    Figure Legend Snippet: Blocking of EC nectin-2 and/or PVR inhibits TCR-dependent TEM of EM CD4 + T cells. TNF-treated CIITA-transduced HDMECs overlaid with TSST-1 were preincubated with isotype control (control), anti–nectin-2 (nectin-2), anti-PVR (PVR), and both blocking Abs (nectin-2+PVR) prior to flow TEM. Panel on the left shows TEM of Vβ2TCR − cells at 15 min. Panel on the right shows TEM of Vβ2TCR + cells at 60 min. Graphs display data from one representative of eight different experiments, testing each condition with T cells from at least three different donors. *** p

    Techniques Used: Blocking Assay, Transmission Electron Microscopy, Flow Cytometry

    Knockdown of EC nectin-2 and PVR inhibits TCR-dependent TEM of EM CD4 + T cells. CIITA HDMECs were transfected with control siRNA or siRNA targeting nectin-2 (nectin-2 siRNA), PVR (PVR siRNA), or both (nectin-2+PVR siRNA), treated with TNF, either harvested for FACS analysis ( A ) or overlaid with TSST-1 superantigen and used in flow TEM assays with EM CD4 + T cells ( B ). A , Histograms show FACS analysis of cells stained with isotype control IgG (thin line) or anti–nectin-2 or anti-PVR (thick lines). B , Graphs display data combined from three separate experiments using T cells from different donors. *** p
    Figure Legend Snippet: Knockdown of EC nectin-2 and PVR inhibits TCR-dependent TEM of EM CD4 + T cells. CIITA HDMECs were transfected with control siRNA or siRNA targeting nectin-2 (nectin-2 siRNA), PVR (PVR siRNA), or both (nectin-2+PVR siRNA), treated with TNF, either harvested for FACS analysis ( A ) or overlaid with TSST-1 superantigen and used in flow TEM assays with EM CD4 + T cells ( B ). A , Histograms show FACS analysis of cells stained with isotype control IgG (thin line) or anti–nectin-2 or anti-PVR (thick lines). B , Graphs display data combined from three separate experiments using T cells from different donors. *** p

    Techniques Used: Transmission Electron Microscopy, Transfection, FACS, Flow Cytometry, Staining

    39) Product Images from "Identification of Endothelial Cell Junctional Proteins and Lymphocyte Receptors Involved in Transendothelial Migration of Human Effector Memory CD4+ T Cells"

    Article Title: Identification of Endothelial Cell Junctional Proteins and Lymphocyte Receptors Involved in Transendothelial Migration of Human Effector Memory CD4+ T Cells

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1002835

    Knockdown of EC JAM-1 inhibits chemokine-dependent TEM of EM CD4 + T cells. CIITA HDMECs were transfected with control siRNA or two different siRNAs targeting JAM-1 (JAM-1 siRNA-5 and JAM-1 siRNA-6), treated with TNF, either harvested for FACS analysis ( A ) or overlaid with TSST-1 superantigen, and used in flow TEM assays with EM CD4 + T cells ( B ). A , Histograms show FACS analysis of cells stained with isotype control IgG (thin line) or anti–JAM-1 (thick lines) demonstrating effective knockdown. B , Left lower panel (VB2 − ) shows TEM of Vβ2TCR − cells at 15 min. Right lower panel on (VB2 + ) shows TEM of Vβ2TCR + cells at 60 min. Graphs display data from one representative experiment of three (VB2 − ) and two (VB2 + ) separate experiments using T cells from different donors. * p
    Figure Legend Snippet: Knockdown of EC JAM-1 inhibits chemokine-dependent TEM of EM CD4 + T cells. CIITA HDMECs were transfected with control siRNA or two different siRNAs targeting JAM-1 (JAM-1 siRNA-5 and JAM-1 siRNA-6), treated with TNF, either harvested for FACS analysis ( A ) or overlaid with TSST-1 superantigen, and used in flow TEM assays with EM CD4 + T cells ( B ). A , Histograms show FACS analysis of cells stained with isotype control IgG (thin line) or anti–JAM-1 (thick lines) demonstrating effective knockdown. B , Left lower panel (VB2 − ) shows TEM of Vβ2TCR − cells at 15 min. Right lower panel on (VB2 + ) shows TEM of Vβ2TCR + cells at 60 min. Graphs display data from one representative experiment of three (VB2 − ) and two (VB2 + ) separate experiments using T cells from different donors. * p

    Techniques Used: Transmission Electron Microscopy, Transfection, FACS, Flow Cytometry, Staining

    Blocking of T cell LFA-1 but not Mac-1 inhibits TCR-dependent TEM of EM CD4 + T cells. A , Mac-1 is expressed on EM CD4 + T cells. Contour plots of FACS analysis of CD45RA − CD4 + T cells stained with isotype-matched control PE- and FITC-conjugated IgG ( left plot ) or PE-conjugated anti-CD11b and FITC-conjugated anti-CCR7 ( right plot ). Note that there is a significant proportion of CD11b + cells in the CCR7 low (i.e., EM) population. B , TEM assays. EM CD4 + T cells were preincubated with isotype control IgG (control), anti–LFA-1 (LFA-1), or anti–Mac-1 (Mac-1) blocking mAb 30 min prior to flow TEM on TNF-treated CIITA-transduced HDMECs overlaid with TSST-1. Panel on the left (VB2 − ) shows TEM of Vβ2TCR − cells at 15 min. Panel on the right (VB2 + ) shows TEM of Vβ2TCR + cells at 60 min. Graphs display data pooled from three separate experiments using T cells isolated from three different donors. *** p
    Figure Legend Snippet: Blocking of T cell LFA-1 but not Mac-1 inhibits TCR-dependent TEM of EM CD4 + T cells. A , Mac-1 is expressed on EM CD4 + T cells. Contour plots of FACS analysis of CD45RA − CD4 + T cells stained with isotype-matched control PE- and FITC-conjugated IgG ( left plot ) or PE-conjugated anti-CD11b and FITC-conjugated anti-CCR7 ( right plot ). Note that there is a significant proportion of CD11b + cells in the CCR7 low (i.e., EM) population. B , TEM assays. EM CD4 + T cells were preincubated with isotype control IgG (control), anti–LFA-1 (LFA-1), or anti–Mac-1 (Mac-1) blocking mAb 30 min prior to flow TEM on TNF-treated CIITA-transduced HDMECs overlaid with TSST-1. Panel on the left (VB2 − ) shows TEM of Vβ2TCR − cells at 15 min. Panel on the right (VB2 + ) shows TEM of Vβ2TCR + cells at 60 min. Graphs display data pooled from three separate experiments using T cells isolated from three different donors. *** p

    Techniques Used: Blocking Assay, Transmission Electron Microscopy, FACS, Staining, Flow Cytometry, Isolation

    Blocking of EC nectin-2 and/or PVR inhibits TCR-dependent TEM of EM CD4 + T cells. TNF-treated CIITA-transduced HDMECs overlaid with TSST-1 were preincubated with isotype control (control), anti–nectin-2 (nectin-2), anti-PVR (PVR), and both blocking Abs (nectin-2+PVR) prior to flow TEM. Panel on the left shows TEM of Vβ2TCR − cells at 15 min. Panel on the right shows TEM of Vβ2TCR + cells at 60 min. Graphs display data from one representative of eight different experiments, testing each condition with T cells from at least three different donors. *** p
    Figure Legend Snippet: Blocking of EC nectin-2 and/or PVR inhibits TCR-dependent TEM of EM CD4 + T cells. TNF-treated CIITA-transduced HDMECs overlaid with TSST-1 were preincubated with isotype control (control), anti–nectin-2 (nectin-2), anti-PVR (PVR), and both blocking Abs (nectin-2+PVR) prior to flow TEM. Panel on the left shows TEM of Vβ2TCR − cells at 15 min. Panel on the right shows TEM of Vβ2TCR + cells at 60 min. Graphs display data from one representative of eight different experiments, testing each condition with T cells from at least three different donors. *** p

    Techniques Used: Blocking Assay, Transmission Electron Microscopy, Flow Cytometry

    Blocking of T cell DNAM-1 and/or Tactile inhibits TCR-dependent TEM of EM CD4 + T cells. A , Contour plots of FACS analysis of total CD4 + T cells stained with FITC-conjugated IgG (IgG–FITC) and PE-conjugated IgG (IgG–PE, upper left ) or FITC-conjugated anti-CCR7 (CCR7–FITC) and PE-conjugated anti–DNAM-1 (DNAM-1–PE, lower left ) or FITC-conjugated anti-CCR7 and Alexa Fluor 647-complexed IgG (IgG-647, upper right ) or FITC-conjugated anti-CCR7 and Alexa Fluor 647-complexed anti-Tactile (Tactile-647, lower right ). Note the distinct population of cells that are DNAM-1 high, CCR7 low or Tactile high, CCR7 low in the middle , left , and right plots , respectively. Lower histograms show overlays of the isotype-matched control IgG and anti–DNAM-1 ( left ) or anti-Tactile ( right ) plots. B , EM CD4 + T cells were preincubated with isotype-matched control IgG (control), anti–DNAM-1 (DNAM-1), anti-Tactile (Tactile), and both anti–DNAM-1 and anti-Tactile blocking mAbs (DNAM-1+Tactile) prior to flow TEM. Left panel shows TEM of Vβ2TCR − cells at 15 min. Right panel shows TEM of Vβ2TCR + cells at 60 min. Graphs display data from one representative experiment of two (VB2 − ) and four (VB2 + ) separate experiments using T cells isolated from different donors. ** p
    Figure Legend Snippet: Blocking of T cell DNAM-1 and/or Tactile inhibits TCR-dependent TEM of EM CD4 + T cells. A , Contour plots of FACS analysis of total CD4 + T cells stained with FITC-conjugated IgG (IgG–FITC) and PE-conjugated IgG (IgG–PE, upper left ) or FITC-conjugated anti-CCR7 (CCR7–FITC) and PE-conjugated anti–DNAM-1 (DNAM-1–PE, lower left ) or FITC-conjugated anti-CCR7 and Alexa Fluor 647-complexed IgG (IgG-647, upper right ) or FITC-conjugated anti-CCR7 and Alexa Fluor 647-complexed anti-Tactile (Tactile-647, lower right ). Note the distinct population of cells that are DNAM-1 high, CCR7 low or Tactile high, CCR7 low in the middle , left , and right plots , respectively. Lower histograms show overlays of the isotype-matched control IgG and anti–DNAM-1 ( left ) or anti-Tactile ( right ) plots. B , EM CD4 + T cells were preincubated with isotype-matched control IgG (control), anti–DNAM-1 (DNAM-1), anti-Tactile (Tactile), and both anti–DNAM-1 and anti-Tactile blocking mAbs (DNAM-1+Tactile) prior to flow TEM. Left panel shows TEM of Vβ2TCR − cells at 15 min. Right panel shows TEM of Vβ2TCR + cells at 60 min. Graphs display data from one representative experiment of two (VB2 − ) and four (VB2 + ) separate experiments using T cells isolated from different donors. ** p

    Techniques Used: Blocking Assay, Transmission Electron Microscopy, FACS, Staining, Flow Cytometry, Isolation

    Knockdown of EC CD99 inhibits TCR-dependent TEM of EM CD4 + T cells. CIITA HDMEC were transfected with control siRNA or two different siRNAs targeting CD99 (CD99 siRNA-2 and CD99 siRNA-5), treated with TNF, analyzed by FACS ( A ) or overlaid with TSST-1 superantigen (recognized by those T cells with the germline-encoded Vβ2 segment in the TCR), and used in flow TEM assays with EM CD4 + T cells ( B ). A , FACS plots of HDMECs stained with isotype-matched control IgG (thin lines) or anti-CD99 or -CD31 (thick lines in left panels and right panels , respectively) demonstrating knockdown of CD99 without reduction of CD31 expression. B , TEM assays. Graph on the left (VB2 − ) shows TEM of Vβ2TCR − cells (i.e., those T cells with TCR that are not activated by TSST-1) at 15 min. Graph on the right (VB2 + ) shows TEM of Vβ2TCR + cells at 60 min. Graphs display data from one representative experiment of two (VB2 − ) or three (VB2 + ) separate experiments using T cells from different donors. *** p
    Figure Legend Snippet: Knockdown of EC CD99 inhibits TCR-dependent TEM of EM CD4 + T cells. CIITA HDMEC were transfected with control siRNA or two different siRNAs targeting CD99 (CD99 siRNA-2 and CD99 siRNA-5), treated with TNF, analyzed by FACS ( A ) or overlaid with TSST-1 superantigen (recognized by those T cells with the germline-encoded Vβ2 segment in the TCR), and used in flow TEM assays with EM CD4 + T cells ( B ). A , FACS plots of HDMECs stained with isotype-matched control IgG (thin lines) or anti-CD99 or -CD31 (thick lines in left panels and right panels , respectively) demonstrating knockdown of CD99 without reduction of CD31 expression. B , TEM assays. Graph on the left (VB2 − ) shows TEM of Vβ2TCR − cells (i.e., those T cells with TCR that are not activated by TSST-1) at 15 min. Graph on the right (VB2 + ) shows TEM of Vβ2TCR + cells at 60 min. Graphs display data from one representative experiment of two (VB2 − ) or three (VB2 + ) separate experiments using T cells from different donors. *** p

    Techniques Used: Transmission Electron Microscopy, Transfection, FACS, Flow Cytometry, Staining, Expressing

    40) Product Images from "KIF21A-Mediated Axonal Transport and Selective Endocytosis Underlie the Polarized Targeting of NCKX2"

    Article Title: KIF21A-Mediated Axonal Transport and Selective Endocytosis Underlie the Polarized Targeting of NCKX2

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.6331-11.2012

    Knockdown of KIF21A inhibits the axonal transport of NCKX2. A , B , shRNA-mediated depletion of KIF21A. A , KIF21A-targeting shRNA (shKIF21A) was expressed by pLentiLox3.7 plasmids (pLL) encoding EGFP or mRFP (pLL-GFP-shKIF21A or pLL-RFP-shKIF21A). FLAG-KIF21A was cotransfected with shKIF21A or empty pLL vectors into HEK293 cells. shKIF21A completely depleted FLAG-KIF21A, but the empty pLL did not. B , Endogenous KIF21A was remarkably depleted in cultured hippocampal neurons infected with lentivirus encoding shKIF21A but not in those with lentivirus encoding nontargeting shRNA (NT control). Time-dependent knockdown of endogenous KIF21A is shown in the right bar graph. The noninfected control is shown in the leftmost bar and the NT control is in the rightmost bar. In A and B , β-actin was detected as a loading control. C , D , The axonal transport of endogenous NCKX2 (green) in the shKIF21A-transfected ( D ) or untransfected control ( C ) neurons. KIF21A-depleted neuron was identified by red fluorescence of mRFP coexpressed with shRNA (insets on DIC images). Dendrites were identified by MAP2 immunofluorescence (red). Neurites that are MAP2 negative but clearly seen in the DIC images (indicated by arrowheads) were regarded as axons. E , Analysis of ADR of endogenous NCKX2. Dendritic ROIs (left; red dotted polygons) were drawn on the endogenous NCKX2 immunofluorescence image (same as in C ). After nullifying pixels that overlap the binary mask of MAP2-positive neurites (middle) from the endogenous NCKX2 image, the axonal ROI was set on the NCKX2 image (right; red dotted polygon). F , The mean ADR (black bars) and DSR (gray bars) of endogenous NCKX2 estimated from the untransfected control ( n = 8) or KIF21A-depleted neurons ( n = 6). ADR of NCKX2 in the KIF21A-depleted group is significantly lower than that in the control group. ** p
    Figure Legend Snippet: Knockdown of KIF21A inhibits the axonal transport of NCKX2. A , B , shRNA-mediated depletion of KIF21A. A , KIF21A-targeting shRNA (shKIF21A) was expressed by pLentiLox3.7 plasmids (pLL) encoding EGFP or mRFP (pLL-GFP-shKIF21A or pLL-RFP-shKIF21A). FLAG-KIF21A was cotransfected with shKIF21A or empty pLL vectors into HEK293 cells. shKIF21A completely depleted FLAG-KIF21A, but the empty pLL did not. B , Endogenous KIF21A was remarkably depleted in cultured hippocampal neurons infected with lentivirus encoding shKIF21A but not in those with lentivirus encoding nontargeting shRNA (NT control). Time-dependent knockdown of endogenous KIF21A is shown in the right bar graph. The noninfected control is shown in the leftmost bar and the NT control is in the rightmost bar. In A and B , β-actin was detected as a loading control. C , D , The axonal transport of endogenous NCKX2 (green) in the shKIF21A-transfected ( D ) or untransfected control ( C ) neurons. KIF21A-depleted neuron was identified by red fluorescence of mRFP coexpressed with shRNA (insets on DIC images). Dendrites were identified by MAP2 immunofluorescence (red). Neurites that are MAP2 negative but clearly seen in the DIC images (indicated by arrowheads) were regarded as axons. E , Analysis of ADR of endogenous NCKX2. Dendritic ROIs (left; red dotted polygons) were drawn on the endogenous NCKX2 immunofluorescence image (same as in C ). After nullifying pixels that overlap the binary mask of MAP2-positive neurites (middle) from the endogenous NCKX2 image, the axonal ROI was set on the NCKX2 image (right; red dotted polygon). F , The mean ADR (black bars) and DSR (gray bars) of endogenous NCKX2 estimated from the untransfected control ( n = 8) or KIF21A-depleted neurons ( n = 6). ADR of NCKX2 in the KIF21A-depleted group is significantly lower than that in the control group. ** p

    Techniques Used: shRNA, Cell Culture, Infection, Transfection, Fluorescence, Immunofluorescence

    Immunocytochemical localization of endogenous ( A ) or exogenous ( B ) NCKX2 in cultured hippocampal neurons. Aa , DIV23 hippocampal neurons were stained for endogenous NCKX2 (green) and dendritic marker MAP2 (red). Ab–Ae , Surface NCKX2 (s-NCKX2) was immunolabeled by incubating live cells with anti-NCKX2 ext (green) at 36°C, and then cells were fixed, permeabilized, and immunostained with antibodies against axonal marker Tau-1 (red; Ab , Ac ) or presynaptic marker synaptophysin (red; Ad ) or dendritic maker MAP2 (red; Ae ). Higher magnification image of the dashed box in Ab is shown in Ac . The sites where endogenous s-NCKX2 was colocalized with Tau-1 ( Ac ) or synaptophysin ( Ad ) are marked with arrows. Af , Endogenous s-NCKX2 (green) was immunolabeled by the same manner as in Ae except incubating live cells with anti-NCKX2 ext at 4°C before fixation. Scale bars: Aa , 10 μm; Ab , Ae , Af , 50 μm; Ac , Ad , 5 μm. Ba , A DIV7 hippocampal neuron transfected with NCKX2-GFP and DsRed. Scale bar, 50 μm. Bb , A DIV13 hippocampal neuron transfected with NCKX2-GFP (green). Surface NCKX2-GFP was visualized by live-cell immunolabeling with antibody against GFP (s-NCKX2-GFP; red), and then stained for MAP2 (blue). Scale bar, 10 μm. The open and solid arrowheads indicate dendrites and axons, respectively. The asterisks show location of somata.
    Figure Legend Snippet: Immunocytochemical localization of endogenous ( A ) or exogenous ( B ) NCKX2 in cultured hippocampal neurons. Aa , DIV23 hippocampal neurons were stained for endogenous NCKX2 (green) and dendritic marker MAP2 (red). Ab–Ae , Surface NCKX2 (s-NCKX2) was immunolabeled by incubating live cells with anti-NCKX2 ext (green) at 36°C, and then cells were fixed, permeabilized, and immunostained with antibodies against axonal marker Tau-1 (red; Ab , Ac ) or presynaptic marker synaptophysin (red; Ad ) or dendritic maker MAP2 (red; Ae ). Higher magnification image of the dashed box in Ab is shown in Ac . The sites where endogenous s-NCKX2 was colocalized with Tau-1 ( Ac ) or synaptophysin ( Ad ) are marked with arrows. Af , Endogenous s-NCKX2 (green) was immunolabeled by the same manner as in Ae except incubating live cells with anti-NCKX2 ext at 4°C before fixation. Scale bars: Aa , 10 μm; Ab , Ae , Af , 50 μm; Ac , Ad , 5 μm. Ba , A DIV7 hippocampal neuron transfected with NCKX2-GFP and DsRed. Scale bar, 50 μm. Bb , A DIV13 hippocampal neuron transfected with NCKX2-GFP (green). Surface NCKX2-GFP was visualized by live-cell immunolabeling with antibody against GFP (s-NCKX2-GFP; red), and then stained for MAP2 (blue). Scale bar, 10 μm. The open and solid arrowheads indicate dendrites and axons, respectively. The asterisks show location of somata.

    Techniques Used: Cell Culture, Staining, Marker, Immunolabeling, Transfection

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    Staining:

    Article Title: In Vivo Role of Focal Adhesion Kinase in Regulating Pancreatic ?-Cell Mass and Function Through Insulin Signaling, Actin Dynamics, and Granule Trafficking
    Article Snippet: .. To detect F-actin, cells were fixed with Z-FIX (Anatech Ltd., Battle Creek, MI) and stained with Alexa Fluor 488–conjugated phalloidin (Invitrogen). β-Cells were identified by insulin immunostaining (Santa Cruz Biotechnology). .. Cell images were captured with a Zeiss AxioCamHRm and acquired with AxioVision 4.8 imaging software (Carl Zeiss MicroImaging).

    Article Title: FAK is required for the assembly of podosome rosettes
    Article Snippet: .. The primary antibodies used in immunofluorescent staining in this study were monoclonal antiacetylated tubulin (1:400), polyclonal anticortactin (1:200), polyclonal anti-FAK (1:200), polyclonal anti–Src pY416 (1:400), monoclonal anti-FAK (1:100), monoclonal antipaxillin (1:200), monoclonal antivinculin (1:200), and monoclonal antivimentin (clone V9 [1:400] and clone VIM13.2 [1:200]). rhodamine-conjugated phalloidin and Alexa Fluor 488–conjugated phalloidin (Invitrogen) were used to stain actin filaments. .. Coverslips were mounted in Anti-Fade DAPI-Fluoromount-G (SouthernBiotech) and viewed using a laser-scanning confocal microscope image system (LSM 510; Carl Zeiss) with a 63× Plan-Apochromat (NA 1.2 W Korr; Carl Zeiss) or a 100× Plan-Apochromat objective (NA 1.4 oil; Carl Zeiss).

    Article Title: Jumu is required for circulating hemocyte differentiation and phagocytosis in Drosophila
    Article Snippet: .. For phalloidin staining, hemocytes were preincubated with PBST (PBS with 0.1% TritonX-100) for 5 min and then incubated with Alexa Fluor 488-labeled phalloidin (Thermo Fisher Scientific) diluted in PBS for 30 min. .. Images were obtained using a Zeiss Axioplan 2 microscope equipped with fluorescence optics.

    Article Title: Acinetobacter baumannii invades epithelial cells and outer membrane protein A mediates interactions with epithelial cells
    Article Snippet: .. Cells were permeabilized with a PBS containing 0.25% Triton X-100 for 10 min. Actin was stained with Alexa Fluor® 488 phalloidin (Molecular Probes). .. A. baumannii was labeled with polyclonal anti-rabbit AbOmpA antibody (1:1,000), followed by Alexa Fluor® 568-conjugated goat anti-rabbit IgG antibody (Molecular Probes).

    Article Title: Engineering Escherichia coli into a Protein Delivery System for Mammalian Cells
    Article Snippet: .. Nuclei were stained with DAPI, and actin was stained with Alexa-Fluor 488 phalloidin (Life Technologies). .. Cell Culture Conditions HeLa and 10T1/2 cells were maintained in high-glucose DMEM (Life Technologies) supplemented with 10% FBS (Atlanta Biologics).

    Incubation:

    Article Title: Jumu is required for circulating hemocyte differentiation and phagocytosis in Drosophila
    Article Snippet: .. For phalloidin staining, hemocytes were preincubated with PBST (PBS with 0.1% TritonX-100) for 5 min and then incubated with Alexa Fluor 488-labeled phalloidin (Thermo Fisher Scientific) diluted in PBS for 30 min. .. Images were obtained using a Zeiss Axioplan 2 microscope equipped with fluorescence optics.

    other:

    Article Title: Adenosine A1 Receptors Promote Vasa Vasorum Endothelial Cell Barrier Integrity via Gi and Akt-Dependent Actin Cytoskeleton Remodeling
    Article Snippet: Alexa Fluor 488 Phalloidin (Cat # A12379) was purchased from Invitrogen.

    Immunostaining:

    Article Title: In Vivo Role of Focal Adhesion Kinase in Regulating Pancreatic ?-Cell Mass and Function Through Insulin Signaling, Actin Dynamics, and Granule Trafficking
    Article Snippet: .. To detect F-actin, cells were fixed with Z-FIX (Anatech Ltd., Battle Creek, MI) and stained with Alexa Fluor 488–conjugated phalloidin (Invitrogen). β-Cells were identified by insulin immunostaining (Santa Cruz Biotechnology). .. Cell images were captured with a Zeiss AxioCamHRm and acquired with AxioVision 4.8 imaging software (Carl Zeiss MicroImaging).

    Labeling:

    Article Title: Localizations of visual cycle components in retinal pigment epithelium
    Article Snippet: .. Alexa Fluor® 488-phalloidin (F-phalloidin) and secondary antibodies labeled with Alexa Fluor® 568 (red) or Alexa Fluor® 488 (green) were obtained from Molecular Probes/Invitrogen, Carlsbad, CA. .. Imaging Immunolabeled mouse or rat retina sections were imaged by confocal microscopy using a Zeiss laser scanning multiphoton confocal microscope (Zeiss LSM 510 MPLSM; Carl Zeiss Microimaging, Inc., Thornwood, NY).

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    Thermo Fisher goat anti rabbit igg secondary antibody conjugated to alexa fluor 568
    Goat Anti Rabbit Igg Secondary Antibody Conjugated To Alexa Fluor 568, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher goat anti rabbit igg alexafluor 568 conjugated secondary antibody
    Twinfilin regulates capping protein localization and dynamics. (A) Localization of EGFP-CP in wild-type and twf1/twf2-KO B16-F1 cells, where F-actin was visualized with <t>AlexaFluor-568</t> phalloidin. Panels in the middle and right are magnifications of lamellipodial regions highlighted in the whole cell images in left. Scale bars = 10 μ m. (B) Examples of line profiles generated across the center of lamellipodia as indicated with dotted lines. Data represent mean of 5 measurements of individual lamellipodia, with standard deviations shown. The ‘0 μ m’ value in x-axis is set to correspond the peak intensity of phalloidin. (C) The ratio of CP and F-actin co-localization widths were detected by measuring the width of localization at 50% of maximum intensity. Data points represent measurements from individual lamellipodia with mean values and standard deviations shown. Statistical significance was calculated with Student’s unpaired, two-tailed t-test. ****, p
    Goat Anti Rabbit Igg Alexafluor 568 Conjugated Secondary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/goat anti rabbit igg alexafluor 568 conjugated secondary antibody/product/Thermo Fisher
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    Thermo Fisher goat anti rabbit igg conjugated to alexa fluor 568
    Twinfilin regulates capping protein localization and dynamics. (A) Localization of EGFP-CP in wild-type and twf1/twf2-KO B16-F1 cells, where F-actin was visualized with <t>AlexaFluor-568</t> phalloidin. Panels in the middle and right are magnifications of lamellipodial regions highlighted in the whole cell images in left. Scale bars = 10 μ m. (B) Examples of line profiles generated across the center of lamellipodia as indicated with dotted lines. Data represent mean of 5 measurements of individual lamellipodia, with standard deviations shown. The ‘0 μ m’ value in x-axis is set to correspond the peak intensity of phalloidin. (C) The ratio of CP and F-actin co-localization widths were detected by measuring the width of localization at 50% of maximum intensity. Data points represent measurements from individual lamellipodia with mean values and standard deviations shown. Statistical significance was calculated with Student’s unpaired, two-tailed t-test. ****, p
    Goat Anti Rabbit Igg Conjugated To Alexa Fluor 568, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/goat anti rabbit igg conjugated to alexa fluor 568/product/Thermo Fisher
    Average 93 stars, based on 19 article reviews
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    Image Search Results


    Twinfilin regulates capping protein localization and dynamics. (A) Localization of EGFP-CP in wild-type and twf1/twf2-KO B16-F1 cells, where F-actin was visualized with AlexaFluor-568 phalloidin. Panels in the middle and right are magnifications of lamellipodial regions highlighted in the whole cell images in left. Scale bars = 10 μ m. (B) Examples of line profiles generated across the center of lamellipodia as indicated with dotted lines. Data represent mean of 5 measurements of individual lamellipodia, with standard deviations shown. The ‘0 μ m’ value in x-axis is set to correspond the peak intensity of phalloidin. (C) The ratio of CP and F-actin co-localization widths were detected by measuring the width of localization at 50% of maximum intensity. Data points represent measurements from individual lamellipodia with mean values and standard deviations shown. Statistical significance was calculated with Student’s unpaired, two-tailed t-test. ****, p

    Journal: bioRxiv

    Article Title: Twinfilin uncaps filament barbed ends to promote turnover of lamellipodial actin networks

    doi: 10.1101/864769

    Figure Lengend Snippet: Twinfilin regulates capping protein localization and dynamics. (A) Localization of EGFP-CP in wild-type and twf1/twf2-KO B16-F1 cells, where F-actin was visualized with AlexaFluor-568 phalloidin. Panels in the middle and right are magnifications of lamellipodial regions highlighted in the whole cell images in left. Scale bars = 10 μ m. (B) Examples of line profiles generated across the center of lamellipodia as indicated with dotted lines. Data represent mean of 5 measurements of individual lamellipodia, with standard deviations shown. The ‘0 μ m’ value in x-axis is set to correspond the peak intensity of phalloidin. (C) The ratio of CP and F-actin co-localization widths were detected by measuring the width of localization at 50% of maximum intensity. Data points represent measurements from individual lamellipodia with mean values and standard deviations shown. Statistical significance was calculated with Student’s unpaired, two-tailed t-test. ****, p

    Article Snippet: Other antibodies used in the study were: Rabbit anti-twinfilin-2 antibody (Sigma-Aldrich #HPA053874, WB, 1:100), rabbit anti-CAPZß antibody (Sigma-Aldrich, #HPA031531, WB, 1:100), mouse anti-α-tubulin antibody (Sigma-Aldrich, #T5168, WB 1:10,000), mouse anti-ß-actin antibody (Sigma-Aldrich, #A5441, WB, 1:10,000), Rabbit anti-p34-Arc/ARPC2 (Merck Millipore, #07-227, dilution in immunofluorescence (IF), 1:200), goat anti-Rabbit IgG AlexaFluor-488 conjugated secondary antibody (Thermo Fisher, #A-11034, IF, 1:400), goat anti-Rabbit IgG AlexaFluor-568 conjugated secondary antibody (Thermo Fisher, #A-11011, IF, 1:400), goat anti-Rabbit IgG AlexaFluor-647 conjugated secondary antibody (Thermo Fisher, #A-32733, IF, 1:400), goat anti-Mouse IgG HRP conjugated secondary antibody (Thermo Fisher, #31430, WB, 1:10,000), goat anti-Rabbit IgG HRP conjugated secondary antibody (Thermo Fisher, #32460, WB, 1:1,000).

    Techniques: Generated, Two Tailed Test

    Examples of high-content and lamellipodia protrusion analysis. (A) Representative images of segmentation procedure in high-content image analysis. Wild-type and twinfilin-deficient B16-F1 cells were stained with DAPI and CellMask Deep Red to segment nuclei (outlined with blue) and cytoplasm (outlined with yellow). The Arp2/3-complex positive structures were detected with anti-p34 antibody staining and used as a mask for the Arp2/3-complex positive F-actin structures (the most-right panel). F-actin was stained with AlexaFluor-568 phalloidin. Cells touching the border of images were excluded from analysis. (B) A representative example of twf1/twf2-KO cell migrating on laminin coated glass imaged with DIC. Direction of migration is indicated with an arrow and kymographs were generated with line drawn across the lamellipodium as indicated with dotted red line. (C) Representative examples of kymographs generated from DIC time-lapse images of wild-type, twf1/twf2 knockout, and EGFP-TWF-1 rescue cells. Protrusions velocities were measured from the overall cell front protrusion as indicated with dotted red lines.

    Journal: bioRxiv

    Article Title: Twinfilin uncaps filament barbed ends to promote turnover of lamellipodial actin networks

    doi: 10.1101/864769

    Figure Lengend Snippet: Examples of high-content and lamellipodia protrusion analysis. (A) Representative images of segmentation procedure in high-content image analysis. Wild-type and twinfilin-deficient B16-F1 cells were stained with DAPI and CellMask Deep Red to segment nuclei (outlined with blue) and cytoplasm (outlined with yellow). The Arp2/3-complex positive structures were detected with anti-p34 antibody staining and used as a mask for the Arp2/3-complex positive F-actin structures (the most-right panel). F-actin was stained with AlexaFluor-568 phalloidin. Cells touching the border of images were excluded from analysis. (B) A representative example of twf1/twf2-KO cell migrating on laminin coated glass imaged with DIC. Direction of migration is indicated with an arrow and kymographs were generated with line drawn across the lamellipodium as indicated with dotted red line. (C) Representative examples of kymographs generated from DIC time-lapse images of wild-type, twf1/twf2 knockout, and EGFP-TWF-1 rescue cells. Protrusions velocities were measured from the overall cell front protrusion as indicated with dotted red lines.

    Article Snippet: Other antibodies used in the study were: Rabbit anti-twinfilin-2 antibody (Sigma-Aldrich #HPA053874, WB, 1:100), rabbit anti-CAPZß antibody (Sigma-Aldrich, #HPA031531, WB, 1:100), mouse anti-α-tubulin antibody (Sigma-Aldrich, #T5168, WB 1:10,000), mouse anti-ß-actin antibody (Sigma-Aldrich, #A5441, WB, 1:10,000), Rabbit anti-p34-Arc/ARPC2 (Merck Millipore, #07-227, dilution in immunofluorescence (IF), 1:200), goat anti-Rabbit IgG AlexaFluor-488 conjugated secondary antibody (Thermo Fisher, #A-11034, IF, 1:400), goat anti-Rabbit IgG AlexaFluor-568 conjugated secondary antibody (Thermo Fisher, #A-11011, IF, 1:400), goat anti-Rabbit IgG AlexaFluor-647 conjugated secondary antibody (Thermo Fisher, #A-32733, IF, 1:400), goat anti-Mouse IgG HRP conjugated secondary antibody (Thermo Fisher, #31430, WB, 1:10,000), goat anti-Rabbit IgG HRP conjugated secondary antibody (Thermo Fisher, #32460, WB, 1:1,000).

    Techniques: Staining, Migration, Generated, Knock-Out

    Representative images of wild-type and twinfilin knockout B16-F1 cells. (A) B16-F1 cells were stained with AlexaFluor-568 phalloidin (F-actin) and anti-p34 antibody (the Arp2/3 complex). Scale bars = 10 μ M. (B) Mean F-actin intensity in B16-F1 wild-type and twinfilin knockout cells. Number of measured cells were: B16-F1 wt = 1,958, twf1-KO-g1 = 1,045, twf2-KO-g3#1 = 1,285, twf1/2-KO-g3 = 1,265, twf1/2-KO-g4 = 1,707. (C) Mean F-actin intensity in the Arp2/3 complex positive regions of B16-F1 wild-type and knockout cells. The Arp2/3 complex positive regions were identified based on p34-antibody staining. Number of measured cells were: B16-F1 wt = 1,658, twf1-KO-g1 = 884, twf2-KO-g3#1 = 1,009, twf1/2-KO-g3 = 1,100, twf1/2-KO-g4 = 1157. Statistical significances in panels B and C were calculated with Mann-Whitney two-tailed test. ****, p

    Journal: bioRxiv

    Article Title: Twinfilin uncaps filament barbed ends to promote turnover of lamellipodial actin networks

    doi: 10.1101/864769

    Figure Lengend Snippet: Representative images of wild-type and twinfilin knockout B16-F1 cells. (A) B16-F1 cells were stained with AlexaFluor-568 phalloidin (F-actin) and anti-p34 antibody (the Arp2/3 complex). Scale bars = 10 μ M. (B) Mean F-actin intensity in B16-F1 wild-type and twinfilin knockout cells. Number of measured cells were: B16-F1 wt = 1,958, twf1-KO-g1 = 1,045, twf2-KO-g3#1 = 1,285, twf1/2-KO-g3 = 1,265, twf1/2-KO-g4 = 1,707. (C) Mean F-actin intensity in the Arp2/3 complex positive regions of B16-F1 wild-type and knockout cells. The Arp2/3 complex positive regions were identified based on p34-antibody staining. Number of measured cells were: B16-F1 wt = 1,658, twf1-KO-g1 = 884, twf2-KO-g3#1 = 1,009, twf1/2-KO-g3 = 1,100, twf1/2-KO-g4 = 1157. Statistical significances in panels B and C were calculated with Mann-Whitney two-tailed test. ****, p

    Article Snippet: Other antibodies used in the study were: Rabbit anti-twinfilin-2 antibody (Sigma-Aldrich #HPA053874, WB, 1:100), rabbit anti-CAPZß antibody (Sigma-Aldrich, #HPA031531, WB, 1:100), mouse anti-α-tubulin antibody (Sigma-Aldrich, #T5168, WB 1:10,000), mouse anti-ß-actin antibody (Sigma-Aldrich, #A5441, WB, 1:10,000), Rabbit anti-p34-Arc/ARPC2 (Merck Millipore, #07-227, dilution in immunofluorescence (IF), 1:200), goat anti-Rabbit IgG AlexaFluor-488 conjugated secondary antibody (Thermo Fisher, #A-11034, IF, 1:400), goat anti-Rabbit IgG AlexaFluor-568 conjugated secondary antibody (Thermo Fisher, #A-11011, IF, 1:400), goat anti-Rabbit IgG AlexaFluor-647 conjugated secondary antibody (Thermo Fisher, #A-32733, IF, 1:400), goat anti-Mouse IgG HRP conjugated secondary antibody (Thermo Fisher, #31430, WB, 1:10,000), goat anti-Rabbit IgG HRP conjugated secondary antibody (Thermo Fisher, #32460, WB, 1:1,000).

    Techniques: Knock-Out, Staining, MANN-WHITNEY, Two Tailed Test

    Knockout of twinfilins leads to abnormal F-actin accumulation in lamellipodia and perinuclear region. (A) Representative images of wild-type and twf1/twf2-KO mouse B16-F1 cells stained with AlexaFluor-568 phalloidin and anti-p34 antibody to visualize F-actin and the Arp2/3 complex, respectively. Scale bar = 10 μ m. (B) F-actin intensities in the cytoplasmic regions of wild-type, twf1/twf2-KO, and knockout cells expressing EGFP-TWF-1 measured by high-content image analysis. Number of cells analyzed were: B16-F1 wt = 4,875, twf1/twf2-KO-g3 = 5,731, twf1/twf2-KO-g3 + EGFP-TWF-1 = 197. (C) Lamellipodia protrusion velocities of wild-type, twf1/twf-2 knockout, and knockout cells expressing EGFP-TWF-1. Data represent individual cells with mean and standard deviations shown. Statistical significances in panels B and D were calculated with Mann-Whitney two-tailed test. ****, p

    Journal: bioRxiv

    Article Title: Twinfilin uncaps filament barbed ends to promote turnover of lamellipodial actin networks

    doi: 10.1101/864769

    Figure Lengend Snippet: Knockout of twinfilins leads to abnormal F-actin accumulation in lamellipodia and perinuclear region. (A) Representative images of wild-type and twf1/twf2-KO mouse B16-F1 cells stained with AlexaFluor-568 phalloidin and anti-p34 antibody to visualize F-actin and the Arp2/3 complex, respectively. Scale bar = 10 μ m. (B) F-actin intensities in the cytoplasmic regions of wild-type, twf1/twf2-KO, and knockout cells expressing EGFP-TWF-1 measured by high-content image analysis. Number of cells analyzed were: B16-F1 wt = 4,875, twf1/twf2-KO-g3 = 5,731, twf1/twf2-KO-g3 + EGFP-TWF-1 = 197. (C) Lamellipodia protrusion velocities of wild-type, twf1/twf-2 knockout, and knockout cells expressing EGFP-TWF-1. Data represent individual cells with mean and standard deviations shown. Statistical significances in panels B and D were calculated with Mann-Whitney two-tailed test. ****, p

    Article Snippet: Other antibodies used in the study were: Rabbit anti-twinfilin-2 antibody (Sigma-Aldrich #HPA053874, WB, 1:100), rabbit anti-CAPZß antibody (Sigma-Aldrich, #HPA031531, WB, 1:100), mouse anti-α-tubulin antibody (Sigma-Aldrich, #T5168, WB 1:10,000), mouse anti-ß-actin antibody (Sigma-Aldrich, #A5441, WB, 1:10,000), Rabbit anti-p34-Arc/ARPC2 (Merck Millipore, #07-227, dilution in immunofluorescence (IF), 1:200), goat anti-Rabbit IgG AlexaFluor-488 conjugated secondary antibody (Thermo Fisher, #A-11034, IF, 1:400), goat anti-Rabbit IgG AlexaFluor-568 conjugated secondary antibody (Thermo Fisher, #A-11011, IF, 1:400), goat anti-Rabbit IgG AlexaFluor-647 conjugated secondary antibody (Thermo Fisher, #A-32733, IF, 1:400), goat anti-Mouse IgG HRP conjugated secondary antibody (Thermo Fisher, #31430, WB, 1:10,000), goat anti-Rabbit IgG HRP conjugated secondary antibody (Thermo Fisher, #32460, WB, 1:1,000).

    Techniques: Knock-Out, Staining, Expressing, MANN-WHITNEY, Two Tailed Test