mouse anti β1  (Thermo Fisher)


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    Structured Review

    Thermo Fisher mouse anti β1
    Alteration of N -glycosylation in GOLPH3-knockdown cells. A, total expression levels of <t>β1</t> integrin were analyzed by Western blotting ( WB ). The same amounts of cell lysates (200 μg) were obtained from control and KD cells, which were picked up from HeLa cells expressed as shRNA for control ( Ctrl ) and GOLPH3 (KD1,2) using the Phoenix system. Cell lysates were immunoprecipitated ( IP ) with anti-β1 antibody. The immunoprecipitates of β1 integrin were treated with (+) or without peptide: N -glycosidase F ( PNGase ) (−), and then immunoblotted with anti-β1 antibody ( upper panel ). The knockdown efficiency of GOLPH3 was confirmed by immunoblotting with anti-GOLPH3 antibody ( middle panel ). The α-tubulin was used as a loading control to warrant the same amounts of proteins to be used ( lower panel ). B, analysis of PA- N -glycans was by reversed-phase HPLC. Then N -glycans released from control or KD cells with peptide: N -glycosidase F were pyridylaminated as described under “Experimental Procedures.” The PA- N -glycans ( upper panel ), sequentially digested with sialidase ( middle panel ) and β-galactosidase ( lower panel ), were subjected to reversed phase HPLC. The asterisk indicates the peaks for sialylated N -glycans. C, RT-PCR for mRNA expression of several sialyltransferases and sialidase as indicated. The β-actin was used as a loading control. Ctrl, control shRNA; KD , shRNA for GOLPH3 -knockdown; β 1, integrin β1.
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    Images

    1) Product Images from "An Oncogenic Protein Golgi Phosphoprotein 3 Up-regulates Cell Migration via Sialylation *"

    Article Title: An Oncogenic Protein Golgi Phosphoprotein 3 Up-regulates Cell Migration via Sialylation *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.542688

    Alteration of N -glycosylation in GOLPH3-knockdown cells. A, total expression levels of β1 integrin were analyzed by Western blotting ( WB ). The same amounts of cell lysates (200 μg) were obtained from control and KD cells, which were picked up from HeLa cells expressed as shRNA for control ( Ctrl ) and GOLPH3 (KD1,2) using the Phoenix system. Cell lysates were immunoprecipitated ( IP ) with anti-β1 antibody. The immunoprecipitates of β1 integrin were treated with (+) or without peptide: N -glycosidase F ( PNGase ) (−), and then immunoblotted with anti-β1 antibody ( upper panel ). The knockdown efficiency of GOLPH3 was confirmed by immunoblotting with anti-GOLPH3 antibody ( middle panel ). The α-tubulin was used as a loading control to warrant the same amounts of proteins to be used ( lower panel ). B, analysis of PA- N -glycans was by reversed-phase HPLC. Then N -glycans released from control or KD cells with peptide: N -glycosidase F were pyridylaminated as described under “Experimental Procedures.” The PA- N -glycans ( upper panel ), sequentially digested with sialidase ( middle panel ) and β-galactosidase ( lower panel ), were subjected to reversed phase HPLC. The asterisk indicates the peaks for sialylated N -glycans. C, RT-PCR for mRNA expression of several sialyltransferases and sialidase as indicated. The β-actin was used as a loading control. Ctrl, control shRNA; KD , shRNA for GOLPH3 -knockdown; β 1, integrin β1.
    Figure Legend Snippet: Alteration of N -glycosylation in GOLPH3-knockdown cells. A, total expression levels of β1 integrin were analyzed by Western blotting ( WB ). The same amounts of cell lysates (200 μg) were obtained from control and KD cells, which were picked up from HeLa cells expressed as shRNA for control ( Ctrl ) and GOLPH3 (KD1,2) using the Phoenix system. Cell lysates were immunoprecipitated ( IP ) with anti-β1 antibody. The immunoprecipitates of β1 integrin were treated with (+) or without peptide: N -glycosidase F ( PNGase ) (−), and then immunoblotted with anti-β1 antibody ( upper panel ). The knockdown efficiency of GOLPH3 was confirmed by immunoblotting with anti-GOLPH3 antibody ( middle panel ). The α-tubulin was used as a loading control to warrant the same amounts of proteins to be used ( lower panel ). B, analysis of PA- N -glycans was by reversed-phase HPLC. Then N -glycans released from control or KD cells with peptide: N -glycosidase F were pyridylaminated as described under “Experimental Procedures.” The PA- N -glycans ( upper panel ), sequentially digested with sialidase ( middle panel ) and β-galactosidase ( lower panel ), were subjected to reversed phase HPLC. The asterisk indicates the peaks for sialylated N -glycans. C, RT-PCR for mRNA expression of several sialyltransferases and sialidase as indicated. The β-actin was used as a loading control. Ctrl, control shRNA; KD , shRNA for GOLPH3 -knockdown; β 1, integrin β1.

    Techniques Used: Expressing, Western Blot, shRNA, Immunoprecipitation, High Performance Liquid Chromatography, Reverse Transcription Polymerase Chain Reaction

    GOLPH3 was associated with sialyltransferases through the cytoplasmic domain of sialyltransferase. A, schematic diagram of sialyltransferases and chimeric constructs. GOLPH3 and the chimera of ST3GAL4 and β4GALT1 ( B ) or the chimera of ST6GAL1 and β4GALT1 ( C ) were transiently expressed in 293T cells. The cell lysates were immunoprecipitated ( IP ) with anti-FLAG and immunoblotted with anti-HA or anti-FLAG antibody. D, WT or GOLPH3 mutants (R171A/R174A, W81A/R90A, Δ190–201) shRNA-resistant in a Tet-inducible expression system were introduced into HeLa cells that expressed the Tet-inducible shRNA GOLPH3 -292 (KD), as described under “Experimental Procedures.” Cells were treated with 1 μg/ml of doxycycline for 72 h, lysed, and immunoprecipitated with SSA-agarose and immunoblotted with anti-β1 integrin. E, to examine the effects of GOLPH3 knockdown on localization of ST6GAL1, those ST6GAL1-GFP cells expressed with the doxycycline ( DOX )-inducible GOLPH3 knockdown system were cultured for 72 h in the presence (KD) or absence ( Ctrl ) of DOX. Cells were stained with anti-GM130 primary antibody, TO-PRO-3, and fluorescent secondary antibodies. The cells were analyzed using an Olympus fluorescence microscope with 60×/1.35 NA oil immersion objective lens (FV1000 system). Scale bar, 10 μm.
    Figure Legend Snippet: GOLPH3 was associated with sialyltransferases through the cytoplasmic domain of sialyltransferase. A, schematic diagram of sialyltransferases and chimeric constructs. GOLPH3 and the chimera of ST3GAL4 and β4GALT1 ( B ) or the chimera of ST6GAL1 and β4GALT1 ( C ) were transiently expressed in 293T cells. The cell lysates were immunoprecipitated ( IP ) with anti-FLAG and immunoblotted with anti-HA or anti-FLAG antibody. D, WT or GOLPH3 mutants (R171A/R174A, W81A/R90A, Δ190–201) shRNA-resistant in a Tet-inducible expression system were introduced into HeLa cells that expressed the Tet-inducible shRNA GOLPH3 -292 (KD), as described under “Experimental Procedures.” Cells were treated with 1 μg/ml of doxycycline for 72 h, lysed, and immunoprecipitated with SSA-agarose and immunoblotted with anti-β1 integrin. E, to examine the effects of GOLPH3 knockdown on localization of ST6GAL1, those ST6GAL1-GFP cells expressed with the doxycycline ( DOX )-inducible GOLPH3 knockdown system were cultured for 72 h in the presence (KD) or absence ( Ctrl ) of DOX. Cells were stained with anti-GM130 primary antibody, TO-PRO-3, and fluorescent secondary antibodies. The cells were analyzed using an Olympus fluorescence microscope with 60×/1.35 NA oil immersion objective lens (FV1000 system). Scale bar, 10 μm.

    Techniques Used: Construct, Immunoprecipitation, shRNA, Expressing, Cell Culture, Staining, Fluorescence, Microscopy

    2) Product Images from "Role of macrophage sialoadhesin in host defense against the sialylated pathogen group B Streptococcus"

    Article Title: Role of macrophage sialoadhesin in host defense against the sialylated pathogen group B Streptococcus

    Journal: Journal of Molecular Medicine (Berlin, Germany)

    doi: 10.1007/s00109-014-1157-y

    Sn facilitates marginal metallophilic macrophage trapping of GBS to limit pathogen dissemination. WT and Sn-deficient mice were intravenously injected with 5-(and-6)-carboxyfluorescein labeled GBS, and kidney, lung, and spleen were collected 1 h post-infection. Sections were stained with mAb anti-mouse Sn, F4/80, and B220. Representative images of spleen sections are shown in a and b . Scale bar is 100 μm for all panels except 20 μm for the right panel of a . Comparison of bacterial counts (expressed in CFU) recovered from the kidney ( c ), lung ( d ), and spleen ( e ) of WT mice and Sn-deficient mice 1 h post-infection. Difference between two groups was calculated by unpaired t test. Representative data ( n = 3–4 mice each group) were shown from two independent experiments
    Figure Legend Snippet: Sn facilitates marginal metallophilic macrophage trapping of GBS to limit pathogen dissemination. WT and Sn-deficient mice were intravenously injected with 5-(and-6)-carboxyfluorescein labeled GBS, and kidney, lung, and spleen were collected 1 h post-infection. Sections were stained with mAb anti-mouse Sn, F4/80, and B220. Representative images of spleen sections are shown in a and b . Scale bar is 100 μm for all panels except 20 μm for the right panel of a . Comparison of bacterial counts (expressed in CFU) recovered from the kidney ( c ), lung ( d ), and spleen ( e ) of WT mice and Sn-deficient mice 1 h post-infection. Difference between two groups was calculated by unpaired t test. Representative data ( n = 3–4 mice each group) were shown from two independent experiments

    Techniques Used: Mouse Assay, Injection, Labeling, Infection, Staining

    3) Product Images from "The Use of Spinning-Disk Confocal Microscopy for the Intravital Analysis of Platelet Dynamics in Response to Systemic and Local Inflammation"

    Article Title: The Use of Spinning-Disk Confocal Microscopy for the Intravital Analysis of Platelet Dynamics in Response to Systemic and Local Inflammation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0025109

    Quantification of platelet aggregation in the livers of mice treated with intravenous LPS using PE-conjugated anti-CD49b. Intravital microscopy of livers from untreated mice ( Ai–Aiv ), LPS treated mice ( Bi–Biv ) and mice pre-treated with an anti-CD18 blocking mAb followed by LPS ( Ci–Civ ). Representative fields of view at low power (10× objective) magnification ( Ai, Bi, Ci ) illustrating platelet aggregation in response to LPS and inhibition of aggregation following pre-treatment with an anti-CD18 blocking Ab. Platelets labelled with PE-conjugated anti-CD49b (red); neutrophils labelled with Alexa Fluor 647-conjugated anti-Gr-1 (blue). Arrows denote large platelet aggregates. Higher power (20× objective) extended focus images ( Aii, Bii, Cii ) and 3D opacity models ( Aiii, Biii, Ciii ) rendered from 29–30 z planes illustrating the extent of platelet aggregation in LPS treated mice. All scale bars, 20 µm; grid 25.7 µm. Select models of neutrophil-platelet aggregates ( Aiv, Biv, Civ ) extracted (as indicated by the white arrows) and enlarged from panel (iii). ( Di, Dii ) Quantification of the number of platelet aggregates equal to, or larger than the indicated sizes. *** p
    Figure Legend Snippet: Quantification of platelet aggregation in the livers of mice treated with intravenous LPS using PE-conjugated anti-CD49b. Intravital microscopy of livers from untreated mice ( Ai–Aiv ), LPS treated mice ( Bi–Biv ) and mice pre-treated with an anti-CD18 blocking mAb followed by LPS ( Ci–Civ ). Representative fields of view at low power (10× objective) magnification ( Ai, Bi, Ci ) illustrating platelet aggregation in response to LPS and inhibition of aggregation following pre-treatment with an anti-CD18 blocking Ab. Platelets labelled with PE-conjugated anti-CD49b (red); neutrophils labelled with Alexa Fluor 647-conjugated anti-Gr-1 (blue). Arrows denote large platelet aggregates. Higher power (20× objective) extended focus images ( Aii, Bii, Cii ) and 3D opacity models ( Aiii, Biii, Ciii ) rendered from 29–30 z planes illustrating the extent of platelet aggregation in LPS treated mice. All scale bars, 20 µm; grid 25.7 µm. Select models of neutrophil-platelet aggregates ( Aiv, Biv, Civ ) extracted (as indicated by the white arrows) and enlarged from panel (iii). ( Di, Dii ) Quantification of the number of platelet aggregates equal to, or larger than the indicated sizes. *** p

    Techniques Used: Mouse Assay, Intravital Microscopy, Blocking Assay, Inhibition

    4) Product Images from "Cyclin-Dependent Kinase Activity Controls the Onset of the HCMV Lytic Cycle"

    Article Title: Cyclin-Dependent Kinase Activity Controls the Onset of the HCMV Lytic Cycle

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1001096

    CDK inhibition relieves the block of MIE gene expression in undifferentiated NT2 cells. (A) Experimental setup. NT2 cells were infected with HCMV-TB40/e (MOI = 5) in the presence of the cyclin-dependent kinase (CDK) inhibitors roscovitine (Rosco), CVT313, SU9515, the histone deacetylase inhibitor trichostatine A (TSA) or solvent (DMSO). CDK inhibitors were used at concentrations from 10 to 50 µM (Rosco, CVT313) and from 2 to 10 µM (SU9516) as indicated. The different compounds were left on the cells for the indicated lengths of time. In addition, NT2 cells were infected after a 7-day exposure to retinoic acid (RA). All cells were harvested at 24 hpi. (B) Cells were stained for IE1/2 and Oct3/4 expression and analyzed by flow cytometry. Shown are dot plots where cells were divided into four subpopulations: MIE − Oct − (lower left quadrant), MIE + Oct − (upper left quadrant), MIE + Oct + (upper right quadrant), MIE − Oct + (lower right quadrant). The proportion of each subpopulation is given as percent of total cells. (C) Cells were stained for IE1/2 expression and DNA content and analyzed by flow cytometry. The depicted cells were divided into the following subpopulations: MIE − G1 (lower left quadrant), MIE + G1 (upper left quadrant), MIE + S/G2/M (upper right quadrant) and MIE - S/G2/M (lower right quadrant) cells. The proportion of each subpopulation is given in percent of total cells.
    Figure Legend Snippet: CDK inhibition relieves the block of MIE gene expression in undifferentiated NT2 cells. (A) Experimental setup. NT2 cells were infected with HCMV-TB40/e (MOI = 5) in the presence of the cyclin-dependent kinase (CDK) inhibitors roscovitine (Rosco), CVT313, SU9515, the histone deacetylase inhibitor trichostatine A (TSA) or solvent (DMSO). CDK inhibitors were used at concentrations from 10 to 50 µM (Rosco, CVT313) and from 2 to 10 µM (SU9516) as indicated. The different compounds were left on the cells for the indicated lengths of time. In addition, NT2 cells were infected after a 7-day exposure to retinoic acid (RA). All cells were harvested at 24 hpi. (B) Cells were stained for IE1/2 and Oct3/4 expression and analyzed by flow cytometry. Shown are dot plots where cells were divided into four subpopulations: MIE − Oct − (lower left quadrant), MIE + Oct − (upper left quadrant), MIE + Oct + (upper right quadrant), MIE − Oct + (lower right quadrant). The proportion of each subpopulation is given as percent of total cells. (C) Cells were stained for IE1/2 expression and DNA content and analyzed by flow cytometry. The depicted cells were divided into the following subpopulations: MIE − G1 (lower left quadrant), MIE + G1 (upper left quadrant), MIE + S/G2/M (upper right quadrant) and MIE - S/G2/M (lower right quadrant) cells. The proportion of each subpopulation is given in percent of total cells.

    Techniques Used: Inhibition, Blocking Assay, Expressing, Infection, Histone Deacetylase Assay, Staining, Flow Cytometry, Cytometry

    Roscovitine treatment efficiently abrogates the block of MIE gene expression in S/G2. (A) Experimental setup of B. Proliferating HEL fibroblasts were infected with HCMV-AD169. Roscovitine, at the indicated concentrations, was added to the cells at the time of infection. It was left on the cells until harvest at 4 hpi. (C) Experimental setup of D. This time, roscovitine treatment was started 30 min before the fibroblasts were infected with HCMV. Treatment was stopped at 2 hpi and infected cells were maintained for further 3 h in regular growth medium. (B, D) MIE gene expression and DNA content of roscovitine treated cells were analyzed by flow cytometry. DMSO treated cells were also analyzed to control for solvent effects. (E, F) Proliferating U373 and HUVEC cells were incubated with EdU for 60 min to label cells undergoing DNA synthesis. After removal of EdU, cells were infected with HCMV and harvested 5 h later. Where indicated, roscovitine was transiently added to the cells according to the schedule described in C. After harvest, cells were analyzed for EdU incorporation and IE1/2-expression by flow cytometry, as detailed in the legend to figure 1 .
    Figure Legend Snippet: Roscovitine treatment efficiently abrogates the block of MIE gene expression in S/G2. (A) Experimental setup of B. Proliferating HEL fibroblasts were infected with HCMV-AD169. Roscovitine, at the indicated concentrations, was added to the cells at the time of infection. It was left on the cells until harvest at 4 hpi. (C) Experimental setup of D. This time, roscovitine treatment was started 30 min before the fibroblasts were infected with HCMV. Treatment was stopped at 2 hpi and infected cells were maintained for further 3 h in regular growth medium. (B, D) MIE gene expression and DNA content of roscovitine treated cells were analyzed by flow cytometry. DMSO treated cells were also analyzed to control for solvent effects. (E, F) Proliferating U373 and HUVEC cells were incubated with EdU for 60 min to label cells undergoing DNA synthesis. After removal of EdU, cells were infected with HCMV and harvested 5 h later. Where indicated, roscovitine was transiently added to the cells according to the schedule described in C. After harvest, cells were analyzed for EdU incorporation and IE1/2-expression by flow cytometry, as detailed in the legend to figure 1 .

    Techniques Used: Blocking Assay, Expressing, Infection, Flow Cytometry, Cytometry, Incubation, DNA Synthesis

    5) Product Images from "Quantification and phenotypic characterisation of peripheral IFN-γ producing leucocytes in chickens vaccinated against Newcastle disease"

    Article Title: Quantification and phenotypic characterisation of peripheral IFN-γ producing leucocytes in chickens vaccinated against Newcastle disease

    Journal: Veterinary Immunology and Immunopathology

    doi: 10.1016/j.vetimm.2017.10.001

    Intracellular staining of IFN-γ in transfected CHO cells. A) CHO cells were either transfected with plasmids containing the chicken IFN-γ gene or empty plasmids (mock controls). Cells (day 22 after transfection) were cultured with or without BFA for 18 hours and subsequently stained intracellularly using monoclonal anti-chicken IFN-γ antibody (Mab80) conjugated with APC. Representative samples are show with frequency of IFN-γ+ cells indicated above gate. B) Comparison of transfected CHO cells (day 30 after transfection) cultured with 10 μg/ml BFA for 18 hours and subsequently stained intracellularly with either commercial ELISA capture antibody (5C.123.08) A647 conjugated secondary anti-mouse IgG1 or monoclonal anti-chicken IFN-γ antibody directly conjugated with APC (Mab80) or commercial ELISA detection antibody (5C.123.02) directly conjugated with APC or polyclonal rabbit anti-chicken IFN-γ antibody FITC-conjugated secondary goat anti-rabbit IgG. Representative samples are show with frequency of IFN-γ+ cells indicated above gate.
    Figure Legend Snippet: Intracellular staining of IFN-γ in transfected CHO cells. A) CHO cells were either transfected with plasmids containing the chicken IFN-γ gene or empty plasmids (mock controls). Cells (day 22 after transfection) were cultured with or without BFA for 18 hours and subsequently stained intracellularly using monoclonal anti-chicken IFN-γ antibody (Mab80) conjugated with APC. Representative samples are show with frequency of IFN-γ+ cells indicated above gate. B) Comparison of transfected CHO cells (day 30 after transfection) cultured with 10 μg/ml BFA for 18 hours and subsequently stained intracellularly with either commercial ELISA capture antibody (5C.123.08) A647 conjugated secondary anti-mouse IgG1 or monoclonal anti-chicken IFN-γ antibody directly conjugated with APC (Mab80) or commercial ELISA detection antibody (5C.123.02) directly conjugated with APC or polyclonal rabbit anti-chicken IFN-γ antibody FITC-conjugated secondary goat anti-rabbit IgG. Representative samples are show with frequency of IFN-γ+ cells indicated above gate.

    Techniques Used: Staining, Transfection, Cell Culture, Enzyme-linked Immunosorbent Assay

    6) Product Images from "Targeting miR‐34a/Pdgfra interactions partially corrects alveologenesis in experimental bronchopulmonary dysplasia"

    Article Title: Targeting miR‐34a/Pdgfra interactions partially corrects alveologenesis in experimental bronchopulmonary dysplasia

    Journal: EMBO Molecular Medicine

    doi: 10.15252/emmm.201809448

    The primary cell type in the normally and aberrantly developing septa are type I alveolar epithelial cells The impact of administration of scrambled antimiR (S) or an antimiR directed against miR‐34a (A34a) on the abundance of type I alveolar epithelial cells (marked by aquaporin 5, Aqp5) and type II alveolar epithelial cells (marked by pro‐surfactant protein C, Sftpc) was assessed in 3‐μm sections of paraffin‐embedded lung tissue from P5 mice undergoing normal (21% O 2 ) or aberrant (85% O 2 ) lung alveolarization. DAPI, 4′,6‐diamidino‐2‐phenylindole. In the DAPI images, white lines delineate tissue from airspaces, and in the 85% O 2  groups demarcate septa. Antibody specificity was validated by rabbit IgG isotype control primary antibodies. The control experiments for the Aqp5 and Sftpc staining runs are illustrated here. Scale bars, 50 μm.
    Figure Legend Snippet: The primary cell type in the normally and aberrantly developing septa are type I alveolar epithelial cells The impact of administration of scrambled antimiR (S) or an antimiR directed against miR‐34a (A34a) on the abundance of type I alveolar epithelial cells (marked by aquaporin 5, Aqp5) and type II alveolar epithelial cells (marked by pro‐surfactant protein C, Sftpc) was assessed in 3‐μm sections of paraffin‐embedded lung tissue from P5 mice undergoing normal (21% O 2 ) or aberrant (85% O 2 ) lung alveolarization. DAPI, 4′,6‐diamidino‐2‐phenylindole. In the DAPI images, white lines delineate tissue from airspaces, and in the 85% O 2 groups demarcate septa. Antibody specificity was validated by rabbit IgG isotype control primary antibodies. The control experiments for the Aqp5 and Sftpc staining runs are illustrated here. Scale bars, 50 μm.

    Techniques Used: Mouse Assay, Staining

    7) Product Images from "Interlocked positive and negative feedback network motifs regulate β-catenin activity in the adherens junction pathway"

    Article Title: Interlocked positive and negative feedback network motifs regulate β-catenin activity in the adherens junction pathway

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E15-02-0083

    B16F0 cells as a model for E-cadherin–based adherens junctions and their functional response to proteolytic disruption of adherens junctions. (A) Bright-field image of spheroids formed by B16F0 at day 13. (B) Fluorescence microscope image of B16F0 spheroids; metabolically active cells were stained with fluorescein diacetate (green), and dead cells were stained with propidium iodide (red). (C) In 2D culture, WISP1 was assayed by ELISA in B16F0 conditioned medium at the indicated times after trypsinization (mean ± SD, n = 3 biological replicates; representative of at least two independent experiments). A WISP1 concentration profile that corresponds to a constant cellular production rate was determined by linear regression (gray dotted line). (D) Total cellular levels of E-cadherin and β-catenin assayed by flow cytometry (left: unstained B16F0 cells as a negative control; right: B16F0 cells stained with both Alexa Fluor 647–conjugated E-cadherin mAb and Alexa Fluor 488–conjugated β-catenin mAb). Red lines correspond to data-driven fluorescence threshold; 95% of unstained flow cytometric events exhibited a mean fluorescence intensity (MFI) below this value. (E, F) Distribution in MFI associated with E-cadherin (E) and β-catenin (F) represented by symbols (median) and error bars that enclose 95% of the distribution. The results for three biological replicates shown at each data point are representative of at least two independent experiments. In C, E, and F, the 0-h time point corresponds to a sample taken just before trypsinization.
    Figure Legend Snippet: B16F0 cells as a model for E-cadherin–based adherens junctions and their functional response to proteolytic disruption of adherens junctions. (A) Bright-field image of spheroids formed by B16F0 at day 13. (B) Fluorescence microscope image of B16F0 spheroids; metabolically active cells were stained with fluorescein diacetate (green), and dead cells were stained with propidium iodide (red). (C) In 2D culture, WISP1 was assayed by ELISA in B16F0 conditioned medium at the indicated times after trypsinization (mean ± SD, n = 3 biological replicates; representative of at least two independent experiments). A WISP1 concentration profile that corresponds to a constant cellular production rate was determined by linear regression (gray dotted line). (D) Total cellular levels of E-cadherin and β-catenin assayed by flow cytometry (left: unstained B16F0 cells as a negative control; right: B16F0 cells stained with both Alexa Fluor 647–conjugated E-cadherin mAb and Alexa Fluor 488–conjugated β-catenin mAb). Red lines correspond to data-driven fluorescence threshold; 95% of unstained flow cytometric events exhibited a mean fluorescence intensity (MFI) below this value. (E, F) Distribution in MFI associated with E-cadherin (E) and β-catenin (F) represented by symbols (median) and error bars that enclose 95% of the distribution. The results for three biological replicates shown at each data point are representative of at least two independent experiments. In C, E, and F, the 0-h time point corresponds to a sample taken just before trypsinization.

    Techniques Used: Functional Assay, Fluorescence, Microscopy, Metabolic Labelling, Staining, Enzyme-linked Immunosorbent Assay, Concentration Assay, Flow Cytometry, Negative Control

    8) Product Images from "Cerebellar Cells Self-Assemble into Functional Organoids on Synthetic, Chemically Crosslinked ECM-Mimicking Peptide Hydrogels"

    Article Title: Cerebellar Cells Self-Assemble into Functional Organoids on Synthetic, Chemically Crosslinked ECM-Mimicking Peptide Hydrogels

    Journal: Biomolecules

    doi: 10.3390/biom10050754

    Confocal images of cerebellar neuronal-glial cell cultures after seven days in vitro (DIV7) on PEG-CLP and PEG-CLP-RGD hydrogel membranes ( a ), on glass coated with PEG-CLP and PEG-CLP-RGD conjugates ( b ), and on poly-L-lysine-coated glass and plastic ( c ). All nuclei were stained blue with Hoechst33342; neurons (yellow) are immunolabelled for microtubule-associated protein 2 (MAP-2), astrocytes (red) are immunolabelled for glial fibrillary acidic protein (GFAP), and microglia are stained green with isolectin GS-IB4. Scale bar is 100 μm.
    Figure Legend Snippet: Confocal images of cerebellar neuronal-glial cell cultures after seven days in vitro (DIV7) on PEG-CLP and PEG-CLP-RGD hydrogel membranes ( a ), on glass coated with PEG-CLP and PEG-CLP-RGD conjugates ( b ), and on poly-L-lysine-coated glass and plastic ( c ). All nuclei were stained blue with Hoechst33342; neurons (yellow) are immunolabelled for microtubule-associated protein 2 (MAP-2), astrocytes (red) are immunolabelled for glial fibrillary acidic protein (GFAP), and microglia are stained green with isolectin GS-IB4. Scale bar is 100 μm.

    Techniques Used: In Vitro, Staining

    9) Product Images from "Cross-talks of glycosylphosphatidylinositol biosynthesis with glycosphingolipid biosynthesis and ER-associated degradation"

    Article Title: Cross-talks of glycosylphosphatidylinositol biosynthesis with glycosphingolipid biosynthesis and ER-associated degradation

    Journal: Nature Communications

    doi: 10.1038/s41467-020-14678-2

    B3GALT4 localizes at trans-cisternae of the Golgi. a Left: HeLa cells stably expressing FLAG-6His tagged B3GALT4 were fixed and stained as described to detecting the FLAG tag (green) and a cis-Golgi marker (GM130; blue) and a TGN marker (TGN46; red). Scale bar, 10 μm. b Average localizations of the indicated Golgi proteins in HeLa cells (see Supplementary Fig. 6d ) relative to GM130 (cis) and TGN46 (TGN) markers. Error bars indicate mean ± SD the number of measurements by linescan analysis for PGAP4 (41 images), B4GALNT1 (31 images), and B3GALT4 (29 images). c B3GALT4 colocalizes with the Golgi markers in HeLa cells. Left: Cells stably expressing FLAG-6His tagged B3GALT4 were fixed and stained for FLAG tag (green), as well as markers (magenta): Calnexin (ER), GM130 (cis-Golgi), B4GALT1 (Trans Golgi cisterna), and TGN46 (TGN). Scale bar, 10 μm. d Localization of B3GALT4 analyzed with 3D-SIM super-resolution microscopy. Left: a maximum intensity z-projection of B3GALT4 (green) and B4GALT1 or TGN46 (magenta), and a magnified region of Golgi in one of the cells. Right: ten magnified serial z-sections. For ( a ) and ( b ), right panel shows linescan analysis along a portion of the white line overlaying the image (left). Source data for ( b ) are provided as a Source Data file.
    Figure Legend Snippet: B3GALT4 localizes at trans-cisternae of the Golgi. a Left: HeLa cells stably expressing FLAG-6His tagged B3GALT4 were fixed and stained as described to detecting the FLAG tag (green) and a cis-Golgi marker (GM130; blue) and a TGN marker (TGN46; red). Scale bar, 10 μm. b Average localizations of the indicated Golgi proteins in HeLa cells (see Supplementary Fig. 6d ) relative to GM130 (cis) and TGN46 (TGN) markers. Error bars indicate mean ± SD the number of measurements by linescan analysis for PGAP4 (41 images), B4GALNT1 (31 images), and B3GALT4 (29 images). c B3GALT4 colocalizes with the Golgi markers in HeLa cells. Left: Cells stably expressing FLAG-6His tagged B3GALT4 were fixed and stained for FLAG tag (green), as well as markers (magenta): Calnexin (ER), GM130 (cis-Golgi), B4GALT1 (Trans Golgi cisterna), and TGN46 (TGN). Scale bar, 10 μm. d Localization of B3GALT4 analyzed with 3D-SIM super-resolution microscopy. Left: a maximum intensity z-projection of B3GALT4 (green) and B4GALT1 or TGN46 (magenta), and a magnified region of Golgi in one of the cells. Right: ten magnified serial z-sections. For ( a ) and ( b ), right panel shows linescan analysis along a portion of the white line overlaying the image (left). Source data for ( b ) are provided as a Source Data file.

    Techniques Used: Stable Transfection, Expressing, Staining, FLAG-tag, Marker, Microscopy

    LacCer enhances B3GALT4 activity toward GPI-GalNAc. a Western blotting of B3GALT4-3HA transiently overexpressed in PIGS-UGCG-DKO, PIGS-B4GALT5-DKO, and PIGS-B4GALT5-B4GALT6-TKO cells. b Representative fluorescence images of 3HA-tagged B3GALT4 in PIGS-B3GALT4-DKO, PIGS-UGCG-DKO, and PIGS-B4GALT5-B4GALT6-TKO HEK293 cells. GM130, a marker for cis-Golgi. Scale bar, 10 μm. c Left: PIGS-UGCG-DKO, PIGS-B4GALT5-DKO, and PIGS-B4GALT5-B4GALT6-TKO cells transiently expressing pME-B3GALT4-3HA were stained with T5 mAb. Right: Quantitative data of MFI from four independent experiments (mean ± SD, n = 4). P values are from one-way ANOVA followed by Dunnett’s test for multiple comparisons to PIGS-B3GALT4-DKO cells. d Western blotting of FLAG-6His tagged B3GALT4. Lysates of PIGS-UGCG-DKO, PIGS-B4GALT5-DKO, and PIGS-B4GALT5-B4GALT6-TKO cells stably expressing empty vector (Vec) and B3GALT4 were analyzed. TfR, a loading control. e Left: PIGS-UGCG-DKO, PIGS-B4GALT5-DKO, and PIGS-B4GALT5-B4GALT6-TKO cells stably expressing B3GALT4 were stained with T5 mAb. Right: Quantitative data of MFI from two independent experiments (mean ± SD, n = 2). f Schematic of LacCer enhanced galactose modification on GPI-GalNAc by B3GALT4 in the Golgi. Source data for ( c ) and ( e ) are provided as a Source Data file.
    Figure Legend Snippet: LacCer enhances B3GALT4 activity toward GPI-GalNAc. a Western blotting of B3GALT4-3HA transiently overexpressed in PIGS-UGCG-DKO, PIGS-B4GALT5-DKO, and PIGS-B4GALT5-B4GALT6-TKO cells. b Representative fluorescence images of 3HA-tagged B3GALT4 in PIGS-B3GALT4-DKO, PIGS-UGCG-DKO, and PIGS-B4GALT5-B4GALT6-TKO HEK293 cells. GM130, a marker for cis-Golgi. Scale bar, 10 μm. c Left: PIGS-UGCG-DKO, PIGS-B4GALT5-DKO, and PIGS-B4GALT5-B4GALT6-TKO cells transiently expressing pME-B3GALT4-3HA were stained with T5 mAb. Right: Quantitative data of MFI from four independent experiments (mean ± SD, n = 4). P values are from one-way ANOVA followed by Dunnett’s test for multiple comparisons to PIGS-B3GALT4-DKO cells. d Western blotting of FLAG-6His tagged B3GALT4. Lysates of PIGS-UGCG-DKO, PIGS-B4GALT5-DKO, and PIGS-B4GALT5-B4GALT6-TKO cells stably expressing empty vector (Vec) and B3GALT4 were analyzed. TfR, a loading control. e Left: PIGS-UGCG-DKO, PIGS-B4GALT5-DKO, and PIGS-B4GALT5-B4GALT6-TKO cells stably expressing B3GALT4 were stained with T5 mAb. Right: Quantitative data of MFI from two independent experiments (mean ± SD, n = 2). f Schematic of LacCer enhanced galactose modification on GPI-GalNAc by B3GALT4 in the Golgi. Source data for ( c ) and ( e ) are provided as a Source Data file.

    Techniques Used: Activity Assay, Western Blot, Fluorescence, Marker, Expressing, Staining, Stable Transfection, Plasmid Preparation, Modification

    10) Product Images from "Single amino acid substitution in LC-CDR1 induces Russell body phenotype that attenuates cellular protein synthesis through eIF2α phosphorylation and thereby downregulates IgG secretion despite operational secretory pathway traffic"

    Article Title: Single amino acid substitution in LC-CDR1 induces Russell body phenotype that attenuates cellular protein synthesis through eIF2α phosphorylation and thereby downregulates IgG secretion despite operational secretory pathway traffic

    Journal: mAbs

    doi: 10.1080/19420862.2017.1314875

    Single amino acid substitution leads to a marked increase in Russell body phenotype occurrence during immunoglobulin biosynthesis. Fluorescent micrographs of HEK293 cells expressing the parental IgG or its N35W variant. On day-2 post transfection, HEK293 cells were resuspended in fresh cell culture media with or without 15 μg/ml BFA, then immediately seeded onto poly-lysine coated glass coverslips and statically cultured for 24 hr. On day-3, cells were fixed, permeabilized, and immuno-stained. Co-staining was performed by using FITC-conjugated anti-gamma chain and Texas Red-conjugated anti-kappa chain polyclonal antibodies. Green and red image fields were superimposed to create ‘merge’ views. DIC and ‘merge’ were superimposed to generate ‘overlay’ views. (A) Subcellular localization of gamma-chain and kappa-chain was visualized under steady-state normal cell growth conditions. Two representative image fields for the parental IgG expressing cells are shown in the first 2 rows. Two representative image fields for N35W variant IgG expressing cells are shown in rows 3 and 4. (B) Gamma- and kappa-chains of the parental IgG (first 2 rows) and N35W variant IgG (rows 3 to 5) were visualized after 24 hr BFA treatment. The RB phenotype frequency for each mAb under steady-state or after BFA treatment is stated in the text. Unlabeled scale bar represents 10 μm.
    Figure Legend Snippet: Single amino acid substitution leads to a marked increase in Russell body phenotype occurrence during immunoglobulin biosynthesis. Fluorescent micrographs of HEK293 cells expressing the parental IgG or its N35W variant. On day-2 post transfection, HEK293 cells were resuspended in fresh cell culture media with or without 15 μg/ml BFA, then immediately seeded onto poly-lysine coated glass coverslips and statically cultured for 24 hr. On day-3, cells were fixed, permeabilized, and immuno-stained. Co-staining was performed by using FITC-conjugated anti-gamma chain and Texas Red-conjugated anti-kappa chain polyclonal antibodies. Green and red image fields were superimposed to create ‘merge’ views. DIC and ‘merge’ were superimposed to generate ‘overlay’ views. (A) Subcellular localization of gamma-chain and kappa-chain was visualized under steady-state normal cell growth conditions. Two representative image fields for the parental IgG expressing cells are shown in the first 2 rows. Two representative image fields for N35W variant IgG expressing cells are shown in rows 3 and 4. (B) Gamma- and kappa-chains of the parental IgG (first 2 rows) and N35W variant IgG (rows 3 to 5) were visualized after 24 hr BFA treatment. The RB phenotype frequency for each mAb under steady-state or after BFA treatment is stated in the text. Unlabeled scale bar represents 10 μm.

    Techniques Used: Expressing, Variant Assay, Transfection, Cell Culture, Staining

    with red indicating high hydrophobicity and white indicating low hydrophobicity. The N35W variant has more exposed hydrophobic characteristics in the CDRs than does the parental mAb. LC-CDR1 (L1) and HC-CDR3 (H3) are long and are predicted to be highly dynamic in solution. The Trp residue of the N35W variant is likely to be solvent-exposed, presenting a hydrophobic patch that could render the antibody prone to non-specific interactions. (B, C, D) Cell culture media and whole cell lysate samples were prepared on day-7 post transfection and were subjected to SDS-PAGE followed by Western blot using rabbit anti-human IgG (H+L) polyclonal antibody. Samples in lanes 1–2 and lanes 3–5 were analyzed in the non-contiguous lanes of a single gel, but the intervening lanes were digitally removed to create the figures B–D. (B) A sample volume corresponding to the 5 μl of harvested cell culture medium was loaded per lane and analyzed under reducing conditions. Expected band for heavy chain (HC) and light chain (LC) subunits are marked by arrowhead. Determined secretion titers for this mAb production run are shown in lanes 1 and 2. (C) Whole cell lysates equivalent to 12,000–12,500 cells were loaded per lane under reducing conditions. Expected band for HC and LC subunits are marked by arrowhead. A faint band marked by asterisk in lane 5 is apparently a LC-N35W dimer species that was resistant to SDS treatment or disulfide reduction. Anti-GAPDH blot is shown at the bottom as a loading reference. (D) Cell culture media were analyzed under non-reducing conditions. LCs are secreted as a mixture of monomers (mono.) and covalent dimers (di.). Trimer and tetramer species are faintly detectable. (E) On day-2 post transfection, transfected cells were resuspended in fresh growth media with or without 15 μg/ml BFA and maintained in suspension format until day-3 when cell culture media and cell pellets were harvested and analyzed under reducing conditions. The amount of IgGs secreted to the culture medium during the 24 hr period is shown in the left panel. The amount of IgGs detected in the cell lysates is shown in the right panel. Anti-GAPDH blot is shown as a loading reference for cell lysate samples. (F, G) Cell viability and viable cell density were monitored daily by using automated cell counters. Data compiled from 6 independent experiments for the parental IgG and 4 independent runs for the N35W variant were used to calculate the average and standard deviation.
    Figure Legend Snippet: with red indicating high hydrophobicity and white indicating low hydrophobicity. The N35W variant has more exposed hydrophobic characteristics in the CDRs than does the parental mAb. LC-CDR1 (L1) and HC-CDR3 (H3) are long and are predicted to be highly dynamic in solution. The Trp residue of the N35W variant is likely to be solvent-exposed, presenting a hydrophobic patch that could render the antibody prone to non-specific interactions. (B, C, D) Cell culture media and whole cell lysate samples were prepared on day-7 post transfection and were subjected to SDS-PAGE followed by Western blot using rabbit anti-human IgG (H+L) polyclonal antibody. Samples in lanes 1–2 and lanes 3–5 were analyzed in the non-contiguous lanes of a single gel, but the intervening lanes were digitally removed to create the figures B–D. (B) A sample volume corresponding to the 5 μl of harvested cell culture medium was loaded per lane and analyzed under reducing conditions. Expected band for heavy chain (HC) and light chain (LC) subunits are marked by arrowhead. Determined secretion titers for this mAb production run are shown in lanes 1 and 2. (C) Whole cell lysates equivalent to 12,000–12,500 cells were loaded per lane under reducing conditions. Expected band for HC and LC subunits are marked by arrowhead. A faint band marked by asterisk in lane 5 is apparently a LC-N35W dimer species that was resistant to SDS treatment or disulfide reduction. Anti-GAPDH blot is shown at the bottom as a loading reference. (D) Cell culture media were analyzed under non-reducing conditions. LCs are secreted as a mixture of monomers (mono.) and covalent dimers (di.). Trimer and tetramer species are faintly detectable. (E) On day-2 post transfection, transfected cells were resuspended in fresh growth media with or without 15 μg/ml BFA and maintained in suspension format until day-3 when cell culture media and cell pellets were harvested and analyzed under reducing conditions. The amount of IgGs secreted to the culture medium during the 24 hr period is shown in the left panel. The amount of IgGs detected in the cell lysates is shown in the right panel. Anti-GAPDH blot is shown as a loading reference for cell lysate samples. (F, G) Cell viability and viable cell density were monitored daily by using automated cell counters. Data compiled from 6 independent experiments for the parental IgG and 4 independent runs for the N35W variant were used to calculate the average and standard deviation.

    Techniques Used: Variant Assay, Cell Culture, Transfection, SDS Page, Western Blot, Standard Deviation

    Transfected cells were first labeled for 30 min using Alexa Fluor 488 Click-iT® Plus OPP reagent to detect actively ongoing protein translation in situ (see Materials and Methods). The click-labeled cells were then immuno-stained with (A) Alexa Fluor 594-conjugated anti-human IgG (H+L) or (B) Texas Red-conjugated anti-gamma chain polyclonal antibodies. Green and red image fields were superimposed to create ‘merge’ views. DIC and green or DIC and red images were superimposed to generate ‘overlay’ views.
    Figure Legend Snippet: Transfected cells were first labeled for 30 min using Alexa Fluor 488 Click-iT® Plus OPP reagent to detect actively ongoing protein translation in situ (see Materials and Methods). The click-labeled cells were then immuno-stained with (A) Alexa Fluor 594-conjugated anti-human IgG (H+L) or (B) Texas Red-conjugated anti-gamma chain polyclonal antibodies. Green and red image fields were superimposed to create ‘merge’ views. DIC and green or DIC and red images were superimposed to generate ‘overlay’ views.

    Techniques Used: Transfection, Labeling, In Situ, Staining

    11) Product Images from "Equine Herpesvirus 1 Multiply Inserted Transmembrane Protein pUL43 Cooperates with pUL56 in Downregulation of Cell Surface Major Histocompatibility Complex Class I"

    Article Title: Equine Herpesvirus 1 Multiply Inserted Transmembrane Protein pUL43 Cooperates with pUL56 in Downregulation of Cell Surface Major Histocompatibility Complex Class I

    Journal: Journal of Virology

    doi: 10.1128/JVI.00032-15

    pUL43 induces downregulation of cell surface MHC-I. (A) Equine NBL6 cells were infected with vAb4G or vUL43STOP virus at an MOI of 3. At 6 h p.i., cells were analyzed by flow cytometry after incubation with mouse anti-MHC-I (CZ3) MAb and Alexa Fluor 647-labeled goat anti-mouse IgG. Representative dot plots are derived from three independent experiments. (B) vAb4G, vUL43STOP, or vUL43STOP_R virus was used to infect NBL6 cells at an MOI of 3. Surface MHC-I levels were measured after 6 h p.i., as described above. (C) At 16 h p.i., HEK293 and HeLa cells infected with vAb4G or vUL43STOP virus were probed with mouse anti-HLA class I (W6/32) MAb and subjected to flow cytometry. (D) NBL6 cells were exposed to infection with the vHA-UL43 mutant. At 6 h p.i., cells were analyzed after incubation with mouse anti-MHC-I (CZ3) MAb and staining with Alexa Fluor 647-labeled goat anti-mouse IgG. All experiments were independently performed in triplicate and analyzed by using the Student t test. Data are presented as means ± standard deviations (error bars). Asterisks represent statistical significance ( P
    Figure Legend Snippet: pUL43 induces downregulation of cell surface MHC-I. (A) Equine NBL6 cells were infected with vAb4G or vUL43STOP virus at an MOI of 3. At 6 h p.i., cells were analyzed by flow cytometry after incubation with mouse anti-MHC-I (CZ3) MAb and Alexa Fluor 647-labeled goat anti-mouse IgG. Representative dot plots are derived from three independent experiments. (B) vAb4G, vUL43STOP, or vUL43STOP_R virus was used to infect NBL6 cells at an MOI of 3. Surface MHC-I levels were measured after 6 h p.i., as described above. (C) At 16 h p.i., HEK293 and HeLa cells infected with vAb4G or vUL43STOP virus were probed with mouse anti-HLA class I (W6/32) MAb and subjected to flow cytometry. (D) NBL6 cells were exposed to infection with the vHA-UL43 mutant. At 6 h p.i., cells were analyzed after incubation with mouse anti-MHC-I (CZ3) MAb and staining with Alexa Fluor 647-labeled goat anti-mouse IgG. All experiments were independently performed in triplicate and analyzed by using the Student t test. Data are presented as means ± standard deviations (error bars). Asterisks represent statistical significance ( P

    Techniques Used: Infection, Flow Cytometry, Cytometry, Incubation, Labeling, Derivative Assay, Mutagenesis, Staining

    ). Cells were infected with vHA-UL43 or mutant vHA-ΔN-UL43 virus. At 6 h p.i., cells were incubated with anti-HA MAb and visualized after staining with Alex Fluor 568-conjugated goat anti-mouse IgG (red) and DAPI (blue). Coverslips were inspected with a 100× oil objective. Bar, 5 μm. (B) vHA-UL43, vHA-ΔN-UL43, or vUL43STOP was used to infect NBL6 cells for 6 h. Levels of cell surface MHC-I were measured by flow cytometry, and triplicate assays were performed independently. Data are expressed as means ± standard deviations (error bars). Differences between various treatments were evaluated by Student's t test. The asterisk represents a significant level ( P
    Figure Legend Snippet: ). Cells were infected with vHA-UL43 or mutant vHA-ΔN-UL43 virus. At 6 h p.i., cells were incubated with anti-HA MAb and visualized after staining with Alex Fluor 568-conjugated goat anti-mouse IgG (red) and DAPI (blue). Coverslips were inspected with a 100× oil objective. Bar, 5 μm. (B) vHA-UL43, vHA-ΔN-UL43, or vUL43STOP was used to infect NBL6 cells for 6 h. Levels of cell surface MHC-I were measured by flow cytometry, and triplicate assays were performed independently. Data are expressed as means ± standard deviations (error bars). Differences between various treatments were evaluated by Student's t test. The asterisk represents a significant level ( P

    Techniques Used: Infection, Mutagenesis, Incubation, Staining, Flow Cytometry, Cytometry

    12) Product Images from "Humanization and Characterization of an Anti-Human TNF-? Murine Monoclonal Antibody"

    Article Title: Humanization and Characterization of an Anti-Human TNF-? Murine Monoclonal Antibody

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0016373

    Binding affinities of h357 IgG to soluble and membrane form TNF-α. (A) Binding affinities of different TNF-α anatagonists to soluble TNF-α. Microtiter plates with the anti-human IgG Fcγ fragment captured h357 (•), etanercept (▾) and adalimumab (△) and the anti-mouse IgG Fcγ fragment captured m357 (○) were incubated with various concentrations of soluble TNF-α for 1 hr at 37°C. After washing, the bound TNF-α was detected by a mouse monoclonal anti-TNF-α antibody conjugated with horseradish peroxidase. Absorbance was read at 450 nm on a microplate reader. (B) The saturation binding curves of m357 and h357 IgGs to transmembrane TNF-α. Transmembrane TNF-α-transfected NS0 cells were incubated with serial log dilutions of m357 (○) or h357 (•) antibody for 1 hr at 4°C. Cell were washed and incubated with Alexa Fluor 488 goat anti-mouse IgG (H+L) for m357 and Alexa Fluor 647 goat anti-human IgG (H+L) for h357, respectively for 1 hr at 4°C. Cells were then washed and analyzed by FACSCalibur flow cytometer.
    Figure Legend Snippet: Binding affinities of h357 IgG to soluble and membrane form TNF-α. (A) Binding affinities of different TNF-α anatagonists to soluble TNF-α. Microtiter plates with the anti-human IgG Fcγ fragment captured h357 (•), etanercept (▾) and adalimumab (△) and the anti-mouse IgG Fcγ fragment captured m357 (○) were incubated with various concentrations of soluble TNF-α for 1 hr at 37°C. After washing, the bound TNF-α was detected by a mouse monoclonal anti-TNF-α antibody conjugated with horseradish peroxidase. Absorbance was read at 450 nm on a microplate reader. (B) The saturation binding curves of m357 and h357 IgGs to transmembrane TNF-α. Transmembrane TNF-α-transfected NS0 cells were incubated with serial log dilutions of m357 (○) or h357 (•) antibody for 1 hr at 4°C. Cell were washed and incubated with Alexa Fluor 488 goat anti-mouse IgG (H+L) for m357 and Alexa Fluor 647 goat anti-human IgG (H+L) for h357, respectively for 1 hr at 4°C. Cells were then washed and analyzed by FACSCalibur flow cytometer.

    Techniques Used: Binding Assay, Incubation, Transfection, Flow Cytometry, Cytometry

    13) Product Images from "4D intravital microscopy uncovers critical strain differences for the roles of PECAM and CD99 in leukocyte diapedesis"

    Article Title: 4D intravital microscopy uncovers critical strain differences for the roles of PECAM and CD99 in leukocyte diapedesis

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    doi: 10.1152/ajpheart.00289.2016

    PECAM and CD99 expression are not different between FVB/n and C57BL/6 mice. A : peripheral blood leukocytes were prepared from FVB/n and C57BL/6 mice and labeled with antibodies against lymphocyte antigen 6 complex G6D [Ly6G (Gr-1)], PECAM, and CD99 or isotype controls. Neutrophils were identified by expression of Gr-1 and examined for expression of PECAM and CD99 (filled curves) compared with isotype controls (open curves) for each strain. Four mice were examined for each group; representative plots are shown. B : cremaster muscles of FVB/n or C57BL/6 mice were inflamed with IL-1β and prepared for 4D intravital imaging, except fluorescence-conjugated antibodies against PECAM and CD99 were injected intravenously 30 min before visualization. C : mouse cremaster muscles were prepared as described in B , except tissue was fixed, removed, and processed for immunofluorescence to detect α-smooth muscle actin (α-SMA). Scale bar = 25 μm.
    Figure Legend Snippet: PECAM and CD99 expression are not different between FVB/n and C57BL/6 mice. A : peripheral blood leukocytes were prepared from FVB/n and C57BL/6 mice and labeled with antibodies against lymphocyte antigen 6 complex G6D [Ly6G (Gr-1)], PECAM, and CD99 or isotype controls. Neutrophils were identified by expression of Gr-1 and examined for expression of PECAM and CD99 (filled curves) compared with isotype controls (open curves) for each strain. Four mice were examined for each group; representative plots are shown. B : cremaster muscles of FVB/n or C57BL/6 mice were inflamed with IL-1β and prepared for 4D intravital imaging, except fluorescence-conjugated antibodies against PECAM and CD99 were injected intravenously 30 min before visualization. C : mouse cremaster muscles were prepared as described in B , except tissue was fixed, removed, and processed for immunofluorescence to detect α-smooth muscle actin (α-SMA). Scale bar = 25 μm.

    Techniques Used: Expressing, Mouse Assay, Labeling, Imaging, Fluorescence, Injection, Immunofluorescence

    PECAM and CD99 function past the level of the endothelium in C57BL/6 mice. A : wild-type FVB/n and C57BL/6 mice were pretreated with control IgG, anti-PECAM, or anti-CD99 (3 mg/kg) prior to ear stimulation with croton oil (1% solution, 20 μl/ear) or carrier alone. After 4 h, ear tissue was collected and immunohistochemical staining was performed for PECAM [endothelial cells (ECs), red], myeloid-related protein 14 (neutrophils, green), and collagen IV [basement membrane (BM), purple]. 3-Dimensional confocal images were acquired from each sample and subsequently analyzed. Insets : z -orthogonal view along the indicated line to demonstrate leukocyte arrest in relation to the endothelium and basement membrane at 1.5 times the xy image. Scale bar = 50 μm. B : quantification of findings in A (percent leukocyte extravasation within 50 μm of postcapillary venules in FVB/n mice). C : schematic of quantification of site of leukocyte blockade. Leukocytes were scored as being in 1 of 6 positions: 1 ) luminal, 2 ) apically arrested, 3 ) arrested partway through the endothelium, 4 ) arrested at the level of the basement membrane, 5 ) actively migrating through the basement membrane, or 6 ) fully extravasated. D : quantification of the site of leukocyte arrest in FVB/n mice. E : quantification of findings in A (percent leukocyte extravasation within 50 μm of postcapillary venules in C57BL/6 mice). F : quantification of the site of leukocyte arrest in C57BL/6 mice. G : schematic model of anti-PECAM and anti-CD99 arrest in FVB/n and C57BL/6 mice. One hundred to 200 cells were analyzed per ear. Carrier-alone stimulation resulted in minimal leukocyte recruitment (data not shown). Total leukocytes per field of view, vessel length, and vessel diameter were equivalent for all treatment groups (data not shown). Data are representative of recordings from ≥4 mice per group with ≥8 images quantified per mouse. * P
    Figure Legend Snippet: PECAM and CD99 function past the level of the endothelium in C57BL/6 mice. A : wild-type FVB/n and C57BL/6 mice were pretreated with control IgG, anti-PECAM, or anti-CD99 (3 mg/kg) prior to ear stimulation with croton oil (1% solution, 20 μl/ear) or carrier alone. After 4 h, ear tissue was collected and immunohistochemical staining was performed for PECAM [endothelial cells (ECs), red], myeloid-related protein 14 (neutrophils, green), and collagen IV [basement membrane (BM), purple]. 3-Dimensional confocal images were acquired from each sample and subsequently analyzed. Insets : z -orthogonal view along the indicated line to demonstrate leukocyte arrest in relation to the endothelium and basement membrane at 1.5 times the xy image. Scale bar = 50 μm. B : quantification of findings in A (percent leukocyte extravasation within 50 μm of postcapillary venules in FVB/n mice). C : schematic of quantification of site of leukocyte blockade. Leukocytes were scored as being in 1 of 6 positions: 1 ) luminal, 2 ) apically arrested, 3 ) arrested partway through the endothelium, 4 ) arrested at the level of the basement membrane, 5 ) actively migrating through the basement membrane, or 6 ) fully extravasated. D : quantification of the site of leukocyte arrest in FVB/n mice. E : quantification of findings in A (percent leukocyte extravasation within 50 μm of postcapillary venules in C57BL/6 mice). F : quantification of the site of leukocyte arrest in C57BL/6 mice. G : schematic model of anti-PECAM and anti-CD99 arrest in FVB/n and C57BL/6 mice. One hundred to 200 cells were analyzed per ear. Carrier-alone stimulation resulted in minimal leukocyte recruitment (data not shown). Total leukocytes per field of view, vessel length, and vessel diameter were equivalent for all treatment groups (data not shown). Data are representative of recordings from ≥4 mice per group with ≥8 images quantified per mouse. * P

    Techniques Used: Mouse Assay, Immunohistochemistry, Staining

    14) Product Images from "SPIN90 Knockdown Attenuates the Formation and Movement of Endosomal Vesicles in the Early Stages of Epidermal Growth Factor Receptor Endocytosis"

    Article Title: SPIN90 Knockdown Attenuates the Formation and Movement of Endosomal Vesicles in the Early Stages of Epidermal Growth Factor Receptor Endocytosis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0082610

    SPIN90 knockdown suppresses the formation of EEA1-positive endosomes. A. Cells were treated with Alexa Fluor 647-conjugated EGF (40 ng/ml) for 10 min and fixed, prior to immunostaining with anti-EEA1 antibody. B. Bar graphs were generated using Sigmaplot. In total, 120 – 170 endosomes per cell were measured using three criteria (vesicle width: 0 – 300 nm, 300 – 700 nm and > 700) in 60 control and SPIN90 knockdown cells (KD). Overall, 150.70±5.82 vesicles (91.89%) in control cells and 121.36±7.65 (96.32%) vesicles in SPIN90 knockdown cells (KD) were 0 – 300 nm in width; 8.77±2.34 (5.35%) in control cells, 3.69±0.16 (2.93%) in KD cells were 300 – 700 nm in width; 4.53±0.67 (2.76%) in control cells, 0.93±0.16 (0.74%) in KD cells were over 700 nm in width. C. Total numbers of EEA1 endosomes (a), internalized EGF (b), and numbers of EEA1-endosomes containing EGF (c) were analyzed in control and SPIN90 knockdown HeLa cells. All values were determined using Metamorph Software.
    Figure Legend Snippet: SPIN90 knockdown suppresses the formation of EEA1-positive endosomes. A. Cells were treated with Alexa Fluor 647-conjugated EGF (40 ng/ml) for 10 min and fixed, prior to immunostaining with anti-EEA1 antibody. B. Bar graphs were generated using Sigmaplot. In total, 120 – 170 endosomes per cell were measured using three criteria (vesicle width: 0 – 300 nm, 300 – 700 nm and > 700) in 60 control and SPIN90 knockdown cells (KD). Overall, 150.70±5.82 vesicles (91.89%) in control cells and 121.36±7.65 (96.32%) vesicles in SPIN90 knockdown cells (KD) were 0 – 300 nm in width; 8.77±2.34 (5.35%) in control cells, 3.69±0.16 (2.93%) in KD cells were 300 – 700 nm in width; 4.53±0.67 (2.76%) in control cells, 0.93±0.16 (0.74%) in KD cells were over 700 nm in width. C. Total numbers of EEA1 endosomes (a), internalized EGF (b), and numbers of EEA1-endosomes containing EGF (c) were analyzed in control and SPIN90 knockdown HeLa cells. All values were determined using Metamorph Software.

    Techniques Used: Immunostaining, Generated, Software

    Overexpression of SPIN90 variants alters the morphology and movement of early endosomes. A. HeLa cells transfected with CFP-tagged Rab5 WT and full-length SPIN90 or empty vector were starved for 16 h, treated with Alexa Fluor 647-conjugated EGF (40 ng/ml), and live images acquired. B. Schematic diagram of SPIN90 representing functional domains. The SPIN90 N-terminal region includes SH3 and PRD (Proline-rich Domain), while the C-terminus is divided into three sections: CN, CM, and CC. C . (a) Moving images of CFP-Rab5-positive vesicles were acquired every 5 sec for 5 min, and the frames merged into one image to trace the movement of Rab5-positive vesicles. (b) The total lengths of the moving paths were measured and divided into 300 sec to determine the velocity of vesicles using Metamorph Software.
    Figure Legend Snippet: Overexpression of SPIN90 variants alters the morphology and movement of early endosomes. A. HeLa cells transfected with CFP-tagged Rab5 WT and full-length SPIN90 or empty vector were starved for 16 h, treated with Alexa Fluor 647-conjugated EGF (40 ng/ml), and live images acquired. B. Schematic diagram of SPIN90 representing functional domains. The SPIN90 N-terminal region includes SH3 and PRD (Proline-rich Domain), while the C-terminus is divided into three sections: CN, CM, and CC. C . (a) Moving images of CFP-Rab5-positive vesicles were acquired every 5 sec for 5 min, and the frames merged into one image to trace the movement of Rab5-positive vesicles. (b) The total lengths of the moving paths were measured and divided into 300 sec to determine the velocity of vesicles using Metamorph Software.

    Techniques Used: Over Expression, Transfection, Plasmid Preparation, Functional Assay, Size-exclusion Chromatography, Software

    15) Product Images from "Trafficking of the amino acid transporter B0,+ (SLC6A14) to the plasma membrane involves an exclusive interaction with SEC24C for its exit from the endoplasmic reticulum"

    Article Title: Trafficking of the amino acid transporter B0,+ (SLC6A14) to the plasma membrane involves an exclusive interaction with SEC24C for its exit from the endoplasmic reticulum

    Journal: Biochimica et biophysica acta. Molecular cell research

    doi: 10.1016/j.bbamcr.2018.11.005

    Localization of ATB 0,+ and SEC24C in MCF7 cells. Cells were fixed with methanol, as described in Materials and methods . They were incubated, as indicated, with either anti-SEC24C or anti-SEC24D antibodies, detected with Alexa Fluor ® 488-conjugated AffinityPure Fab Fragment Goat Anti-Rabbit IgG (H + L) (green). Further treatment was performed as described in Materials and methods Section 2.5 . Next the cells were incubated with anti-ATB 0,+ antibodies conjugated with biotin, followed by incubation with AlexaFluor568 conjugated streptavidin (red). Nuclei are visualized with DAPI. NC, negative control without the primary antibodies. B 0,+ , analysis of B 0,+ localization without anti-SEC24 antibodies. Bar 20 μm.
    Figure Legend Snippet: Localization of ATB 0,+ and SEC24C in MCF7 cells. Cells were fixed with methanol, as described in Materials and methods . They were incubated, as indicated, with either anti-SEC24C or anti-SEC24D antibodies, detected with Alexa Fluor ® 488-conjugated AffinityPure Fab Fragment Goat Anti-Rabbit IgG (H + L) (green). Further treatment was performed as described in Materials and methods Section 2.5 . Next the cells were incubated with anti-ATB 0,+ antibodies conjugated with biotin, followed by incubation with AlexaFluor568 conjugated streptavidin (red). Nuclei are visualized with DAPI. NC, negative control without the primary antibodies. B 0,+ , analysis of B 0,+ localization without anti-SEC24 antibodies. Bar 20 μm.

    Techniques Used: Incubation, Negative Control

    16) Product Images from "Binding of WIP to Actin Is Essential for T Cell Actin Cytoskeleton Integrity and Tissue Homing"

    Article Title: Binding of WIP to Actin Is Essential for T Cell Actin Cytoskeleton Integrity and Tissue Homing

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00533-14

    Defective homing of adoptively transferred WIPΔABD CD4 + T cells to cutaneous sites of Ag challenge in WT recipient mice. (A) Tritiated thymidine ( 3 H-Td) proliferation and IL-2 and IFN-γ production by splenocytes from WIPΔABD and WT mice on the DO11.10 background after stimulation with OVA ( n = 5 for each group). n.d., not detected. (B and C) Representative FACS analysis of OVA-stimulated splenic CD4 + T cells from WIPΔABD and WT mice on the DO11.10 background for surface expression of CCR4, CD43, and PSGL-1 (B) and integrins LFA-1 and VLA-4 (C) ( n = 5 for each group). The solid gray curve in the histogram represents the isotype control. (D) Adhesion of OVA-stimulated splenic CD4 + T cells from WIPΔABD and WT mice on the DO11.10 background to ICAM-1 and VCAM-1 under conditions of physiologic shear stress flow (1 dyn/cm 2 ; n = 2 for each group). (E) Representative FACS analysis of CD4 + KJ1-26 + cells in OVA- and PBS-challenged ears from WT recipients of OVA-stimulated CD4 + cells derived from DO11.10 WIPΔABD or WT mice. (F) Percentages and numbers of CD4 + KJ1-26 + cells in OVA- and PBS-challenged ears from WT recipients of OVA-stimulated CD4 + cells derived from DO11.10 WIPΔABD or WT mice ( n = 5 for each group). (G) Percentages of cells that have proliferated, as determined by dilution of CellTrace Violet (left panel), and of annexin V + cells (right panel) among CD4 + KJ1-26 + cells in OVA-challenged ears from WT recipients of OVA-stimulated CD4 + cells derived from DO11.10 WIPΔABD or WT mice ( n = 4 for each group). Columns and bars represent means ± the SEM. **, P
    Figure Legend Snippet: Defective homing of adoptively transferred WIPΔABD CD4 + T cells to cutaneous sites of Ag challenge in WT recipient mice. (A) Tritiated thymidine ( 3 H-Td) proliferation and IL-2 and IFN-γ production by splenocytes from WIPΔABD and WT mice on the DO11.10 background after stimulation with OVA ( n = 5 for each group). n.d., not detected. (B and C) Representative FACS analysis of OVA-stimulated splenic CD4 + T cells from WIPΔABD and WT mice on the DO11.10 background for surface expression of CCR4, CD43, and PSGL-1 (B) and integrins LFA-1 and VLA-4 (C) ( n = 5 for each group). The solid gray curve in the histogram represents the isotype control. (D) Adhesion of OVA-stimulated splenic CD4 + T cells from WIPΔABD and WT mice on the DO11.10 background to ICAM-1 and VCAM-1 under conditions of physiologic shear stress flow (1 dyn/cm 2 ; n = 2 for each group). (E) Representative FACS analysis of CD4 + KJ1-26 + cells in OVA- and PBS-challenged ears from WT recipients of OVA-stimulated CD4 + cells derived from DO11.10 WIPΔABD or WT mice. (F) Percentages and numbers of CD4 + KJ1-26 + cells in OVA- and PBS-challenged ears from WT recipients of OVA-stimulated CD4 + cells derived from DO11.10 WIPΔABD or WT mice ( n = 5 for each group). (G) Percentages of cells that have proliferated, as determined by dilution of CellTrace Violet (left panel), and of annexin V + cells (right panel) among CD4 + KJ1-26 + cells in OVA-challenged ears from WT recipients of OVA-stimulated CD4 + cells derived from DO11.10 WIPΔABD or WT mice ( n = 4 for each group). Columns and bars represent means ± the SEM. **, P

    Techniques Used: Mouse Assay, FACS, Expressing, Flow Cytometry, Derivative Assay

    17) Product Images from "Infectious Lassa Virus, but Not Filoviruses, Is Restricted by BST-2/Tetherin ▿"

    Article Title: Infectious Lassa Virus, but Not Filoviruses, Is Restricted by BST-2/Tetherin ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.00103-10

    Human BST-2 overexpression inhibits infectious LASV, but not ZEBOV, release and spread. (A) Human 293 cells stably expressing human BST-2 (FLP-BST-2), CAT (FLP-CAT), or an empty plasmid (FLP) were infected with LASV at the indicated MOIs. After 72 h, viral RNA was extracted from the media, and the viral copy number was determined by qRT-PCR. (B to D) Alternatively, cells were infected with ZEBOV (B), CPXV-GFP (C), or RVFV (D), fixed in formalin and stained for high-content quantitative image-based analysis with virus-specific antibodies 24 or 48 h (C and D) or 72 h (B) after infection. Error bars indicate standard deviations.
    Figure Legend Snippet: Human BST-2 overexpression inhibits infectious LASV, but not ZEBOV, release and spread. (A) Human 293 cells stably expressing human BST-2 (FLP-BST-2), CAT (FLP-CAT), or an empty plasmid (FLP) were infected with LASV at the indicated MOIs. After 72 h, viral RNA was extracted from the media, and the viral copy number was determined by qRT-PCR. (B to D) Alternatively, cells were infected with ZEBOV (B), CPXV-GFP (C), or RVFV (D), fixed in formalin and stained for high-content quantitative image-based analysis with virus-specific antibodies 24 or 48 h (C and D) or 72 h (B) after infection. Error bars indicate standard deviations.

    Techniques Used: Over Expression, Stable Transfection, Expressing, Plasmid Preparation, Infection, Quantitative RT-PCR, Staining

    BST-2 localization in relation to filoviral GP 1,2 . (A) Human 293 cells stably expressing human BST-2 were infected with ZEBOV. Cells were fixed in formalin 24 h later and stained with antibodies against BST-2 and ZEBOV GP 1,2 . (B and C) Alternatively, cells were infected with MARV for 24 h (B) or 48 h (C) and stained with antibodies against BST-2 and MARV GP 1,2 . Immunofluorescence of anti-filoviral GP 1,2 (green) and anti-BST-2 (red) in a single confocal plain is shown as indicated. Right columns show the merged images for triple staining of BST-2 (red), filoviral GP 1,2 (green), and the nucleus (blue), as well as bright-field images.
    Figure Legend Snippet: BST-2 localization in relation to filoviral GP 1,2 . (A) Human 293 cells stably expressing human BST-2 were infected with ZEBOV. Cells were fixed in formalin 24 h later and stained with antibodies against BST-2 and ZEBOV GP 1,2 . (B and C) Alternatively, cells were infected with MARV for 24 h (B) or 48 h (C) and stained with antibodies against BST-2 and MARV GP 1,2 . Immunofluorescence of anti-filoviral GP 1,2 (green) and anti-BST-2 (red) in a single confocal plain is shown as indicated. Right columns show the merged images for triple staining of BST-2 (red), filoviral GP 1,2 (green), and the nucleus (blue), as well as bright-field images.

    Techniques Used: Stable Transfection, Expressing, Infection, Staining, Immunofluorescence

    BST-2 inhibits budding of arenaviral, filoviral, and paramyxoviral VLPs. (A) Human 293T cells were cotransfected with plasmids encoding hemagglutinin (HA)-tagged matrix proteins of LASV, MACV, ZEBOV, MARV, or NiV, together with an empty plasmid, or plasmid encoding VPS4A-E228Q, human BST-2 (hBST-2), or murine BST-2 (mBST-2). Cell lysates and supernatants were harvested 48 h after transfection. VLPs in clarified medium were pelleted through a sucrose cushion, and HA-tagged matrix proteins in VLPs were analyzed by Western blotting. Numbers below each lane indicate values obtained with densitometric scanning using the ImageJ program (NIH). (B) Expression of the HA-tagged matrix proteins, actin, VPS4A-E228Q, hBST-2, or mBST-2 in cell lysates was determined by Western blotting. Shown is a representative Western blot from three independent experiments.
    Figure Legend Snippet: BST-2 inhibits budding of arenaviral, filoviral, and paramyxoviral VLPs. (A) Human 293T cells were cotransfected with plasmids encoding hemagglutinin (HA)-tagged matrix proteins of LASV, MACV, ZEBOV, MARV, or NiV, together with an empty plasmid, or plasmid encoding VPS4A-E228Q, human BST-2 (hBST-2), or murine BST-2 (mBST-2). Cell lysates and supernatants were harvested 48 h after transfection. VLPs in clarified medium were pelleted through a sucrose cushion, and HA-tagged matrix proteins in VLPs were analyzed by Western blotting. Numbers below each lane indicate values obtained with densitometric scanning using the ImageJ program (NIH). (B) Expression of the HA-tagged matrix proteins, actin, VPS4A-E228Q, hBST-2, or mBST-2 in cell lysates was determined by Western blotting. Shown is a representative Western blot from three independent experiments.

    Techniques Used: Plasmid Preparation, Transfection, Western Blot, Expressing

    Knockdown of human BST-2 expression enhances infectious LASV release, but not ZEBOV or MARV spread. (A) HeLa cells were transfected with siRNA targeting BST-2 or with a control siRNA. After 24, 48, or 72 h, cellular RNA was extracted, and the relative BST-2 expression levels were determined by qRT-PCR. BST-2 protein expression levels in cell lysates were also determined by Western blotting. (B) Cells were infected with LASV (MOI = 0.1) 24 h after transfection. Viral RNA was extracted from the medium 72 h later, and the viral copy number was determined by qRT-PCR. (C) Alternatively, cells were infected with ZEBOV-GFP (MOI = 10) or MARV (MOI = 3). Cells were fixed in formalin 72 h later and stained for high-content quantitative image-based analysis with virus-specific antibodies. Error bars indicate standard deviations.
    Figure Legend Snippet: Knockdown of human BST-2 expression enhances infectious LASV release, but not ZEBOV or MARV spread. (A) HeLa cells were transfected with siRNA targeting BST-2 or with a control siRNA. After 24, 48, or 72 h, cellular RNA was extracted, and the relative BST-2 expression levels were determined by qRT-PCR. BST-2 protein expression levels in cell lysates were also determined by Western blotting. (B) Cells were infected with LASV (MOI = 0.1) 24 h after transfection. Viral RNA was extracted from the medium 72 h later, and the viral copy number was determined by qRT-PCR. (C) Alternatively, cells were infected with ZEBOV-GFP (MOI = 10) or MARV (MOI = 3). Cells were fixed in formalin 72 h later and stained for high-content quantitative image-based analysis with virus-specific antibodies. Error bars indicate standard deviations.

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

    18) Product Images from "DDR1 autophosphorylation is a result of aggregation into dense clusters"

    Article Title: DDR1 autophosphorylation is a result of aggregation into dense clusters

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-53176-4

    Signalling-defective DDR1 mutants bind triple-helical DDR1 selective peptide but do not phosphorylate with peptide stimulation. ( A ) COS-7 cells transiently expressing wild-type DDR1 or the indicated DDR1 mutant were incubated with or without a biotinylated DDR-selective collagen-mimetic peptide for 60 minutes on ice, followed by incubation with anti-DDR1 mAb 7A9 on ice. Cells were then fixed and incubated with Alexa Fluor-488 goat-anti-mouse IgG and Alexa Fluor-546 conjugated streptavidin. Cells were imaged by widefield microscopy. The graph shows mean fluorescence intensity, normalised to respective DDR1 expression levels. N = 27–31 fields of view from 3 independent experiments. Scale bar, 20 μm. ( B ) HEK293 transiently expressing wild-type DDR1 or the indicated DDR1 mutant were stimulated with collagen I (C), or with DDR-selective collagen-mimetic peptide (P) for 60 minutes at 37 °C or were left unstimulated. Cell lysates were analysed by Western blot using an Ab against phosphorylated Tyr-513 (anti-pY). The blot was stripped and re-probed with anti-DDR1. The positions of molecular mass markers are indicated on the left in kDa. The bar chart shows the densitometry analysis of pY513 band intensities after normalization to total DDR1. Each value is a percentage of the sum of all the pY513/DDR1 signals on the blot. The graph shows mean band intensities + SEM (N = 3). NS, no significance; *p
    Figure Legend Snippet: Signalling-defective DDR1 mutants bind triple-helical DDR1 selective peptide but do not phosphorylate with peptide stimulation. ( A ) COS-7 cells transiently expressing wild-type DDR1 or the indicated DDR1 mutant were incubated with or without a biotinylated DDR-selective collagen-mimetic peptide for 60 minutes on ice, followed by incubation with anti-DDR1 mAb 7A9 on ice. Cells were then fixed and incubated with Alexa Fluor-488 goat-anti-mouse IgG and Alexa Fluor-546 conjugated streptavidin. Cells were imaged by widefield microscopy. The graph shows mean fluorescence intensity, normalised to respective DDR1 expression levels. N = 27–31 fields of view from 3 independent experiments. Scale bar, 20 μm. ( B ) HEK293 transiently expressing wild-type DDR1 or the indicated DDR1 mutant were stimulated with collagen I (C), or with DDR-selective collagen-mimetic peptide (P) for 60 minutes at 37 °C or were left unstimulated. Cell lysates were analysed by Western blot using an Ab against phosphorylated Tyr-513 (anti-pY). The blot was stripped and re-probed with anti-DDR1. The positions of molecular mass markers are indicated on the left in kDa. The bar chart shows the densitometry analysis of pY513 band intensities after normalization to total DDR1. Each value is a percentage of the sum of all the pY513/DDR1 signals on the blot. The graph shows mean band intensities + SEM (N = 3). NS, no significance; *p

    Techniques Used: Expressing, Mutagenesis, Incubation, Microscopy, Fluorescence, Western Blot

    19) Product Images from "A New and Robust Method of Tethering IgG Surrogate Antigens on Lipid Bilayer Membranes to Facilitate the TIRFM Based Live Cell and Single Molecule Imaging Experiments"

    Article Title: A New and Robust Method of Tethering IgG Surrogate Antigens on Lipid Bilayer Membranes to Facilitate the TIRFM Based Live Cell and Single Molecule Imaging Experiments

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0063735

    H12-D-domain mediated PLB membranes only tether whole IgG, but not Fab, F(ab’) 2 or IgM molecules. (A) Shown are representative TIRFM images of Alexa 568-conjugated goat whole IgG, F(ab’) 2 or Fab fragments of goat IgG molecules, or the biotin-conjugated mouse IgM molecules tethered on the surface of PLB membranes with pre-attached H12-D-domain. The PLB membranes tethering the biotin-conjugated mouse IgM molecules were further incubated with the Alexa 568-conjugated streptavidin for TIRFM imaging. Bar is 1.5 µm. (B) Statistical quantification for the mFI of Alexa 568-conjugated goat whole IgG, F(ab’) 2 or Fab fragments of goat IgG molecules tethered on the surface of PLB membranes with pre-attached H12-D-domain. (C) Statistical quantification for the mFI of biotin-conjugated mouse IgM molecules tethered on the surface of PLB membranes with or without pre-attached H12-D-domain. The PLB membranes tethering the biotin-conjugated mouse IgM molecules were further incubated with the Alexa 568-conjugated streptavidin for TIRFM imaging. In both B and C, each dot represents a single measurement for the mFI of the tethered IgG surrogate antigens by Image J software. Bars represent means±SD. Two-tailed t tests were performed for statistical comparisons.
    Figure Legend Snippet: H12-D-domain mediated PLB membranes only tether whole IgG, but not Fab, F(ab’) 2 or IgM molecules. (A) Shown are representative TIRFM images of Alexa 568-conjugated goat whole IgG, F(ab’) 2 or Fab fragments of goat IgG molecules, or the biotin-conjugated mouse IgM molecules tethered on the surface of PLB membranes with pre-attached H12-D-domain. The PLB membranes tethering the biotin-conjugated mouse IgM molecules were further incubated with the Alexa 568-conjugated streptavidin for TIRFM imaging. Bar is 1.5 µm. (B) Statistical quantification for the mFI of Alexa 568-conjugated goat whole IgG, F(ab’) 2 or Fab fragments of goat IgG molecules tethered on the surface of PLB membranes with pre-attached H12-D-domain. (C) Statistical quantification for the mFI of biotin-conjugated mouse IgM molecules tethered on the surface of PLB membranes with or without pre-attached H12-D-domain. The PLB membranes tethering the biotin-conjugated mouse IgM molecules were further incubated with the Alexa 568-conjugated streptavidin for TIRFM imaging. In both B and C, each dot represents a single measurement for the mFI of the tethered IgG surrogate antigens by Image J software. Bars represent means±SD. Two-tailed t tests were performed for statistical comparisons.

    Techniques Used: Incubation, Imaging, Software, Two Tailed Test

    H12-D-domain construct efficiently tethers the IgG surrogate antigens on PLB membranes and these tethered IgG surrogate antigens induce the formation of BCR and surrogate antigen microclusters. (A) Shown are representative TIRFM images of Alexa 647-conjugated goat IgG anti human IgM surrogate antigens tethered on the surface of PLB membranes through H12-D-domain. The Alexa 647-conjugated goat IgG anti human IgM surrogate molecules were pre-incubated (100 or 0 nM) with the PLB membranes containing H12-D-domain. Bar is 1.5 µm. (B) Statistical quantification for the mean fluorescence intensity (mFI) of Alexa 647-conjugated goat IgG anti human IgM surrogate antigens tethered on the surface of PLB membranes. Each dot represents a single measurement for the mFI of the tethered IgG surrogate antigens by Image J software. Bars represent means ± SD. Two-tailed t tests were performed for statistical comparisons. (C) Shown are representative two-color TIRFM images of BCR (green) or the surrogate antigen (red) microclusters within the contact interface of human Ramos B cell with the PLB membranes tethering the Alexa 647-conjugated goat IgG anti human IgM surrogate antigens. Also shown are the merged images. Bar is 1.5 µm. (D) Statistical quantification for the mFI of BCR microclusters (top panel) or surrogate antigen microclusters (lower panel) within the B cell immunological synapse. Each dot shows one measurement from a single cell. Bars represent means ± SD. Two-tailed t tests were performed for statistical comparisons.
    Figure Legend Snippet: H12-D-domain construct efficiently tethers the IgG surrogate antigens on PLB membranes and these tethered IgG surrogate antigens induce the formation of BCR and surrogate antigen microclusters. (A) Shown are representative TIRFM images of Alexa 647-conjugated goat IgG anti human IgM surrogate antigens tethered on the surface of PLB membranes through H12-D-domain. The Alexa 647-conjugated goat IgG anti human IgM surrogate molecules were pre-incubated (100 or 0 nM) with the PLB membranes containing H12-D-domain. Bar is 1.5 µm. (B) Statistical quantification for the mean fluorescence intensity (mFI) of Alexa 647-conjugated goat IgG anti human IgM surrogate antigens tethered on the surface of PLB membranes. Each dot represents a single measurement for the mFI of the tethered IgG surrogate antigens by Image J software. Bars represent means ± SD. Two-tailed t tests were performed for statistical comparisons. (C) Shown are representative two-color TIRFM images of BCR (green) or the surrogate antigen (red) microclusters within the contact interface of human Ramos B cell with the PLB membranes tethering the Alexa 647-conjugated goat IgG anti human IgM surrogate antigens. Also shown are the merged images. Bar is 1.5 µm. (D) Statistical quantification for the mFI of BCR microclusters (top panel) or surrogate antigen microclusters (lower panel) within the B cell immunological synapse. Each dot shows one measurement from a single cell. Bars represent means ± SD. Two-tailed t tests were performed for statistical comparisons.

    Techniques Used: Construct, Incubation, Fluorescence, Software, Two Tailed Test

    The IgG surrogate antigens tethered by H12-D-domain show more uniform distribution with better lateral mobility than the ones tethered by streptavidin. (A) Shown is the distribution of fluorescence intensities (FI) of 10905 IgG surrogate antigen molecules tethered by H12-D-domain (red color) versus 13323 IgG surrogate antigen molecule tethered by streptavidin (blue color) on PLB membranes with a Gaussian fit (red or blue colored curve respectively) to the histogram plot. (B) Cumulative diffusion probability plot of all the calculated instant diffusion coefficients of 10905 IgG surrogate antigen molecules tethered by H12-D-domain (red color) versus 13323 IgG surrogate antigen molecule tethered by streptavidin (blue color) on PLB membranes.
    Figure Legend Snippet: The IgG surrogate antigens tethered by H12-D-domain show more uniform distribution with better lateral mobility than the ones tethered by streptavidin. (A) Shown is the distribution of fluorescence intensities (FI) of 10905 IgG surrogate antigen molecules tethered by H12-D-domain (red color) versus 13323 IgG surrogate antigen molecule tethered by streptavidin (blue color) on PLB membranes with a Gaussian fit (red or blue colored curve respectively) to the histogram plot. (B) Cumulative diffusion probability plot of all the calculated instant diffusion coefficients of 10905 IgG surrogate antigen molecules tethered by H12-D-domain (red color) versus 13323 IgG surrogate antigen molecule tethered by streptavidin (blue color) on PLB membranes.

    Techniques Used: Diffusion-based Assay

    IgG surrogate antigens tethered by H12-D-domain enhance the accumulation of BCR and pSyk into B cell immunological synapse than the ones tethered by streptavidin. (A) Statistical quantification for the mean fluorescence intensity (mFI) of biotin and Alexa 568-conjugated goat IgG anti human IgM surrogate antigens tethered on the surface of PLB membranes by either H12-D-domain (red color) or streptavidin (blue color). Each dot represents a single measurement for the mFI of the tethered IgG surrogate antigens by Image J software. Bars represent means ± SD. Two-tailed t tests were performed for statistical comparisons. (B) Statistical quantification for the accumulation of human IgM-BCRs into the immunological synapse as measured by the mFI of BCR within the immunological synapse from Ramos human B cells that were placed on PLB membranes presenting the same amount of IgG surrogate antigen as shown in A that were tethered by either H12-D-domain or streptavidin. Each dot represents a single measurement for the mFI of IgM-BCRs by Image J software. Bars represent means ± SD. Two-tailed t tests were performed for statistical comparisons. (C) Statistical quantification for the accumulation of pSyk signaling molecules into the immunological synapse as measured by the mFI of BCR within the immunological synapse from Ramos human B cells that were placed on PLB membranes presenting the same amount of IgG surrogate antigen as shown in A that were tethered by either H12-D-domain or streptavidin. Each dot represents a single measurement for the mFI of pSyk by Image J software. Bars represent means ± SD. Two-tailed t tests were performed for statistical comparisons.
    Figure Legend Snippet: IgG surrogate antigens tethered by H12-D-domain enhance the accumulation of BCR and pSyk into B cell immunological synapse than the ones tethered by streptavidin. (A) Statistical quantification for the mean fluorescence intensity (mFI) of biotin and Alexa 568-conjugated goat IgG anti human IgM surrogate antigens tethered on the surface of PLB membranes by either H12-D-domain (red color) or streptavidin (blue color). Each dot represents a single measurement for the mFI of the tethered IgG surrogate antigens by Image J software. Bars represent means ± SD. Two-tailed t tests were performed for statistical comparisons. (B) Statistical quantification for the accumulation of human IgM-BCRs into the immunological synapse as measured by the mFI of BCR within the immunological synapse from Ramos human B cells that were placed on PLB membranes presenting the same amount of IgG surrogate antigen as shown in A that were tethered by either H12-D-domain or streptavidin. Each dot represents a single measurement for the mFI of IgM-BCRs by Image J software. Bars represent means ± SD. Two-tailed t tests were performed for statistical comparisons. (C) Statistical quantification for the accumulation of pSyk signaling molecules into the immunological synapse as measured by the mFI of BCR within the immunological synapse from Ramos human B cells that were placed on PLB membranes presenting the same amount of IgG surrogate antigen as shown in A that were tethered by either H12-D-domain or streptavidin. Each dot represents a single measurement for the mFI of pSyk by Image J software. Bars represent means ± SD. Two-tailed t tests were performed for statistical comparisons.

    Techniques Used: Fluorescence, Software, Two Tailed Test

    20) Product Images from "Fibroblastic niches prime T cell alloimmunity through Delta-like Notch ligands"

    Article Title: Fibroblastic niches prime T cell alloimmunity through Delta-like Notch ligands

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI89535

    Fibroblastic niches in spleen express DLL1/4 Notch ligands and localize next to alloreactive T cells. ( A and B ) Immunofluorescence microscopy of splenic cryosections from Tg Ccl19-Cre+ Dll1 +/+ Dll4 +/+ ROSA26 eYFP and Tg Ccl19-Cre+ Dll1 Δ/Δ Dll4 Δ/Δ ROSA26 eYFP mice stained for GFP, CD35, and DLL4 ( A ) or GFP, CD157, and DLL4 ( B ). ( C – E ) Immunofluorescence microscopy of splenic cryosections from lethally irradiated (8.5 Gy) BALB/c mice transplanted with CMTMR-labeled alloantigen-specific CD4 + 4C TCR–transgenic cells and pulsed with EdU 12 hours prior to organ collection to label proliferating cells. Cryosections were incubated with Alexa Fluor 488 picolyl azide to reveal EdU, along with anti-DLL4 ( C ), anti-CD35 ( D ), or anti-CD157 ( E ). Organs were collected on day 1.5 after transplantation. Data are representative of at least 2 experiments.
    Figure Legend Snippet: Fibroblastic niches in spleen express DLL1/4 Notch ligands and localize next to alloreactive T cells. ( A and B ) Immunofluorescence microscopy of splenic cryosections from Tg Ccl19-Cre+ Dll1 +/+ Dll4 +/+ ROSA26 eYFP and Tg Ccl19-Cre+ Dll1 Δ/Δ Dll4 Δ/Δ ROSA26 eYFP mice stained for GFP, CD35, and DLL4 ( A ) or GFP, CD157, and DLL4 ( B ). ( C – E ) Immunofluorescence microscopy of splenic cryosections from lethally irradiated (8.5 Gy) BALB/c mice transplanted with CMTMR-labeled alloantigen-specific CD4 + 4C TCR–transgenic cells and pulsed with EdU 12 hours prior to organ collection to label proliferating cells. Cryosections were incubated with Alexa Fluor 488 picolyl azide to reveal EdU, along with anti-DLL4 ( C ), anti-CD35 ( D ), or anti-CD157 ( E ). Organs were collected on day 1.5 after transplantation. Data are representative of at least 2 experiments.

    Techniques Used: Immunofluorescence, Microscopy, Mouse Assay, Staining, Irradiation, Labeling, Transgenic Assay, Incubation, Transplantation Assay

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

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

    Journal: Journal of Virology

    doi: 10.1128/JVI.02227-18

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

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

    22) Product Images from "Sequential Roles for Mash1 and Ngn2 in the Generation of Dorsal Spinal Cord Interneurons"

    Article Title: Sequential Roles for Mash1 and Ngn2 in the Generation of Dorsal Spinal Cord Interneurons

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.01859

    Mash1 is required for dI3 and dI5 neuron formation while Ngn2 represses formation of these populations Immunofluorescence on transverse sections of neural tubes in wild type (A,E,I), Mash1 − / − (B,F,J), Ngn2 − / − (C,G,K) and Mash1 − / − ;Ngn2 − / − (D,H,L) E10.5 mouse embryos. (A-D) Co-labeling with Brn3a and Lhx1/5 (yellow) marks dI2 neurons. There is a significant increase in dI2 neurons in the Mash1 − / − and Mash1 − / − ;Ngn2 − / − embryos but not Ngn2 − / − relative to wild type. (E-H) Isl1 identifies dI3 neurons (green). In the Mash1 − / − and Mash1 − / − ;Ngn2 − / − mutant embryos, there is a dramatic decrease in dI3 neurons relative to wild type. In contrast, an increase in dI3 neurons in Ngn2 − / − is detected. (I-L) Pax2 marks dI4 and dI6 neurons (green), and Lmx1b labels dI5 neurons (red). In the Mash1 − / − and Mash1 − / − ;Ngn2 − / − embryos, there is a complete loss of dI5. This is opposite in Ngn2 − / − embryos where there is an increase in dI5 relative to wild type. dI4/6 neurons increase in the Mash1 − / −, are unchanged in the Ngn2 − / −, and are dramatically reduced in the Mash1 − / − ;Ngn2 − / − relative to wild type embryos. Individual populations in each mutant were counted on at least three sections from at least three embryos each and cell counts are shown in the table. Because dI4 and dI6 neuronal populations cannot be distinguished in the Mash1 null, all Pax2 cells dorsal to the boundary where the first ventral Pax2 cell was found within the ventricular zone were counted, and as such, they are labeled as dI4/6. Scale bar is 50 μm.
    Figure Legend Snippet: Mash1 is required for dI3 and dI5 neuron formation while Ngn2 represses formation of these populations Immunofluorescence on transverse sections of neural tubes in wild type (A,E,I), Mash1 − / − (B,F,J), Ngn2 − / − (C,G,K) and Mash1 − / − ;Ngn2 − / − (D,H,L) E10.5 mouse embryos. (A-D) Co-labeling with Brn3a and Lhx1/5 (yellow) marks dI2 neurons. There is a significant increase in dI2 neurons in the Mash1 − / − and Mash1 − / − ;Ngn2 − / − embryos but not Ngn2 − / − relative to wild type. (E-H) Isl1 identifies dI3 neurons (green). In the Mash1 − / − and Mash1 − / − ;Ngn2 − / − mutant embryos, there is a dramatic decrease in dI3 neurons relative to wild type. In contrast, an increase in dI3 neurons in Ngn2 − / − is detected. (I-L) Pax2 marks dI4 and dI6 neurons (green), and Lmx1b labels dI5 neurons (red). In the Mash1 − / − and Mash1 − / − ;Ngn2 − / − embryos, there is a complete loss of dI5. This is opposite in Ngn2 − / − embryos where there is an increase in dI5 relative to wild type. dI4/6 neurons increase in the Mash1 − / −, are unchanged in the Ngn2 − / −, and are dramatically reduced in the Mash1 − / − ;Ngn2 − / − relative to wild type embryos. Individual populations in each mutant were counted on at least three sections from at least three embryos each and cell counts are shown in the table. Because dI4 and dI6 neuronal populations cannot be distinguished in the Mash1 null, all Pax2 cells dorsal to the boundary where the first ventral Pax2 cell was found within the ventricular zone were counted, and as such, they are labeled as dI4/6. Scale bar is 50 μm.

    Techniques Used: Immunofluorescence, Labeling, Mutagenesis

    Excess Mash1 promotes dI3 and dI5 populations while excess Ngn2 represses them in the chick neural tube Immunofluorescence on transverse sections of E4 (HH13-14) chick neural tubes electroporated with pMiWIII-Mash1 (A,D,G,J) or pMiWIII-Ngn2 (B,E,H,K) at E3 (HH23-24). The arrow above each row of panels indicates the electroporated side of the neural tube. Neuronal population dI2 was labeled by Brn3a;Lhx1/5 (yellow) (A,B), dI3 by Isl1 (C,D), dI4 by Pax2 (E,F), and dI5 by Lmx1b (G,H). Each panel is representative of the phenotype seen in at least three sections from at least three electroporated embryos. Relative induction or repression is expressed as the number of cells on the injected side divided by the number of cells on the control side and plotted in the graph on a logarithmic scale. ** indicates p
    Figure Legend Snippet: Excess Mash1 promotes dI3 and dI5 populations while excess Ngn2 represses them in the chick neural tube Immunofluorescence on transverse sections of E4 (HH13-14) chick neural tubes electroporated with pMiWIII-Mash1 (A,D,G,J) or pMiWIII-Ngn2 (B,E,H,K) at E3 (HH23-24). The arrow above each row of panels indicates the electroporated side of the neural tube. Neuronal population dI2 was labeled by Brn3a;Lhx1/5 (yellow) (A,B), dI3 by Isl1 (C,D), dI4 by Pax2 (E,F), and dI5 by Lmx1b (G,H). Each panel is representative of the phenotype seen in at least three sections from at least three electroporated embryos. Relative induction or repression is expressed as the number of cells on the injected side divided by the number of cells on the control side and plotted in the graph on a logarithmic scale. ** indicates p

    Techniques Used: Immunofluorescence, Labeling, Injection

    Distinct functions for Mash1 and Ngn2 in specification of dorsal neurons Immunofluorescence on cross sections of E11.5 mouse neural tubes where Mash1 and Ngn2 have been swapped into the Ngn2 and Mash1 locus, respectively: wild type (A,F,K), Mash1 KINgn2/+ (B,G,L), Mash1 KINgn2 (C,H,M), Ngn2 KIMash1/+ (D,I,N), and Ngn2 KIMash1 (E,J,O). (A-E) Co-localization of Brn3a (red) and Lhx1/5 (green) detects dI2 neurons (yellow). (F-J) Isl1 (green) detects dI3 neurons. (K-O) Pax2 (green) and Lmx1b (red) detect dI4 and dI5 neurons, respectively. Cell counts for each marker on at least three sections from at least three embryos of each genotype are shown in the table. Because dI4 and dI6 populations cannot be distinguished in the Mash1 null, all Pax2 counts were completed by drawing a boundary line at the first Pax2 expressing cell found nearest the ventricular zone, and as such, they are labeled as dI4/6. ** indicates p
    Figure Legend Snippet: Distinct functions for Mash1 and Ngn2 in specification of dorsal neurons Immunofluorescence on cross sections of E11.5 mouse neural tubes where Mash1 and Ngn2 have been swapped into the Ngn2 and Mash1 locus, respectively: wild type (A,F,K), Mash1 KINgn2/+ (B,G,L), Mash1 KINgn2 (C,H,M), Ngn2 KIMash1/+ (D,I,N), and Ngn2 KIMash1 (E,J,O). (A-E) Co-localization of Brn3a (red) and Lhx1/5 (green) detects dI2 neurons (yellow). (F-J) Isl1 (green) detects dI3 neurons. (K-O) Pax2 (green) and Lmx1b (red) detect dI4 and dI5 neurons, respectively. Cell counts for each marker on at least three sections from at least three embryos of each genotype are shown in the table. Because dI4 and dI6 populations cannot be distinguished in the Mash1 null, all Pax2 counts were completed by drawing a boundary line at the first Pax2 expressing cell found nearest the ventricular zone, and as such, they are labeled as dI4/6. ** indicates p

    Techniques Used: Immunofluorescence, Marker, Expressing, Labeling

    23) 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

    24) Product Images from "Abnormal Accumulation of Desmin in Gastrocnemius Myofibers of Patients with Peripheral Artery Disease"

    Article Title: Abnormal Accumulation of Desmin in Gastrocnemius Myofibers of Patients with Peripheral Artery Disease

    Journal: Journal of Histochemistry and Cytochemistry

    doi: 10.1369/0022155415569348

    Schematic representation of the desmin intermediate filament network and its association with myofibrils, sarcolemma, mitochondria and the nucleus, in a skeletal myofiber. Desmin is a key protein of the intermediate filaments of the cytoskeleton in myofibers
    Figure Legend Snippet: Schematic representation of the desmin intermediate filament network and its association with myofibrils, sarcolemma, mitochondria and the nucleus, in a skeletal myofiber. Desmin is a key protein of the intermediate filaments of the cytoskeleton in myofibers

    Techniques Used:

    Abnormal desmin accumu-lation in the myofibers of Peripheral Artery Disease (PAD) gastrocnemius (A) and associated changes in myofiber morphology and extracellular matrix (B). Sections from the gastrocnemius of a PAD subject with moderate to severe myopathy
    Figure Legend Snippet: Abnormal desmin accumu-lation in the myofibers of Peripheral Artery Disease (PAD) gastrocnemius (A) and associated changes in myofiber morphology and extracellular matrix (B). Sections from the gastrocnemius of a PAD subject with moderate to severe myopathy

    Techniques Used:

    Myofiber Morphology and Density Change in association with Desmin Accumulation in PAD Myofibers
    Figure Legend Snippet: Myofiber Morphology and Density Change in association with Desmin Accumulation in PAD Myofibers

    Techniques Used:

    25) Product Images from "Microfluidic isolation of platelet-covered circulating tumor cells"

    Article Title: Microfluidic isolation of platelet-covered circulating tumor cells

    Journal: Lab on a chip

    doi: 10.1039/c7lc00654c

    Lung CTC isolation using platelet- and EpCAM-targeted capture. (A) CTC counts in blood samples obtained from healthy donor (HD) and metastatic lung cancer patients (Lung pt) that were captured using CD41 or EpCAM antibodies. (B) Fluorescent images of CTCs captured using CD41 (top) and EpCAM antibodies (bottom). The cells were immunostained for EpCAM/cadherin-11 (CTC marker; Alexa Fluor 488; green), CD45 (leukocyte marker; Alexa Fluor 647; red) and CD61 (platelet marker; Alexa Fluor 568; gold) respectively. Scale bar, 10 μm.
    Figure Legend Snippet: Lung CTC isolation using platelet- and EpCAM-targeted capture. (A) CTC counts in blood samples obtained from healthy donor (HD) and metastatic lung cancer patients (Lung pt) that were captured using CD41 or EpCAM antibodies. (B) Fluorescent images of CTCs captured using CD41 (top) and EpCAM antibodies (bottom). The cells were immunostained for EpCAM/cadherin-11 (CTC marker; Alexa Fluor 488; green), CD45 (leukocyte marker; Alexa Fluor 647; red) and CD61 (platelet marker; Alexa Fluor 568; gold) respectively. Scale bar, 10 μm.

    Techniques Used: Isolation, Marker

    26) Product Images from "Multivalent Conjugates of Sonic Hedgehog Accelerate Diabetic Wound Healing"

    Article Title: Multivalent Conjugates of Sonic Hedgehog Accelerate Diabetic Wound Healing

    Journal: Tissue Engineering. Part A

    doi: 10.1089/ten.tea.2014.0281

    mvShh activated canonical Shh signaling at lower concentrations relative to unconjugated Shh. (A) ShhLight II fibroblasts exhibit valency-dependent increases in Gli1-mediated transcriptional activity. The dashed line represents an arbitrary threshold
    Figure Legend Snippet: mvShh activated canonical Shh signaling at lower concentrations relative to unconjugated Shh. (A) ShhLight II fibroblasts exhibit valency-dependent increases in Gli1-mediated transcriptional activity. The dashed line represents an arbitrary threshold

    Techniques Used: Activity Assay

    27) Product Images from "Recognition of Double-Stranded RNA and Regulation of Interferon Pathway by Toll-Like Receptor 10"

    Article Title: Recognition of Double-Stranded RNA and Regulation of Interferon Pathway by Toll-Like Receptor 10

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.00516

    Myeloid differentiation primary response gene 88 (MyD88) is the adaptor protein for toll-like receptor (TLR)-10 signaling following stimulation by dsRNA. (A) Alignment of toll/interleukin-1 receptor domain sequences of human TLRs. Sequence logo (top) represents the conserved motif identified by MEME. Sequence in black box is the BB-loop sequence in TLR10. The alanine/proline residues highlighted in green determine the adaptor protein bound by TLRs. All human TLRs, except TLR3, have proline in the BB-loop. (B) Confocal micrograph of THP-1 cells stimulated with 10 µg/ml poly(I:C) stained at different time points for TLR10 (red), MyD88 (green), with nuclei stained with DNA-binding dye 4′,6-Diamidin-2-phenylindol (DAPI) (blue). Unstimulated (US) cells were included as a control. Arrows indicate the co-localization of TLR10 and MyD88 (yellow). Inset (at 10 min post-challenge) is an enlargement of the white square box. Scale bars, 5 µm. (C) TLR10 interacts with MyD88 upon poly(I:C) stimulation. Cell lysates of THP-1 cells stimulated with 10 µg/ml poly(I:C) at different time points were immunoprecipitated using anti-TLR10 antibody and then analyzed by Western blotting using anti-MyD88 or anti-TRIF antibodies. β-ACTIN was the input control. Data shown are representative of at least two independent experiments.
    Figure Legend Snippet: Myeloid differentiation primary response gene 88 (MyD88) is the adaptor protein for toll-like receptor (TLR)-10 signaling following stimulation by dsRNA. (A) Alignment of toll/interleukin-1 receptor domain sequences of human TLRs. Sequence logo (top) represents the conserved motif identified by MEME. Sequence in black box is the BB-loop sequence in TLR10. The alanine/proline residues highlighted in green determine the adaptor protein bound by TLRs. All human TLRs, except TLR3, have proline in the BB-loop. (B) Confocal micrograph of THP-1 cells stimulated with 10 µg/ml poly(I:C) stained at different time points for TLR10 (red), MyD88 (green), with nuclei stained with DNA-binding dye 4′,6-Diamidin-2-phenylindol (DAPI) (blue). Unstimulated (US) cells were included as a control. Arrows indicate the co-localization of TLR10 and MyD88 (yellow). Inset (at 10 min post-challenge) is an enlargement of the white square box. Scale bars, 5 µm. (C) TLR10 interacts with MyD88 upon poly(I:C) stimulation. Cell lysates of THP-1 cells stimulated with 10 µg/ml poly(I:C) at different time points were immunoprecipitated using anti-TLR10 antibody and then analyzed by Western blotting using anti-MyD88 or anti-TRIF antibodies. β-ACTIN was the input control. Data shown are representative of at least two independent experiments.

    Techniques Used: Sequencing, Staining, Binding Assay, Immunoprecipitation, Western Blot

    Proposed model for the novel dual functions of toll-like receptor (TLR)-10 in regulating IFN signaling. (A) Sensing of dsRNA by TLR10 in endosomes. If TLR10 forms homodimer/heterodimer or co-factors needed for signaling is not clear. (B) Activation of TLR10 recruits myeloid differentiation primary response gene 88 (MyD88), subsequently leading to decrease in interferon regulatory factor (IRF)-7 phosphorylation and suppress IFN β expression. (C) Ligand sequestration: TLR10 competes with TLR3 for dsRNA, attenuates TLR3 mediated IFN β expression. (D) Signaling of TLR10 negatively regulates TLR3 expression and promotes expression of negative regulator of the signaling, sterile alpha and TIR motif-containing protein 1 (SARM1) to further suppress TLR3 signaling and the subsequent IFNβ expression.
    Figure Legend Snippet: Proposed model for the novel dual functions of toll-like receptor (TLR)-10 in regulating IFN signaling. (A) Sensing of dsRNA by TLR10 in endosomes. If TLR10 forms homodimer/heterodimer or co-factors needed for signaling is not clear. (B) Activation of TLR10 recruits myeloid differentiation primary response gene 88 (MyD88), subsequently leading to decrease in interferon regulatory factor (IRF)-7 phosphorylation and suppress IFN β expression. (C) Ligand sequestration: TLR10 competes with TLR3 for dsRNA, attenuates TLR3 mediated IFN β expression. (D) Signaling of TLR10 negatively regulates TLR3 expression and promotes expression of negative regulator of the signaling, sterile alpha and TIR motif-containing protein 1 (SARM1) to further suppress TLR3 signaling and the subsequent IFNβ expression.

    Techniques Used: Activation Assay, Expressing

    Crosstalk between toll-like receptor (TLR)-10 and TLR3. (A) THP-1 cells were challenged with fluorophore-conjugated poly(I:C) (cyan) and stained for TLR10 (red) and TLR3 (green). Arrow indicates co-localization of TLR10, TLR3, and poly(I:C) (white). Scale bars, 5 µm. (B,C) The ectodomains (ECD) of TLR10 or TLR3 recombinant proteins were incubated with biotin-conjugated poly(I:C) alone or together for 1 h at pH 5.5. The biotin-poly(I:C) bound complexes were pulled-down by streptavidin beads and analyzed by immunoblotting using anti-TLR10 (B) or anti-TLR3 (C) antibodies. (D) Expression of IFN β in wild-type (WT), TLR3 knockdown (KD), TLR10 KD, and TLR3/10 double KD THP-1 cells upon poly(I:C) challenge. (E,F) Expression of TLR3 (E) and sterile alpha and TIR motif-containing protein 1 (F) in WT and TLR10 overexpressed (OE) cells in response to poly(I:C) challenge (10 µg/ml, 6 h post-stimulation). The mRNA expression was quantitated using RT-qPCR and denoted as fold change compared with corresponding unstimulated cells. Data are mean with SEM from three independent experiments. * p
    Figure Legend Snippet: Crosstalk between toll-like receptor (TLR)-10 and TLR3. (A) THP-1 cells were challenged with fluorophore-conjugated poly(I:C) (cyan) and stained for TLR10 (red) and TLR3 (green). Arrow indicates co-localization of TLR10, TLR3, and poly(I:C) (white). Scale bars, 5 µm. (B,C) The ectodomains (ECD) of TLR10 or TLR3 recombinant proteins were incubated with biotin-conjugated poly(I:C) alone or together for 1 h at pH 5.5. The biotin-poly(I:C) bound complexes were pulled-down by streptavidin beads and analyzed by immunoblotting using anti-TLR10 (B) or anti-TLR3 (C) antibodies. (D) Expression of IFN β in wild-type (WT), TLR3 knockdown (KD), TLR10 KD, and TLR3/10 double KD THP-1 cells upon poly(I:C) challenge. (E,F) Expression of TLR3 (E) and sterile alpha and TIR motif-containing protein 1 (F) in WT and TLR10 overexpressed (OE) cells in response to poly(I:C) challenge (10 µg/ml, 6 h post-stimulation). The mRNA expression was quantitated using RT-qPCR and denoted as fold change compared with corresponding unstimulated cells. Data are mean with SEM from three independent experiments. * p

    Techniques Used: Staining, Recombinant, Incubation, Expressing, Quantitative RT-PCR

    Toll-like receptor (TLR)-10 regulates dsRNA-mediated type I IFN expression. (A) Basal expression of TLR10 in unstimulated wild-type (WT), TLR10 overexpressed (OE), and knockdown (KD) THP-1 cells. (B) Expression of IFN β in WT, TLR10 OE, and TLR10 KD THP-1 cells upon challenge by 10 µg/ml poly(I:C) at 4 h post-stimulation. Intracellular: poly(I:C) transfected by cationic lipid delivery; surface: poly(I:C) added to cell culture medium directly. (C,D) Expression of IFN β in WT, TLR10 OE, and KD THP-1 cells at different time points (C) and concentrations (D) upon poly(I:C) stimulation. (E) Basal expression of TLR3 and RIG-I compared with TLR10 in WT, TLR10 OE, and KD THP-1 cells. (F) Expression of IFNβ in WT, TLR10 OE, and KD THP-1 cells upon stimulation by 2′3 ′-cGAMP, 5′pppdsRNA synthesized in vitro (dsRNA WT) or its variant (dsRNA M5). Data are mean with SEM from three independent experiments. * p
    Figure Legend Snippet: Toll-like receptor (TLR)-10 regulates dsRNA-mediated type I IFN expression. (A) Basal expression of TLR10 in unstimulated wild-type (WT), TLR10 overexpressed (OE), and knockdown (KD) THP-1 cells. (B) Expression of IFN β in WT, TLR10 OE, and TLR10 KD THP-1 cells upon challenge by 10 µg/ml poly(I:C) at 4 h post-stimulation. Intracellular: poly(I:C) transfected by cationic lipid delivery; surface: poly(I:C) added to cell culture medium directly. (C,D) Expression of IFN β in WT, TLR10 OE, and KD THP-1 cells at different time points (C) and concentrations (D) upon poly(I:C) stimulation. (E) Basal expression of TLR3 and RIG-I compared with TLR10 in WT, TLR10 OE, and KD THP-1 cells. (F) Expression of IFNβ in WT, TLR10 OE, and KD THP-1 cells upon stimulation by 2′3 ′-cGAMP, 5′pppdsRNA synthesized in vitro (dsRNA WT) or its variant (dsRNA M5). Data are mean with SEM from three independent experiments. * p

    Techniques Used: Expressing, Transfection, Cell Culture, Synthesized, In Vitro, Variant Assay

    Toll-like receptor (TLR)-10 binds dsRNA in vitro . (A,B) Cell lysates of THP-1 cells were incubated with biotin-poly(I:C) (5 ng/ml) at (A) pH 5.5 or (B) pH 7.4, with or without addition of competitive unlabeled poly(I:C) (50 ng/ml) for 1 h. Complexes were pulled-down using streptavidin beads and analyzed by Western blotting using anti-TLR10 antibody. Data shown are representative of at least three independent experiments.
    Figure Legend Snippet: Toll-like receptor (TLR)-10 binds dsRNA in vitro . (A,B) Cell lysates of THP-1 cells were incubated with biotin-poly(I:C) (5 ng/ml) at (A) pH 5.5 or (B) pH 7.4, with or without addition of competitive unlabeled poly(I:C) (50 ng/ml) for 1 h. Complexes were pulled-down using streptavidin beads and analyzed by Western blotting using anti-TLR10 antibody. Data shown are representative of at least three independent experiments.

    Techniques Used: In Vitro, Incubation, Western Blot

    Interaction between toll-like receptor (TLR)-10 and poly(I:C) was observed by fluorescent resonance energy transfer (FRET) after acceptor photo bleaching. (A) Confocal micrograph of THP-1 stained TLR10 (red), organelle marker (green), and transfected fluorophore-conjugated poly(I:C) (cyan). Arrows indicate the co-localization of TLR10 and poly(I:C) in corresponding endosomal compartments (white). (B) Fluorophore-conjugated poly(I:C) were transfected to THP-1 cells. Channels corresponding to TLR10 (red) and poly(I:C) (green). Gradual photo-bleaching of the acceptor by 561 nm laser followed by signal capture from both channels starts after the fifth frame. Merged images depicting co-localization of TLR10 and poly(I:C) with the region of interest for acceptor and donor images before and after bleaching circled. Quantification of FRET for the circled region is displayed graphically as fluorescence intensity over frame. Scale bars, 5 µm. Data are mean with SEM of eight individual samples.
    Figure Legend Snippet: Interaction between toll-like receptor (TLR)-10 and poly(I:C) was observed by fluorescent resonance energy transfer (FRET) after acceptor photo bleaching. (A) Confocal micrograph of THP-1 stained TLR10 (red), organelle marker (green), and transfected fluorophore-conjugated poly(I:C) (cyan). Arrows indicate the co-localization of TLR10 and poly(I:C) in corresponding endosomal compartments (white). (B) Fluorophore-conjugated poly(I:C) were transfected to THP-1 cells. Channels corresponding to TLR10 (red) and poly(I:C) (green). Gradual photo-bleaching of the acceptor by 561 nm laser followed by signal capture from both channels starts after the fifth frame. Merged images depicting co-localization of TLR10 and poly(I:C) with the region of interest for acceptor and donor images before and after bleaching circled. Quantification of FRET for the circled region is displayed graphically as fluorescence intensity over frame. Scale bars, 5 µm. Data are mean with SEM of eight individual samples.

    Techniques Used: Förster Resonance Energy Transfer, Staining, Marker, Transfection, Fluorescence

    Toll-like receptor (TLR)-10 stimulated by dsRNA regulates type I IFN responses through phosphorylation of interferon regulatory factor (IRF)-7. (A,B) Phosphorylation level of (A) IRF7 and (B) IRF3 in wild-type (WT) and TLR10 overexpressed (OE) THP-1 cells upon stimulation by 10 µg/ml poly(I:C) at different time points post-challenge was analyzed by Western blotting with anti-phospho-IRF7 (Ser477) and anti-phospho-IRF3 (Ser396) antibodies, respectively. A representative blot (left) and mean (with SEM, right) from three independent experiments are shown. (C) Augmented type I IFN signaling in TLR10 knockdown THP-1 cells through an IRF-inducible luciferase reporter. Luciferase activity measured in THP-1 reporter cells upon transfection with 10 µg/ml poly(I:C) in TLR10 small interfering RNA (siRNA) (si-TLR10) or a non-targeting control siRNA (NC) treated THP-1 cells. Data are mean with SEM from at least three independent experiments. * p
    Figure Legend Snippet: Toll-like receptor (TLR)-10 stimulated by dsRNA regulates type I IFN responses through phosphorylation of interferon regulatory factor (IRF)-7. (A,B) Phosphorylation level of (A) IRF7 and (B) IRF3 in wild-type (WT) and TLR10 overexpressed (OE) THP-1 cells upon stimulation by 10 µg/ml poly(I:C) at different time points post-challenge was analyzed by Western blotting with anti-phospho-IRF7 (Ser477) and anti-phospho-IRF3 (Ser396) antibodies, respectively. A representative blot (left) and mean (with SEM, right) from three independent experiments are shown. (C) Augmented type I IFN signaling in TLR10 knockdown THP-1 cells through an IRF-inducible luciferase reporter. Luciferase activity measured in THP-1 reporter cells upon transfection with 10 µg/ml poly(I:C) in TLR10 small interfering RNA (siRNA) (si-TLR10) or a non-targeting control siRNA (NC) treated THP-1 cells. Data are mean with SEM from at least three independent experiments. * p

    Techniques Used: Western Blot, Luciferase, Activity Assay, Transfection, Small Interfering RNA

    Sub-cellular localization of toll-like receptor (TLR)-10 in THP-1 cells. (A) Confocal micrograph of resting wild-type THP-1 cells stained for TLR10 (red), organelle markers (green), and nuclei (blue) stained with DNA-binding dye 4′,6-Diamidin-2-phenylindol (DAPI). Co-localization of TLR10 and respective organelle marker (yellow). Scale bars, 5 µm. (B) Relative expression of TLR10 in different organelles. The expression level of TLR10 in different organelles was compared with those expressed in the Golgi apparatus (GIANTIN + ). Signals of more than 30 randomly picked cells from three independent experiments were computed using ImageJ with the co-localization plug-in. Data are presented as mean with SEM.
    Figure Legend Snippet: Sub-cellular localization of toll-like receptor (TLR)-10 in THP-1 cells. (A) Confocal micrograph of resting wild-type THP-1 cells stained for TLR10 (red), organelle markers (green), and nuclei (blue) stained with DNA-binding dye 4′,6-Diamidin-2-phenylindol (DAPI). Co-localization of TLR10 and respective organelle marker (yellow). Scale bars, 5 µm. (B) Relative expression of TLR10 in different organelles. The expression level of TLR10 in different organelles was compared with those expressed in the Golgi apparatus (GIANTIN + ). Signals of more than 30 randomly picked cells from three independent experiments were computed using ImageJ with the co-localization plug-in. Data are presented as mean with SEM.

    Techniques Used: Staining, Binding Assay, Marker, Expressing

    28) Product Images from "The PD-L1:B7-1 pathway restrains diabetogenic effector T cells in vivo"

    Article Title: The PD-L1:B7-1 pathway restrains diabetogenic effector T cells in vivo

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

    doi: 10.4049/jimmunol.1003496

    Both 10F.2H11 and 10F.9G2 antibodies precipitate diabetes transferred by islet antigen-specific transgenic CD4 + or CD8 + effector T cells
    Figure Legend Snippet: Both 10F.2H11 and 10F.9G2 antibodies precipitate diabetes transferred by islet antigen-specific transgenic CD4 + or CD8 + effector T cells

    Techniques Used: Transgenic Assay

    10F.2H11 and 10F.9G2 mAbs increase CD8 + effector T cell expansion, activation and cytokine production
    Figure Legend Snippet: 10F.2H11 and 10F.9G2 mAbs increase CD8 + effector T cell expansion, activation and cytokine production

    Techniques Used: Activation Assay

    10F.9G2 and 10F.2H11 recognize distinct epitopes of PD-L1
    Figure Legend Snippet: 10F.9G2 and 10F.2H11 recognize distinct epitopes of PD-L1

    Techniques Used:

    10F.2H11 and 10F.9G2 mAb treatment increases islet infiltration by CD8 + effector T cells
    Figure Legend Snippet: 10F.2H11 and 10F.9G2 mAb treatment increases islet infiltration by CD8 + effector T cells

    Techniques Used:

    Anti-PD-L1 mAb 10F.2H11 does not interfere with functional PD-L1:PD-1 signaling, whereas 10F.9G2 does
    Figure Legend Snippet: Anti-PD-L1 mAb 10F.2H11 does not interfere with functional PD-L1:PD-1 signaling, whereas 10F.9G2 does

    Techniques Used: Functional Assay

    29) Product Images from "Characterization of the novel mitochondrial genome replication factor MiRF172 in Trypanosoma brucei"

    Article Title: Characterization of the novel mitochondrial genome replication factor MiRF172 in Trypanosoma brucei

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.211730

    MiRF172 and TAC102 after p197 RNAi depletion and recovery after removal of tet in γL262P p197 RNAi BSF T. brucei cells. (A) Colocalization of MiRF172–PTP with TAC102 in γL262P p197 RNAi BSF cells. Localization of MiRF172–PTP (magenta) and TAC102 (green) is represented by maximum intensity projections from immunofluorescence microscopy image stacks of γL262P p197 RNAi BSF T. brucei cells. MiRF172–PTP was detected with anti-Protein A antibody. TAC102 was detected with anti-TAC102 monoclonal mouse antibody. The kDNA and the nucleus were stained with DAPI (cyan). The inset shows a higher magnification view. (B) TAC recovery experiment in γL262P p197 RNAi BSF T. brucei cells. To detect MiRF172–PTP, TAC102 and DNA the same antibodies and reagents as in A were used. The pictures were obtained under the same conditions as in A. The basal bodies (red) were detected with the YL1/2 monoclonal antibody. - tet, uninduced cells; d5 post induction, MiRF172-depleted cells at day 5 of RNAi (RNAi was induced by addition of tet); d4 post recovery, after 5 days of RNAi, tet was removed and cells were grown for 4 additional days. (C) Quantification of the relative occurrence of kDNA discs and nuclei in γL262P p197 RNAi induced and uninduced cells ( n ≥113 for each time point). K, kDNA; N, nucleus. (D) Quantitative analysis of TAC102 in γL262P p197 RNAi cells without tet (no tet), with tet at day five (d5 p.i.) as well as 2 days after removal of tet (post recovery; d2 p.r.) and at day 4 post recovery (d4 p.r.) ( n ≥105 for each time point). (E) Quantitative analysis of the MiRF172–PTP signal in in γL262P p197 RNAi cells as in D ( n ≥105 for each time point). (F) Western blot analysis of γL262P p197 RNAi BSF cells. Total protein isolated from uninduced cells (−tet), cells induced with tet for 5 days (d5 p.i.) and cells released from p197 RNAi at day 2 (d2 p.r.) and day 4 post recovery (d4 p.r.) was used. C-terminally PTP-tagged MiRF172 was detected with the anti-PAP antibody and TAC102 with the anti-TAC102 monoclonal mouse antibody. EF1α serves as a loading control. Arrowheads point to the TAC102 and MiRF172 signals. PH, phase contrast. Scale bars: 5 µm.
    Figure Legend Snippet: MiRF172 and TAC102 after p197 RNAi depletion and recovery after removal of tet in γL262P p197 RNAi BSF T. brucei cells. (A) Colocalization of MiRF172–PTP with TAC102 in γL262P p197 RNAi BSF cells. Localization of MiRF172–PTP (magenta) and TAC102 (green) is represented by maximum intensity projections from immunofluorescence microscopy image stacks of γL262P p197 RNAi BSF T. brucei cells. MiRF172–PTP was detected with anti-Protein A antibody. TAC102 was detected with anti-TAC102 monoclonal mouse antibody. The kDNA and the nucleus were stained with DAPI (cyan). The inset shows a higher magnification view. (B) TAC recovery experiment in γL262P p197 RNAi BSF T. brucei cells. To detect MiRF172–PTP, TAC102 and DNA the same antibodies and reagents as in A were used. The pictures were obtained under the same conditions as in A. The basal bodies (red) were detected with the YL1/2 monoclonal antibody. - tet, uninduced cells; d5 post induction, MiRF172-depleted cells at day 5 of RNAi (RNAi was induced by addition of tet); d4 post recovery, after 5 days of RNAi, tet was removed and cells were grown for 4 additional days. (C) Quantification of the relative occurrence of kDNA discs and nuclei in γL262P p197 RNAi induced and uninduced cells ( n ≥113 for each time point). K, kDNA; N, nucleus. (D) Quantitative analysis of TAC102 in γL262P p197 RNAi cells without tet (no tet), with tet at day five (d5 p.i.) as well as 2 days after removal of tet (post recovery; d2 p.r.) and at day 4 post recovery (d4 p.r.) ( n ≥105 for each time point). (E) Quantitative analysis of the MiRF172–PTP signal in in γL262P p197 RNAi cells as in D ( n ≥105 for each time point). (F) Western blot analysis of γL262P p197 RNAi BSF cells. Total protein isolated from uninduced cells (−tet), cells induced with tet for 5 days (d5 p.i.) and cells released from p197 RNAi at day 2 (d2 p.r.) and day 4 post recovery (d4 p.r.) was used. C-terminally PTP-tagged MiRF172 was detected with the anti-PAP antibody and TAC102 with the anti-TAC102 monoclonal mouse antibody. EF1α serves as a loading control. Arrowheads point to the TAC102 and MiRF172 signals. PH, phase contrast. Scale bars: 5 µm.

    Techniques Used: Immunofluorescence, Microscopy, Staining, Western Blot, Isolation

    Quantification of TAC102 in MiRF172 RNAi BSF cells. (A) MiRF172 RNAi BSF cells stained for TAC102 (green) and basal bodies (red) from either uninduced (-tet) or RNAi induced [day (d)3] cells. Pictures show maximum intensity projections from immunofluorescence microscopy image stacks of MiRF172 RNAi BSF T. brucei cells. TAC102 was detected with the anti-TAC102 polyclonal rat antibody and the basal bodies with the monoclonal mouse antibody BBA4. The kDNA and the nucleus were stained with DAPI (cyan). (B) γL262P MiF172 RNAi BSF cells stained for MiRF172–PTP (magenta), TAC102 (green), basal bodies (red) and DAPI (cyan). Proteins and DNA were detected with the same antibodies and reagents as in A. MiRF172–PTP was detected with the anti-Protein A antibody. The pictures show maximum intensity projections as in A. (C) Western blot analysis of γL262P MiRF172 RNAi BSF cells. Total protein isolated from uninduced cells (d0) and cells induced with tet for 3 days (d3). C-terminally PTP-tagged MiRF172 was detected with an anti-PAP antibody and TAC102 with the anti-TAC102 monoclonal mouse antibody. Tubulin serves as a loading control. (D) Quantification of the relative occurrence of kDNA discs and nuclei in γL262P MiRF172 RNAi induced and uninduced cells ( n ≥180 for each time point). K, kDNA; N, nucleus. (E) Quantification of TAC102 in γL262P MiRF172 RNAi uninduced (−tet) and cells induced for three days with tet (d3 tet). Black represents the wild-type TAC102 signal and gray stands for a weak TAC102 signal. (F) Quantification of the relative occurrence of the TAC102 signal in γL262P MiRF172 RNAi cells with different kDNA and nucleus DNA content. PH, phase contrast. Scale bars: 5 µm.
    Figure Legend Snippet: Quantification of TAC102 in MiRF172 RNAi BSF cells. (A) MiRF172 RNAi BSF cells stained for TAC102 (green) and basal bodies (red) from either uninduced (-tet) or RNAi induced [day (d)3] cells. Pictures show maximum intensity projections from immunofluorescence microscopy image stacks of MiRF172 RNAi BSF T. brucei cells. TAC102 was detected with the anti-TAC102 polyclonal rat antibody and the basal bodies with the monoclonal mouse antibody BBA4. The kDNA and the nucleus were stained with DAPI (cyan). (B) γL262P MiF172 RNAi BSF cells stained for MiRF172–PTP (magenta), TAC102 (green), basal bodies (red) and DAPI (cyan). Proteins and DNA were detected with the same antibodies and reagents as in A. MiRF172–PTP was detected with the anti-Protein A antibody. The pictures show maximum intensity projections as in A. (C) Western blot analysis of γL262P MiRF172 RNAi BSF cells. Total protein isolated from uninduced cells (d0) and cells induced with tet for 3 days (d3). C-terminally PTP-tagged MiRF172 was detected with an anti-PAP antibody and TAC102 with the anti-TAC102 monoclonal mouse antibody. Tubulin serves as a loading control. (D) Quantification of the relative occurrence of kDNA discs and nuclei in γL262P MiRF172 RNAi induced and uninduced cells ( n ≥180 for each time point). K, kDNA; N, nucleus. (E) Quantification of TAC102 in γL262P MiRF172 RNAi uninduced (−tet) and cells induced for three days with tet (d3 tet). Black represents the wild-type TAC102 signal and gray stands for a weak TAC102 signal. (F) Quantification of the relative occurrence of the TAC102 signal in γL262P MiRF172 RNAi cells with different kDNA and nucleus DNA content. PH, phase contrast. Scale bars: 5 µm.

    Techniques Used: Staining, Immunofluorescence, Microscopy, Western Blot, Isolation

    30) Product Images from "The RNA Methyltransferase Complex of WTAP, METTL3, and METTL14 Regulates Mitotic Clonal Expansion in Adipogenesis"

    Article Title: The RNA Methyltransferase Complex of WTAP, METTL3, and METTL14 Regulates Mitotic Clonal Expansion in Adipogenesis

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00116-18

    WTAP may recruit METTL3 and METTL14 to RNA in adipocyte differentiation in vitro . (A) 3T3-L1 cells 48 h after the induction by DMI were harvested and treated with RNase or left untreated. Their cytosolic, nuclear, and total proteins were extracted and immunoblotted with anti-WTAP antibody. Note that the increase of nuclear WTAP by DMI was canceled by RNase treatment. The fractionation of nuclear and cytosolic protein was validated by immunoblotting of lamin A/C and α-tubulin, respectively. (B and C) The effect of knockdown of METTL3, METTL14, and WTAP 24 h after DMI treatment in 3T3-L1 cells. (B) The nuclear protein levels analyzed by Western blotting. (C) The immunofluorescence study, shown with METTL3-Alexa Fluor 488 (AF488) (green), METTL14-AF594 (red), WTAP-AF647 (blue), and DNA-DAPI (cyan). Scale bars, 50 μm (left) and 10 μm (right).
    Figure Legend Snippet: WTAP may recruit METTL3 and METTL14 to RNA in adipocyte differentiation in vitro . (A) 3T3-L1 cells 48 h after the induction by DMI were harvested and treated with RNase or left untreated. Their cytosolic, nuclear, and total proteins were extracted and immunoblotted with anti-WTAP antibody. Note that the increase of nuclear WTAP by DMI was canceled by RNase treatment. The fractionation of nuclear and cytosolic protein was validated by immunoblotting of lamin A/C and α-tubulin, respectively. (B and C) The effect of knockdown of METTL3, METTL14, and WTAP 24 h after DMI treatment in 3T3-L1 cells. (B) The nuclear protein levels analyzed by Western blotting. (C) The immunofluorescence study, shown with METTL3-Alexa Fluor 488 (AF488) (green), METTL14-AF594 (red), WTAP-AF647 (blue), and DNA-DAPI (cyan). Scale bars, 50 μm (left) and 10 μm (right).

    Techniques Used: In Vitro, Fractionation, Western Blot, Immunofluorescence

    Immunofluorescence staining of WTAP, METTL3, and METTL14 in nuclei is enhanced in adipocyte differentiation of 3T3-L1 cells. Immunofluorescence study in 3T3-L1 cells, shown with METTL3-Alexa Fluor 488 (AF488) (green), METTL14-AF594 (red), WTAP-AF647 (blue), and DNA-DAPI (cyan). (A and B) The time course of METTL3, METTL14, and WTAP staining during adipocyte differentiation by DMI induction. (C) The merged images of DAPI with METTL3, METTL14, and WTAP in the cell without DMI induction. Scale bars, 50 μm (A) and 10 μm (B and C).
    Figure Legend Snippet: Immunofluorescence staining of WTAP, METTL3, and METTL14 in nuclei is enhanced in adipocyte differentiation of 3T3-L1 cells. Immunofluorescence study in 3T3-L1 cells, shown with METTL3-Alexa Fluor 488 (AF488) (green), METTL14-AF594 (red), WTAP-AF647 (blue), and DNA-DAPI (cyan). (A and B) The time course of METTL3, METTL14, and WTAP staining during adipocyte differentiation by DMI induction. (C) The merged images of DAPI with METTL3, METTL14, and WTAP in the cell without DMI induction. Scale bars, 50 μm (A) and 10 μm (B and C).

    Techniques Used: Immunofluorescence, Staining

    31) Product Images from "The RNA Methyltransferase Complex of WTAP, METTL3, and METTL14 Regulates Mitotic Clonal Expansion in Adipogenesis"

    Article Title: The RNA Methyltransferase Complex of WTAP, METTL3, and METTL14 Regulates Mitotic Clonal Expansion in Adipogenesis

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00116-18

    WTAP may recruit METTL3 and METTL14 to RNA in adipocyte differentiation in vitro . (A) 3T3-L1 cells 48 h after the induction by DMI were harvested and treated with RNase or left untreated. Their cytosolic, nuclear, and total proteins were extracted and immunoblotted with anti-WTAP antibody. Note that the increase of nuclear WTAP by DMI was canceled by RNase treatment. The fractionation of nuclear and cytosolic protein was validated by immunoblotting of lamin A/C and α-tubulin, respectively. (B and C) The effect of knockdown of METTL3, METTL14, and WTAP 24 h after DMI treatment in 3T3-L1 cells. (B) The nuclear protein levels analyzed by Western blotting. (C) The immunofluorescence study, shown with METTL3-Alexa Fluor 488 (AF488) (green), METTL14-AF594 (red), WTAP-AF647 (blue), and DNA-DAPI (cyan). Scale bars, 50 μm (left) and 10 μm (right).
    Figure Legend Snippet: WTAP may recruit METTL3 and METTL14 to RNA in adipocyte differentiation in vitro . (A) 3T3-L1 cells 48 h after the induction by DMI were harvested and treated with RNase or left untreated. Their cytosolic, nuclear, and total proteins were extracted and immunoblotted with anti-WTAP antibody. Note that the increase of nuclear WTAP by DMI was canceled by RNase treatment. The fractionation of nuclear and cytosolic protein was validated by immunoblotting of lamin A/C and α-tubulin, respectively. (B and C) The effect of knockdown of METTL3, METTL14, and WTAP 24 h after DMI treatment in 3T3-L1 cells. (B) The nuclear protein levels analyzed by Western blotting. (C) The immunofluorescence study, shown with METTL3-Alexa Fluor 488 (AF488) (green), METTL14-AF594 (red), WTAP-AF647 (blue), and DNA-DAPI (cyan). Scale bars, 50 μm (left) and 10 μm (right).

    Techniques Used: In Vitro, Fractionation, Western Blot, Immunofluorescence

    Immunofluorescence staining of WTAP, METTL3, and METTL14 in nuclei is enhanced in adipocyte differentiation of 3T3-L1 cells. Immunofluorescence study in 3T3-L1 cells, shown with METTL3-Alexa Fluor 488 (AF488) (green), METTL14-AF594 (red), WTAP-AF647 (blue), and DNA-DAPI (cyan). (A and B) The time course of METTL3, METTL14, and WTAP staining during adipocyte differentiation by DMI induction. (C) The merged images of DAPI with METTL3, METTL14, and WTAP in the cell without DMI induction. Scale bars, 50 μm (A) and 10 μm (B and C).
    Figure Legend Snippet: Immunofluorescence staining of WTAP, METTL3, and METTL14 in nuclei is enhanced in adipocyte differentiation of 3T3-L1 cells. Immunofluorescence study in 3T3-L1 cells, shown with METTL3-Alexa Fluor 488 (AF488) (green), METTL14-AF594 (red), WTAP-AF647 (blue), and DNA-DAPI (cyan). (A and B) The time course of METTL3, METTL14, and WTAP staining during adipocyte differentiation by DMI induction. (C) The merged images of DAPI with METTL3, METTL14, and WTAP in the cell without DMI induction. Scale bars, 50 μm (A) and 10 μm (B and C).

    Techniques Used: Immunofluorescence, Staining

    32) Product Images from "The VAR2CSA malaria protein efficiently retrieves circulating tumor cells in an EpCAM-independent manner"

    Article Title: The VAR2CSA malaria protein efficiently retrieves circulating tumor cells in an EpCAM-independent manner

    Journal: Nature Communications

    doi: 10.1038/s41467-018-05793-2

    rVAR2-capture of CTCs from prostate cancer patients. a Number of CK+ CD45− DAPI+ CTCs isolated from 7.5 mL blood from four prostate cancer patients using rVAR2 or anti-EpCAM antibody-coated beads or the CellSearch® CTC platform. b CTC enumeration using rVAR2-coated beads on blood samples from prostate cancer patients with different disease stages ( n = 25) as well as from healthy controls ( n = 16) and patients with non-malignant diseases ( n = 12). ( P = 0.0001 for association between disease severity and CTC number, Kruskal–Wallis test). UTI: urinary tract infection, BPH: benign prostatic hyperplasia
    Figure Legend Snippet: rVAR2-capture of CTCs from prostate cancer patients. a Number of CK+ CD45− DAPI+ CTCs isolated from 7.5 mL blood from four prostate cancer patients using rVAR2 or anti-EpCAM antibody-coated beads or the CellSearch® CTC platform. b CTC enumeration using rVAR2-coated beads on blood samples from prostate cancer patients with different disease stages ( n = 25) as well as from healthy controls ( n = 16) and patients with non-malignant diseases ( n = 12). ( P = 0.0001 for association between disease severity and CTC number, Kruskal–Wallis test). UTI: urinary tract infection, BPH: benign prostatic hyperplasia

    Techniques Used: Isolation, Infection

    rVAR2 binds specifically to a diverse repertoire of cancer cells. a Detection of cancer cells using the CytoTrack platform. Representative confocal microscopy images of indicated cell lines. Cancer cells were mixed with PBMCs in a 1:5000 ratio prior to analysis and stained with His-tagged rVAR2 in combination with anti-penta His Alexa Fluor 488 (green), an anti-CD45 Cy5 antibody (red), and DAPI (blue). Scale bars, 10 µm. b Flow cytometry measured fluorescence intensity of three breast cancer (left panel), three prostate cancer (middle panel), and three colorectal cancer (right panel) cell lines stained by His-tagged rVAR2 in combination with anti-penta His Alexa Fluor 488 ( y -axis) and a PE-conjugated anti-EpCAM antibody ( x -axis)
    Figure Legend Snippet: rVAR2 binds specifically to a diverse repertoire of cancer cells. a Detection of cancer cells using the CytoTrack platform. Representative confocal microscopy images of indicated cell lines. Cancer cells were mixed with PBMCs in a 1:5000 ratio prior to analysis and stained with His-tagged rVAR2 in combination with anti-penta His Alexa Fluor 488 (green), an anti-CD45 Cy5 antibody (red), and DAPI (blue). Scale bars, 10 µm. b Flow cytometry measured fluorescence intensity of three breast cancer (left panel), three prostate cancer (middle panel), and three colorectal cancer (right panel) cell lines stained by His-tagged rVAR2 in combination with anti-penta His Alexa Fluor 488 ( y -axis) and a PE-conjugated anti-EpCAM antibody ( x -axis)

    Techniques Used: Confocal Microscopy, Staining, Flow Cytometry, Cytometry, Fluorescence

    33) Product Images from "Characterization of the cytokine and maturation responses of pure populations of porcine plasmacytoid dendritic cells to porcine viruses and toll-like receptor agonists"

    Article Title: Characterization of the cytokine and maturation responses of pure populations of porcine plasmacytoid dendritic cells to porcine viruses and toll-like receptor agonists

    Journal: Veterinary Immunology and Immunopathology

    doi: 10.1016/j.vetimm.2009.10.026

    Constitutive expression of IRF-7 by porcine PBMC subsets. Stained porcine PBMC were virtually separated into CD4 − CD172 − , CD4 − CD172 hi , CD4 + CD172 − , and CD4 hi CD172 lo subsets in CD4 versus CD172 histograms (comparable to Fig. 1 A, PBMC panel). (A) Fluorescent intensities of electronically gated PBMC subsets reacting with anti-IRF-7 polyclonal IgG (clear areas) or a non-specific counterpart (shaded areas). Prior to analysis, areas defining each respective subset in the two histograms were made spatially identical. (B) Mean fluorescence intensities (MFIs) of electronically gated, IRF-7 stained PBMC subsets. Vertical bars represent the mean ± SEM of a representative experiment ( n = 3).
    Figure Legend Snippet: Constitutive expression of IRF-7 by porcine PBMC subsets. Stained porcine PBMC were virtually separated into CD4 − CD172 − , CD4 − CD172 hi , CD4 + CD172 − , and CD4 hi CD172 lo subsets in CD4 versus CD172 histograms (comparable to Fig. 1 A, PBMC panel). (A) Fluorescent intensities of electronically gated PBMC subsets reacting with anti-IRF-7 polyclonal IgG (clear areas) or a non-specific counterpart (shaded areas). Prior to analysis, areas defining each respective subset in the two histograms were made spatially identical. (B) Mean fluorescence intensities (MFIs) of electronically gated, IRF-7 stained PBMC subsets. Vertical bars represent the mean ± SEM of a representative experiment ( n = 3).

    Techniques Used: Expressing, Staining, Fluorescence

    34) Product Images from "Streptococcal Cysteine Protease-Mediated Cleavage of Desmogleins Is Involved in the Pathogenesis of Cutaneous Infection"

    Article Title: Streptococcal Cysteine Protease-Mediated Cleavage of Desmogleins Is Involved in the Pathogenesis of Cutaneous Infection

    Journal: Frontiers in Cellular and Infection Microbiology

    doi: 10.3389/fcimb.2018.00010

    SpeB-mediated cleavage of desmogleins contributes to epidermal barrier dysfunction. Cutaneous sections obtained from mice infected with the examined S. pyogenes strains were subjected to immunofluorescence staining. Dsg1 and Dsg3 were labeled with anti-Dsg1 and anti-Dsg3 antibodies, respectively, followed by incubation with an Alexa Fluor 647-conjugated antibody. S. pyogenes was labeled with anti-Group A carbohydrate and Alexa Fluor 488-conjugated antibodies, and cell nuclei were stained with Hoechst 33342. Obtained tissue sections were analyzed using a confocal laser microscope. The boxed area is magnified in the box panel below. Data shown are representative of at least three separate experiments.
    Figure Legend Snippet: SpeB-mediated cleavage of desmogleins contributes to epidermal barrier dysfunction. Cutaneous sections obtained from mice infected with the examined S. pyogenes strains were subjected to immunofluorescence staining. Dsg1 and Dsg3 were labeled with anti-Dsg1 and anti-Dsg3 antibodies, respectively, followed by incubation with an Alexa Fluor 647-conjugated antibody. S. pyogenes was labeled with anti-Group A carbohydrate and Alexa Fluor 488-conjugated antibodies, and cell nuclei were stained with Hoechst 33342. Obtained tissue sections were analyzed using a confocal laser microscope. The boxed area is magnified in the box panel below. Data shown are representative of at least three separate experiments.

    Techniques Used: Mouse Assay, Infection, Immunofluorescence, Staining, Labeling, Incubation, Microscopy

    35) Product Images from "FOXL2 Interacts with Steroidogenic Factor-1 (SF-1) and Represses SF-1-Induced CYP17 Transcription in Granulosa Cells"

    Article Title: FOXL2 Interacts with Steroidogenic Factor-1 (SF-1) and Represses SF-1-Induced CYP17 Transcription in Granulosa Cells

    Journal: Molecular Endocrinology

    doi: 10.1210/me.2009-0375

    were coexpressed with or without SF-1 in 293T cells. Immunoprecipitation (IP) experiments were conducted using α-SF-1 or α-Myc antibodies. Equal amounts of total protein from the cell lysates were used in each lane. Lysates were prepared 24 h after transfection, separated into nuclear and cytosolic fractions, and immunoblotted (IB) with the respective antibodies. B, The 293T cells overexpressed WT or mutant FOXL2 with or without SF-1. Lysates were prepared 24 h after transfection, separated into heavy membrane and cytosolic fractions, and immunoblotted with the respective antibodies. C, Myc-tagged WT and mutated FOXL2 were overexpressed in 293T cells, and fluorescence confocal microscopy images are shown. Myc-FOXL2s were visualized using Alexa Fluor 546 goat antimouse IgG. Visualization of nucleus was determined using 4′,6-diamidino-2-phenylindole (DAPI). D, Myc-FOXL2s were coexpressed with SF-1 in 293T cells, and the FOXL2s and Flag-tagged SF-1 proteins were visualized using Alexa Fluor 546 goat antimouse IgG or Alexa Fluor 488 goat antirabbit IgG, respectively.
    Figure Legend Snippet: were coexpressed with or without SF-1 in 293T cells. Immunoprecipitation (IP) experiments were conducted using α-SF-1 or α-Myc antibodies. Equal amounts of total protein from the cell lysates were used in each lane. Lysates were prepared 24 h after transfection, separated into nuclear and cytosolic fractions, and immunoblotted (IB) with the respective antibodies. B, The 293T cells overexpressed WT or mutant FOXL2 with or without SF-1. Lysates were prepared 24 h after transfection, separated into heavy membrane and cytosolic fractions, and immunoblotted with the respective antibodies. C, Myc-tagged WT and mutated FOXL2 were overexpressed in 293T cells, and fluorescence confocal microscopy images are shown. Myc-FOXL2s were visualized using Alexa Fluor 546 goat antimouse IgG. Visualization of nucleus was determined using 4′,6-diamidino-2-phenylindole (DAPI). D, Myc-FOXL2s were coexpressed with SF-1 in 293T cells, and the FOXL2s and Flag-tagged SF-1 proteins were visualized using Alexa Fluor 546 goat antimouse IgG or Alexa Fluor 488 goat antirabbit IgG, respectively.

    Techniques Used: Immunoprecipitation, Transfection, Mutagenesis, Fluorescence, Confocal Microscopy

    36) Product Images from "Specialized attachment structure of the fish pathogenic oomycete Saprolegnia parasitica"

    Article Title: Specialized attachment structure of the fish pathogenic oomycete Saprolegnia parasitica

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0190361

    Protein immunolocalization of bundles of hooked hairs of Saprolegnia parasitica . The figure shows that treatment with PNGase F prevents the unspecific binding of the anti-β-tubulin antibodies. Differential interference contrast (A and D), confocal light fluorescent (B and E) and merged micrographs (C and F) of bundles (Bu) of hooked hairs of secondary cysts. The cysts were enzymatically treated (D, E, and F) or not (A, B and C) with PNGase F, and incubated in the presence of monoclonal anti-β-tubulin antibodies (1:5000) and goat-anti-rabbit Alexa Fluor 488 conjugate as secondary antibodies (shown in green color). Scale bar 1μm.
    Figure Legend Snippet: Protein immunolocalization of bundles of hooked hairs of Saprolegnia parasitica . The figure shows that treatment with PNGase F prevents the unspecific binding of the anti-β-tubulin antibodies. Differential interference contrast (A and D), confocal light fluorescent (B and E) and merged micrographs (C and F) of bundles (Bu) of hooked hairs of secondary cysts. The cysts were enzymatically treated (D, E, and F) or not (A, B and C) with PNGase F, and incubated in the presence of monoclonal anti-β-tubulin antibodies (1:5000) and goat-anti-rabbit Alexa Fluor 488 conjugate as secondary antibodies (shown in green color). Scale bar 1μm.

    Techniques Used: Binding Assay, Incubation

    37) Product Images from "Cag Type IV Secretion System: CagI Independent Bacterial Surface Localization of CagA"

    Article Title: Cag Type IV Secretion System: CagI Independent Bacterial Surface Localization of CagA

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0074620

    Cellular localization of CagI in H. pylori . ( A ) Western blots showing sub-cellular fractionation of wild-type H. pylori . TC, S and TM indicate total-cell lysate, soluble (cytoplasmic/periplasmic) and total membrane fractions respectively. Volume of TM was adjusted corresponding to that of S fraction and then equal volume of each was loaded in to gel. ( B ) Western blots showing osmotic shock analysis of H. pylori . r-TM, r-OS, and Os stand for residual total membrane, residual osmotic shocked content (cytosolic contents) and osmotic shocked fraction (periplasmic contents) respectively. Antibodies used are marked. ( C ) Western blots showing selective biotinylation of CagI from biotin labeled wild-type H. pylori (Hp) and HpΔcagT mutant strains. TM and S stand for total membrane fraction and soluble fraction. Antibodies used are marked. M-indicates molecular size standard. Arrow indicates position of non-biotinylated DnaB. ( D ) Immunofluorescence microscopy showing cellular localization of CagI in wild-type H. pylori (Hp) under permeabilized (P) and non-permeabilized (NP) conditions. H. pylori cells were fixed and permeabilized with 0.2% Triton X-100 and probed with antibodies as indicated. Alexa fluor 488 (green colour) and Alexa fluor 594 (red colour) conjugated secondary antibodies were used for signal generation and finally examined by immunofluorescence microscopy. Permeabilized HpΔcagI cells are showing specificity of α-CagI antibody. Pre-immune serum was used as control antibody. Scale bars indicate 5µM.
    Figure Legend Snippet: Cellular localization of CagI in H. pylori . ( A ) Western blots showing sub-cellular fractionation of wild-type H. pylori . TC, S and TM indicate total-cell lysate, soluble (cytoplasmic/periplasmic) and total membrane fractions respectively. Volume of TM was adjusted corresponding to that of S fraction and then equal volume of each was loaded in to gel. ( B ) Western blots showing osmotic shock analysis of H. pylori . r-TM, r-OS, and Os stand for residual total membrane, residual osmotic shocked content (cytosolic contents) and osmotic shocked fraction (periplasmic contents) respectively. Antibodies used are marked. ( C ) Western blots showing selective biotinylation of CagI from biotin labeled wild-type H. pylori (Hp) and HpΔcagT mutant strains. TM and S stand for total membrane fraction and soluble fraction. Antibodies used are marked. M-indicates molecular size standard. Arrow indicates position of non-biotinylated DnaB. ( D ) Immunofluorescence microscopy showing cellular localization of CagI in wild-type H. pylori (Hp) under permeabilized (P) and non-permeabilized (NP) conditions. H. pylori cells were fixed and permeabilized with 0.2% Triton X-100 and probed with antibodies as indicated. Alexa fluor 488 (green colour) and Alexa fluor 594 (red colour) conjugated secondary antibodies were used for signal generation and finally examined by immunofluorescence microscopy. Permeabilized HpΔcagI cells are showing specificity of α-CagI antibody. Pre-immune serum was used as control antibody. Scale bars indicate 5µM.

    Techniques Used: Western Blot, Cell Fractionation, Labeling, Mutagenesis, Immunofluorescence, Microscopy

    Surface localization of CagA in wild-type and HpΔcagI strains. ( A ) Immunofluorescence microscopy showing surface localization of CagA in wild-type and HpΔcagI strains . Wild-type and HpΔcagI cells were fixed and one set was permeabilized with 0.2% Triton X-100. M, NP and P stand for merge, non-permeabilized and permeabilized cell respectively. Primary antibodies used in IFM are indicated. Secondary antibodies used were Alexa fluor 488 (green colour) and Alexa fluor 594 (red colour) conjugated. ( B and C ) Immunogold electron microscopy (IEM) showing surface localization of CagA in Hp Δ cagI and wild-type H. pylori (Hp) strains . Immunogold labeling of H. pylori strains ultrathin sections were performed as described in Materials and Methods. ( B ) HpΔcagI cells were stained with anti-CagA and gold-labeled secondary antibody. ( C ) Wild-type H. pylori cells were stained with anti-CagA and gold-labeled secondary antibody. ( D ) HpΔcagA cells were stained with anti-CagA and gold-labeled secondary antibody. ( E ) Pre-immune (preimm.) serum was used as negative control. Scale bars indicate 100 nm. Arrowheads indicate location of gold-labeled secondary antibody.
    Figure Legend Snippet: Surface localization of CagA in wild-type and HpΔcagI strains. ( A ) Immunofluorescence microscopy showing surface localization of CagA in wild-type and HpΔcagI strains . Wild-type and HpΔcagI cells were fixed and one set was permeabilized with 0.2% Triton X-100. M, NP and P stand for merge, non-permeabilized and permeabilized cell respectively. Primary antibodies used in IFM are indicated. Secondary antibodies used were Alexa fluor 488 (green colour) and Alexa fluor 594 (red colour) conjugated. ( B and C ) Immunogold electron microscopy (IEM) showing surface localization of CagA in Hp Δ cagI and wild-type H. pylori (Hp) strains . Immunogold labeling of H. pylori strains ultrathin sections were performed as described in Materials and Methods. ( B ) HpΔcagI cells were stained with anti-CagA and gold-labeled secondary antibody. ( C ) Wild-type H. pylori cells were stained with anti-CagA and gold-labeled secondary antibody. ( D ) HpΔcagA cells were stained with anti-CagA and gold-labeled secondary antibody. ( E ) Pre-immune (preimm.) serum was used as negative control. Scale bars indicate 100 nm. Arrowheads indicate location of gold-labeled secondary antibody.

    Techniques Used: Immunofluorescence, Microscopy, Electron Microscopy, Labeling, Staining, Negative Control

    38) Product Images from "Anti-tumor macrophages activated by ferumoxytol combined or surface-functionalized with the TLR3 agonist poly (I : C) promote melanoma regression"

    Article Title: Anti-tumor macrophages activated by ferumoxytol combined or surface-functionalized with the TLR3 agonist poly (I : C) promote melanoma regression

    Journal: Theranostics

    doi: 10.7150/thno.29746

    Therapeutic benefits of FP-NPs for lung metastasis. C57BL/6 mice bearing lung metastasis were prepared by intravenous injection with 5 × 10 5 B16F10 melanoma cells overnight and were intravenously treated with PBS, FP-NPs (FMT-NH 2 200 μg composited with PIC 10 μg), and FMT-NH 2 (200 μg) combined with PIC (10 μg) every other day for three times. (A) Photograph of dissected lung tissues from tumor-bearing mice after the indicated treatment. The blank arrows indicated the metastatic nodules. (B) Corresponding quantitative metastatic nodules displayed in (A). (C) Histopathologic analysis of H E-stained tissue sections of the lung in tumor-bearing mice with the indicated treatment. The blank arrows indicated the metastatic nodules. (D, E) FCM analysis of leukocyte and macrophage populations within lung tissues of tumor-bearing mice with the indicated treatment. (F) Representative immunofluorescence staining for CD86 (red) and CD206 (green) in lung metastatic mice with the indicated treatment, Scale bar: 30 μm. (G-I) Relative gene expression of iNOS and Arg-1, and NO concentration in lung tissues quantified by qRT-PCR and Griess test, respectively. (J) FCM analysis of MDSC populations in the blood of tumor bearing mice with the indicated treatment. All representative data are from three independent experiments. Error bars, SD. *P
    Figure Legend Snippet: Therapeutic benefits of FP-NPs for lung metastasis. C57BL/6 mice bearing lung metastasis were prepared by intravenous injection with 5 × 10 5 B16F10 melanoma cells overnight and were intravenously treated with PBS, FP-NPs (FMT-NH 2 200 μg composited with PIC 10 μg), and FMT-NH 2 (200 μg) combined with PIC (10 μg) every other day for three times. (A) Photograph of dissected lung tissues from tumor-bearing mice after the indicated treatment. The blank arrows indicated the metastatic nodules. (B) Corresponding quantitative metastatic nodules displayed in (A). (C) Histopathologic analysis of H E-stained tissue sections of the lung in tumor-bearing mice with the indicated treatment. The blank arrows indicated the metastatic nodules. (D, E) FCM analysis of leukocyte and macrophage populations within lung tissues of tumor-bearing mice with the indicated treatment. (F) Representative immunofluorescence staining for CD86 (red) and CD206 (green) in lung metastatic mice with the indicated treatment, Scale bar: 30 μm. (G-I) Relative gene expression of iNOS and Arg-1, and NO concentration in lung tissues quantified by qRT-PCR and Griess test, respectively. (J) FCM analysis of MDSC populations in the blood of tumor bearing mice with the indicated treatment. All representative data are from three independent experiments. Error bars, SD. *P

    Techniques Used: Mouse Assay, Injection, Staining, Immunofluorescence, Expressing, Concentration Assay, Quantitative RT-PCR

    39) Product Images from "Independent modes of disease repair by AIM protein distinguished in AIM-felinized mice"

    Article Title: Independent modes of disease repair by AIM protein distinguished in AIM-felinized mice

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-31580-6

    States of inflammation and fibrosis in the liver in the absence of serum IgM-free AIM. ( a ) The mRNA levels of various genes responsive to different types of stresses (i.e., endoplasmic reticulum, mitochondrial, or oxidative stress) addressed by qPCR using RNA from the whole liver of AIM-felinized, WT, AIM −/− , and Δsµ mice before and after being fed an HFD for 12 weeks (n = 4 before HFD and n = 5–6 for 12-week HFD per group). Error bar indicates the SEM. (b,c) The mRNA levels of inflammatory cytokine genes in the liver, as assessed by qPCR using the RNA as in ( a ). (d ) Representative photomicrographs of liver from AIM-felinized, WT, AIM −/− , and Δsμ mice fed an HFD for 12 weeks stained with Sirius red Stain. Scale bars, 100 μm. (e) The mRNA levels of fibrogenetic genes in the liver, as assessed by qPCR using the same RNA as in ( a ). In (a – c) and (e) , *is used to represent the statistical significance between the values of each mouse strain group within the same period, whereas # is attached to the bar of 12 w when the value of 12 w was significantly changed compared with that of 0 w in the same mouse strain group.
    Figure Legend Snippet: States of inflammation and fibrosis in the liver in the absence of serum IgM-free AIM. ( a ) The mRNA levels of various genes responsive to different types of stresses (i.e., endoplasmic reticulum, mitochondrial, or oxidative stress) addressed by qPCR using RNA from the whole liver of AIM-felinized, WT, AIM −/− , and Δsµ mice before and after being fed an HFD for 12 weeks (n = 4 before HFD and n = 5–6 for 12-week HFD per group). Error bar indicates the SEM. (b,c) The mRNA levels of inflammatory cytokine genes in the liver, as assessed by qPCR using the RNA as in ( a ). (d ) Representative photomicrographs of liver from AIM-felinized, WT, AIM −/− , and Δsμ mice fed an HFD for 12 weeks stained with Sirius red Stain. Scale bars, 100 μm. (e) The mRNA levels of fibrogenetic genes in the liver, as assessed by qPCR using the same RNA as in ( a ). In (a – c) and (e) , *is used to represent the statistical significance between the values of each mouse strain group within the same period, whereas # is attached to the bar of 12 w when the value of 12 w was significantly changed compared with that of 0 w in the same mouse strain group.

    Techniques Used: Real-time Polymerase Chain Reaction, Mouse Assay, Staining

    Effect of serum IgM-free AIM on obesity and liver steatosis. ( a,b) Weights from AIM-felinized, WT, AIM −/− , and Δsµ mice before and after being fed an HFD for 12 weeks (n = 4 before HFD and n = 6–9 for 12-week HFD per group). Error bar indicates the SEM. (a) Body weights. (b ) Weights of epidydimal adipose tissues. (c) Representative photomicrographs of epididymal fat tissues from AIM-felinized, WT, AIM −/− , and Δsµ mice fed an HFD for 12 weeks stained with H E. Adipocyte sizes of 50 independent adipocytes in different areas were evaluated. Results are presented as average ± SEM (in μm 2 ). Scale bars, 100 μm. ( d ) Representative photomicrographs of epididymal fat tissues from AIM-felinized, WT, and AIM −/− mice (fed an HFD for 12 weeks) stained for AIM (blue), F4/80 (macrophage marker; green), and IgM (red, WT and AIM −/− mice. See Supplementary Fig. 1 for AIM-felinized mice). Yellow arrows represent where IgM-free AIM signals exist, while red arrows indicate where AIM is co-stained with IgM. Scale bars, 100 μm. (e) The mRNA levels of AIM and F4/80 were assessed by qPCR using RNA isolated from epididymal fat in WT mice before or after being fed an HFD for 12 weeks. Values were normalized to those of GAPDH and presented as the expression relative to that of AIM from lean WT mice liver and of F4/80 from fat tissues before being fed an HFD (n = 4 per group). Error bar indicates the SEM. (f) Representative photomicrographs of liver from AIM-felinized, WT, AIM −/− , and Δsµ mice fed an HFD for 12 weeks stained with H E. Scale bars, 100 µm. (g) Liver weights and TG contents from AIM-felinized, WT, AIM −/− , and Δsµ mice before and after being fed an HFD for 12 weeks (n = 4 before HFD and n = 6–9 for 12-week HFD per group). Error bar indicates the SEM.
    Figure Legend Snippet: Effect of serum IgM-free AIM on obesity and liver steatosis. ( a,b) Weights from AIM-felinized, WT, AIM −/− , and Δsµ mice before and after being fed an HFD for 12 weeks (n = 4 before HFD and n = 6–9 for 12-week HFD per group). Error bar indicates the SEM. (a) Body weights. (b ) Weights of epidydimal adipose tissues. (c) Representative photomicrographs of epididymal fat tissues from AIM-felinized, WT, AIM −/− , and Δsµ mice fed an HFD for 12 weeks stained with H E. Adipocyte sizes of 50 independent adipocytes in different areas were evaluated. Results are presented as average ± SEM (in μm 2 ). Scale bars, 100 μm. ( d ) Representative photomicrographs of epididymal fat tissues from AIM-felinized, WT, and AIM −/− mice (fed an HFD for 12 weeks) stained for AIM (blue), F4/80 (macrophage marker; green), and IgM (red, WT and AIM −/− mice. See Supplementary Fig. 1 for AIM-felinized mice). Yellow arrows represent where IgM-free AIM signals exist, while red arrows indicate where AIM is co-stained with IgM. Scale bars, 100 μm. (e) The mRNA levels of AIM and F4/80 were assessed by qPCR using RNA isolated from epididymal fat in WT mice before or after being fed an HFD for 12 weeks. Values were normalized to those of GAPDH and presented as the expression relative to that of AIM from lean WT mice liver and of F4/80 from fat tissues before being fed an HFD (n = 4 per group). Error bar indicates the SEM. (f) Representative photomicrographs of liver from AIM-felinized, WT, AIM −/− , and Δsµ mice fed an HFD for 12 weeks stained with H E. Scale bars, 100 µm. (g) Liver weights and TG contents from AIM-felinized, WT, AIM −/− , and Δsµ mice before and after being fed an HFD for 12 weeks (n = 4 before HFD and n = 6–9 for 12-week HFD per group). Error bar indicates the SEM.

    Techniques Used: Mouse Assay, Staining, Marker, Real-time Polymerase Chain Reaction, Isolation, Expressing

    40) Product Images from "Type I vs type II spiral ganglion neurons exhibit differential survival and neuritogenesis during cochlear development"

    Article Title: Type I vs type II spiral ganglion neurons exhibit differential survival and neuritogenesis during cochlear development

    Journal: Neural Development

    doi: 10.1186/1749-8104-6-33

    The expression pattern of β-tubulin and peripherin in type I and type II spiral ganglion neurons is maintained in vitro . Maximum intensity projections from confocal z-stacks show immunofluorescence labeling for β-tubulin (green) and peripherin (red) in the mid-apex-mid-turn region of P1 (D,E) and P7 (A-C) mouse cochlea following 48 hour organotypic culture (SGNs and organ of Corti intact). Rhodamine-phalloidin labeling (grey in (B-E)) confirmed the survival of target hair cells. (A,B) Immunolabeling of organotypic cultures of P7 tissue shows the inner spiral plexus (isp), formed by type I fibers, contains β-tubulin protein only (detailed in (B)), whereas the outer spiral bundles (osb) that arise from type II fibers express both β-tubulin and peripherin (arrows in (B,C)). (C) Examination of peripherin immunofluorescence alone confirms that this protein is expressed only in fibers that cross the tunnel of Corti and innervate the outer hair cells (ohc). (D,E) Confocal imaging of organotypic culture of P1 cochlear tissue shows that singularly β-tubulin immunofluorescent fibers beneath the inner hair cells (ihc) are reduced in density compared to the in vivo situation (compare (D) with Figure 1B). A large portion of these fibers co-immunolabel for peripherin (D,E). The outer spiral bundle double immunolabels with β-tubulin and peripherin (D,E). Scale bars: 150 μm (A); 50 μm (C-E).
    Figure Legend Snippet: The expression pattern of β-tubulin and peripherin in type I and type II spiral ganglion neurons is maintained in vitro . Maximum intensity projections from confocal z-stacks show immunofluorescence labeling for β-tubulin (green) and peripherin (red) in the mid-apex-mid-turn region of P1 (D,E) and P7 (A-C) mouse cochlea following 48 hour organotypic culture (SGNs and organ of Corti intact). Rhodamine-phalloidin labeling (grey in (B-E)) confirmed the survival of target hair cells. (A,B) Immunolabeling of organotypic cultures of P7 tissue shows the inner spiral plexus (isp), formed by type I fibers, contains β-tubulin protein only (detailed in (B)), whereas the outer spiral bundles (osb) that arise from type II fibers express both β-tubulin and peripherin (arrows in (B,C)). (C) Examination of peripherin immunofluorescence alone confirms that this protein is expressed only in fibers that cross the tunnel of Corti and innervate the outer hair cells (ohc). (D,E) Confocal imaging of organotypic culture of P1 cochlear tissue shows that singularly β-tubulin immunofluorescent fibers beneath the inner hair cells (ihc) are reduced in density compared to the in vivo situation (compare (D) with Figure 1B). A large portion of these fibers co-immunolabel for peripherin (D,E). The outer spiral bundle double immunolabels with β-tubulin and peripherin (D,E). Scale bars: 150 μm (A); 50 μm (C-E).

    Techniques Used: Expressing, In Vitro, Immunofluorescence, Labeling, Immunolabeling, Imaging, Immunohistochemistry, In Vivo

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    Transfection:

    Article Title: HDAC4 regulates satellite cell proliferation and differentiation by targeting P21 and Sharp1 genes
    Article Snippet: .. To better detect the green fluorescent protein in transfected cells, we used primary anti-GFP antibody (Life technologies) diluted 1:400 in 1% BSA PBS, by following the above protocol and adding a permeabilization step of 5 minutes with 0.25% Triton-X 100 (Sigma-Aldrich) in PBS before blocking in 10% goat serum in PBS. .. For P21 immunofluorescence, cells were fixed with 4% PFA buffered solution for 10 minutes and permeabilized with 0.25% Triton-X 100 (Sigma-Aldrich).

    Flow Cytometry:

    Article Title: An Alternative Phosphorylation Switch in Integrin β2 (CD18) Tail for Dok1 Binding
    Article Snippet: .. Clones expressing Dok1-CFP were screened by flow cytometry analyses and verified by immunoblotting with anti-GFP antibody (Life Technologies, Grand Island, NY). .. Stable K562 cells expressing Dok1-CFP transfected with integrin αL, integrin β2-YFP and CCR5 plasmids (12 μg each) were stained with either 1 μg each of mAb MHM24 (anti-αL) or mAb 2D7 (anti-CCR5, BD Biosciences, San Jose, CA) followed by APC-conjugated secondary antibody (goat anti-mouse IgG, BD Biosciences, 1:500 dilution).

    Immunostaining:

    Article Title: Subunit composition of a DEG/ENaC mechanosensory channel of Caenorhabditis elegans
    Article Snippet: .. The following antibodies were used for immunostaining: anti–MEC-2 N terminus , anti-FLAG (mouse, F1804; Sigma), and anti-GFP (rabbit polyclonal and mouse monoclonal 3E6; Life Technologies) diluted 1:200, Rhodamine Red-X-conjugated and Alexa Fluor 488-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories), and Alexa Fluor 488/555-conjugated goat anti-rabbit/mouse IgG (Life Technologies) diluted 1:700. .. Cross-reactions between secondary antibodies and primary antibodies from different species have been examined and found to be minimal.

    Cytometry:

    Article Title: An Alternative Phosphorylation Switch in Integrin β2 (CD18) Tail for Dok1 Binding
    Article Snippet: .. Clones expressing Dok1-CFP were screened by flow cytometry analyses and verified by immunoblotting with anti-GFP antibody (Life Technologies, Grand Island, NY). .. Stable K562 cells expressing Dok1-CFP transfected with integrin αL, integrin β2-YFP and CCR5 plasmids (12 μg each) were stained with either 1 μg each of mAb MHM24 (anti-αL) or mAb 2D7 (anti-CCR5, BD Biosciences, San Jose, CA) followed by APC-conjugated secondary antibody (goat anti-mouse IgG, BD Biosciences, 1:500 dilution).

    Immunoprecipitation:

    Article Title: DAF-16/FOXO and HLH-30/TFEB function as combinatorial transcription factors to promote stress resistance and longevity
    Article Snippet: .. DAF-16::GFP was immunoprecipitated using anti-GFP antibody (3E6, Invitrogen) coupled to Protein A resin (Biorad). .. Immunoprecipitated proteins were eluted using 100 mM glycine at pH2.6.

    Generated:

    Article Title: PINK1 is degraded through the N-end rule pathway
    Article Snippet: .. The following antibodies were used for immunoblotting: rabbit anti-GFP (Invitrogen, A-11122), mouse anti-SQSTM1 (clone 2C11, Novus Biologicals, H00008878-M01), rabbit anti-LC3B (Sigma, L7543), mouse anti-α-Tubulin (clone B-5-1-2, Invitrogen, 32-2500) and mouse anti-Actin (clone AC-40, Sigma, A4700), rabbit anti-TOMM20 (Santa Cruz Biotechnology, Inc., sc-11415), rabbit anti-PINK1(Novus Biologicals, BC100-494), mouse anti-TIMM23 (clone 32, BD Transduction Laboratories, 611222), mouse anti-Cytochrome c (clone 7H8.2C12, BD Transduction Laboratories, 556433), rabbit anti-HTRA2/Omi (R & D Systems, AF1458), rabbit anti-MFN1 (generated as described previously ), rabbit anti-UBR4 (abcam, ab86738), rabbit anti-UBR1 and chicken anti-UBR2 (kind gifts from Dr Yong Tae Kwon , ). .. The following antibodies were used for immunostaining: guinea pig anti-SQSTM1 (Progen, GP62-C), rabbit anti-HSPA9/GRP75 (Cell Signaling, 3593) and rabbit anti-TOMM20 (Santa Cruz Biotechnology, Inc., sc-11415).

    Blocking Assay:

    Article Title: HDAC4 regulates satellite cell proliferation and differentiation by targeting P21 and Sharp1 genes
    Article Snippet: .. To better detect the green fluorescent protein in transfected cells, we used primary anti-GFP antibody (Life technologies) diluted 1:400 in 1% BSA PBS, by following the above protocol and adding a permeabilization step of 5 minutes with 0.25% Triton-X 100 (Sigma-Aldrich) in PBS before blocking in 10% goat serum in PBS. .. For P21 immunofluorescence, cells were fixed with 4% PFA buffered solution for 10 minutes and permeabilized with 0.25% Triton-X 100 (Sigma-Aldrich).

    Expressing:

    Article Title: An Alternative Phosphorylation Switch in Integrin β2 (CD18) Tail for Dok1 Binding
    Article Snippet: .. Clones expressing Dok1-CFP were screened by flow cytometry analyses and verified by immunoblotting with anti-GFP antibody (Life Technologies, Grand Island, NY). .. Stable K562 cells expressing Dok1-CFP transfected with integrin αL, integrin β2-YFP and CCR5 plasmids (12 μg each) were stained with either 1 μg each of mAb MHM24 (anti-αL) or mAb 2D7 (anti-CCR5, BD Biosciences, San Jose, CA) followed by APC-conjugated secondary antibody (goat anti-mouse IgG, BD Biosciences, 1:500 dilution).

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    Thermo Fisher alexa fluor 647 conjugated f
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    Thermo Fisher alexa fluor 647 conjugated goat anti mouse igg
    Flow cytometric quantification of induced GalNAc-T2. A , cells were induced for 48 h with doxycycline ( Dox ) before being fixed, permeabilized, and stained with anti-GalNAc-T2 primary antibody and <t>Alexa</t> <t>Fluor</t> 647 secondary antibody. B , enlargement of HEK ind T2 induced from 0 to 16 ng/ml doxycycline with a help line centered at the peak of 0 ng/ml. The induction is biphasic with a distinct global right shift of the lower-expressing population. Data are representative of two biological and two technical replicates.
    Alexa Fluor 647 Conjugated Goat Anti Mouse Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher af647 conjugated goat polyclonal anti mouse igg
    Flow cytometric quantification of induced GalNAc-T2. A , cells were induced for 48 h with doxycycline ( Dox ) before being fixed, permeabilized, and stained with anti-GalNAc-T2 primary antibody and <t>Alexa</t> <t>Fluor</t> 647 secondary antibody. B , enlargement of HEK ind T2 induced from 0 to 16 ng/ml doxycycline with a help line centered at the peak of 0 ng/ml. The induction is biphasic with a distinct global right shift of the lower-expressing population. Data are representative of two biological and two technical replicates.
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    Flow cytometric quantification of induced GalNAc-T2. A , cells were induced for 48 h with doxycycline ( Dox ) before being fixed, permeabilized, and stained with anti-GalNAc-T2 primary antibody and Alexa Fluor 647 secondary antibody. B , enlargement of HEK ind T2 induced from 0 to 16 ng/ml doxycycline with a help line centered at the peak of 0 ng/ml. The induction is biphasic with a distinct global right shift of the lower-expressing population. Data are representative of two biological and two technical replicates.

    Journal: The Journal of Biological Chemistry

    Article Title: Probing the contribution of individual polypeptide GalNAc-transferase isoforms to the O-glycoproteome by inducible expression in isogenic cell lines

    doi: 10.1074/jbc.RA118.004516

    Figure Lengend Snippet: Flow cytometric quantification of induced GalNAc-T2. A , cells were induced for 48 h with doxycycline ( Dox ) before being fixed, permeabilized, and stained with anti-GalNAc-T2 primary antibody and Alexa Fluor 647 secondary antibody. B , enlargement of HEK ind T2 induced from 0 to 16 ng/ml doxycycline with a help line centered at the peak of 0 ng/ml. The induction is biphasic with a distinct global right shift of the lower-expressing population. Data are representative of two biological and two technical replicates.

    Article Snippet: After washing in PBS, cells were incubated with Alexa Fluor 647–conjugated goat anti-mouse IgG (A-21235, Thermo Scientific) (1:1000 in PBS, 1% BSA) for 1 h at RT, washed, and stored in PBS, 1% BSA at 4 °C until analysis.

    Techniques: Flow Cytometry, Staining, Expressing