anti rabbit igg  (Thermo Fisher)


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    Name:
    Rabbit anti Rat IgG Biotin
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    Catalog Number:
    pa1-28572
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    Structured Review

    Thermo Fisher anti rabbit igg
    Physical exercise and intracarotid injection of IGF-I produce similar effects in the brain. A , The same brain areas show labeling of neurons with IGF-I after treadmill running ( a–c ) and intracarotid injection of IGF-I ( d–f ). Three representative areas are shown. Nonexercised, saline-injected rats show almost undetectable brain IGF-I staining ( B ). Str , Striatum; Cx , cerebral cortex; RN , red nucleus. <t>Biotinylated</t> anti-rabbit <t>IgG</t> followed by Cy3-streptavidin was used after incubation with a polyclonal anti-IGF-I antibody. B , Digoxigenin ( DIG ) and IGF-I colocalize within the same neurons after intracarotid injection of DIG–IGF-I. A representative field in the brainstem is shown. a , Low magnification (10×) of IGF-I staining in the inferior olive nucleus ( IO ) of a saline-injected rat. Note the absence of signal. Inset , Higher magnification (40×) of the IO field. b , The same field showing IGF-I staining in an IGF-I-injected rat. Inset , High magnification showing IGF-I-positive cells. c , High magnification (40×) of IO neurons stained with a monoclonal anti-DIG antibody ( green ). d , The same field stained with a polyclonal anti-IGF-I antibody ( red ). e , Colocalization of DIG and IGF-I within the same IO neurons. Scale bars: a , b , 500 μm; c – e , 50 μm. Primary antibody incubation was followed by an anti-rabbit Cy2 and anti-mouse Cy5, respectively. C , Exercise or intracarotid injection of IGF-I elicits a similar pattern of increased c-Fos staining throughout the brain. Only the piriform cortex ( Pir ) is shown as a representative area. a , Control animals show no c-Fos staining. b , c-Fos staining after 1 hr of intracarotid injection of IGF-I. c , c-Fos staining after 1 hr of running. Scale bar ( a – c ): 500 μm. d , Higher magnification of the field in c showing nuclear localization of the c-Fos signal. Scale bar, 50 μm. Arrows indicate immunoreactive cells. A monoclonal anti-c-Fos antibody followed by a biotinylated anti-mouse IgG and Cy3-streptavidin was used. D , Blockade of the exercise-induced capture of IGF-I by brain cells results in absence of c-Fos labeling after exercise. a , IGF-I labeling in the hippocampus of a rat that ran for 1 hr. c , Chronic intracerebroventricular delivery of a combination of an anti-IGF-I antibody and an IGF-I receptor antagonist results in absence of IGF-I staining after 1 hr of running exercise. Scale bar ( a , c ): 50 μm. b , c-Fos staining is induced in the hippocampus by 1 hr of running. c , No c-Fos labeling is seen in exercised animals in which brain uptake of IGF-I is blocked. Scale bar ( b , d ): 500 μm. The hippocampus is shown as a representative area, but absence of labeling for IGF-I and c-Fos was found in all brain areas. E , Expression of BDNF in the hippocampus is increased by running and by intracarotid injection of IGF-I. Control: background BDNF RNA staining in brain slices incubated with excess unlabeled probe. Saline: animals injected with saline show weak BDNF expression in the hippocampus. Exercise: running induces increased expression of BDNF in the hippocampus. IGF-I: injection of IGF-I produces a similar increase in hippocampal expression of BDNF. Cx , Cortex; Hy , hippocampus.

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    Images

    1) Product Images from "Circulating Insulin-Like Growth Factor I Mediates Effects of Exercise on the Brain"

    Article Title: Circulating Insulin-Like Growth Factor I Mediates Effects of Exercise on the Brain

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.20-08-02926.2000

    Physical exercise and intracarotid injection of IGF-I produce similar effects in the brain. A , The same brain areas show labeling of neurons with IGF-I after treadmill running ( a–c ) and intracarotid injection of IGF-I ( d–f ). Three representative areas are shown. Nonexercised, saline-injected rats show almost undetectable brain IGF-I staining ( B ). Str , Striatum; Cx , cerebral cortex; RN , red nucleus. Biotinylated anti-rabbit IgG followed by Cy3-streptavidin was used after incubation with a polyclonal anti-IGF-I antibody. B , Digoxigenin ( DIG ) and IGF-I colocalize within the same neurons after intracarotid injection of DIG–IGF-I. A representative field in the brainstem is shown. a , Low magnification (10×) of IGF-I staining in the inferior olive nucleus ( IO ) of a saline-injected rat. Note the absence of signal. Inset , Higher magnification (40×) of the IO field. b , The same field showing IGF-I staining in an IGF-I-injected rat. Inset , High magnification showing IGF-I-positive cells. c , High magnification (40×) of IO neurons stained with a monoclonal anti-DIG antibody ( green ). d , The same field stained with a polyclonal anti-IGF-I antibody ( red ). e , Colocalization of DIG and IGF-I within the same IO neurons. Scale bars: a , b , 500 μm; c – e , 50 μm. Primary antibody incubation was followed by an anti-rabbit Cy2 and anti-mouse Cy5, respectively. C , Exercise or intracarotid injection of IGF-I elicits a similar pattern of increased c-Fos staining throughout the brain. Only the piriform cortex ( Pir ) is shown as a representative area. a , Control animals show no c-Fos staining. b , c-Fos staining after 1 hr of intracarotid injection of IGF-I. c , c-Fos staining after 1 hr of running. Scale bar ( a – c ): 500 μm. d , Higher magnification of the field in c showing nuclear localization of the c-Fos signal. Scale bar, 50 μm. Arrows indicate immunoreactive cells. A monoclonal anti-c-Fos antibody followed by a biotinylated anti-mouse IgG and Cy3-streptavidin was used. D , Blockade of the exercise-induced capture of IGF-I by brain cells results in absence of c-Fos labeling after exercise. a , IGF-I labeling in the hippocampus of a rat that ran for 1 hr. c , Chronic intracerebroventricular delivery of a combination of an anti-IGF-I antibody and an IGF-I receptor antagonist results in absence of IGF-I staining after 1 hr of running exercise. Scale bar ( a , c ): 50 μm. b , c-Fos staining is induced in the hippocampus by 1 hr of running. c , No c-Fos labeling is seen in exercised animals in which brain uptake of IGF-I is blocked. Scale bar ( b , d ): 500 μm. The hippocampus is shown as a representative area, but absence of labeling for IGF-I and c-Fos was found in all brain areas. E , Expression of BDNF in the hippocampus is increased by running and by intracarotid injection of IGF-I. Control: background BDNF RNA staining in brain slices incubated with excess unlabeled probe. Saline: animals injected with saline show weak BDNF expression in the hippocampus. Exercise: running induces increased expression of BDNF in the hippocampus. IGF-I: injection of IGF-I produces a similar increase in hippocampal expression of BDNF. Cx , Cortex; Hy , hippocampus.
    Figure Legend Snippet: Physical exercise and intracarotid injection of IGF-I produce similar effects in the brain. A , The same brain areas show labeling of neurons with IGF-I after treadmill running ( a–c ) and intracarotid injection of IGF-I ( d–f ). Three representative areas are shown. Nonexercised, saline-injected rats show almost undetectable brain IGF-I staining ( B ). Str , Striatum; Cx , cerebral cortex; RN , red nucleus. Biotinylated anti-rabbit IgG followed by Cy3-streptavidin was used after incubation with a polyclonal anti-IGF-I antibody. B , Digoxigenin ( DIG ) and IGF-I colocalize within the same neurons after intracarotid injection of DIG–IGF-I. A representative field in the brainstem is shown. a , Low magnification (10×) of IGF-I staining in the inferior olive nucleus ( IO ) of a saline-injected rat. Note the absence of signal. Inset , Higher magnification (40×) of the IO field. b , The same field showing IGF-I staining in an IGF-I-injected rat. Inset , High magnification showing IGF-I-positive cells. c , High magnification (40×) of IO neurons stained with a monoclonal anti-DIG antibody ( green ). d , The same field stained with a polyclonal anti-IGF-I antibody ( red ). e , Colocalization of DIG and IGF-I within the same IO neurons. Scale bars: a , b , 500 μm; c – e , 50 μm. Primary antibody incubation was followed by an anti-rabbit Cy2 and anti-mouse Cy5, respectively. C , Exercise or intracarotid injection of IGF-I elicits a similar pattern of increased c-Fos staining throughout the brain. Only the piriform cortex ( Pir ) is shown as a representative area. a , Control animals show no c-Fos staining. b , c-Fos staining after 1 hr of intracarotid injection of IGF-I. c , c-Fos staining after 1 hr of running. Scale bar ( a – c ): 500 μm. d , Higher magnification of the field in c showing nuclear localization of the c-Fos signal. Scale bar, 50 μm. Arrows indicate immunoreactive cells. A monoclonal anti-c-Fos antibody followed by a biotinylated anti-mouse IgG and Cy3-streptavidin was used. D , Blockade of the exercise-induced capture of IGF-I by brain cells results in absence of c-Fos labeling after exercise. a , IGF-I labeling in the hippocampus of a rat that ran for 1 hr. c , Chronic intracerebroventricular delivery of a combination of an anti-IGF-I antibody and an IGF-I receptor antagonist results in absence of IGF-I staining after 1 hr of running exercise. Scale bar ( a , c ): 50 μm. b , c-Fos staining is induced in the hippocampus by 1 hr of running. c , No c-Fos labeling is seen in exercised animals in which brain uptake of IGF-I is blocked. Scale bar ( b , d ): 500 μm. The hippocampus is shown as a representative area, but absence of labeling for IGF-I and c-Fos was found in all brain areas. E , Expression of BDNF in the hippocampus is increased by running and by intracarotid injection of IGF-I. Control: background BDNF RNA staining in brain slices incubated with excess unlabeled probe. Saline: animals injected with saline show weak BDNF expression in the hippocampus. Exercise: running induces increased expression of BDNF in the hippocampus. IGF-I: injection of IGF-I produces a similar increase in hippocampal expression of BDNF. Cx , Cortex; Hy , hippocampus.

    Techniques Used: Injection, Labeling, Staining, Incubation, Expressing

    2) Product Images from "Aryl hydrocarbon receptor activation restores filaggrin expression via OVOL1 in atopic dermatitis"

    Article Title: Aryl hydrocarbon receptor activation restores filaggrin expression via OVOL1 in atopic dermatitis

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2017.322

    Nuclear translocation of OVOL1 was likely to be inhibited in AD skin, leading to the reduced FLG expression in AD skin. Normal skin ( a ) and AD skin ( d ) were stained with hematoxylin and eosin. The scale bar is 100 μ m. Expression of FLG and OVOL1 in the epidermis of the same skin lesion was analyzed by IHC staining for FLG (red) or OVOL1 (red). The expression of FLG was observed in normal skin ( b ) and was low in AD skin ( e ). The expression of OVOL1 was observed mainly in the nuclei of keratinocytes in normal skin ( c ); however, nuclear OVOL1 expression was lower in AD skin ( f ). For semiquantitative analysis of IHC staining, microscopic visual fields of the samples from each group were randomly chosen and examined. In a high-power field (× 400 magnification), the nuclear-OVOL1-stained cells of the epidermis were counted, as were all the cells with hematoxylin staining. Nuclear OVOL1 expression was lower in AD skin (AD) compared with normal skin (NS) ( g ). NHEKs treated with DMSO ( h ), FICZ (100 nM) ( i ), IL-4 (10 ng/ml) ( j ), or FICZ plus IL-4 ( k) for 24 h were stained with an anti-OVOL1 antibody (primary antibody) and an Alexa Fluor 488-conjugated anti-rabbit IgG antibody (secondary). The nuclei were counterstained with DAPI (blue). Confocal laser scanning images revealed that OVOL1 expression was noticeable mainly in the cytoplasm in a steady state ( h ) and that the AHR activation by FICZ induced nuclear translocation of OVOL1 ( i ). In contrast, IL-4 did not induce nuclear translocation of OVOL1, and the latter was retained in the cytoplasm ( j ). IL-4-mediated blockade of the nuclear translocation of OVOL1 was overridden by treatment with FICZ ( k ). ( l ) Isotype negative control. The scale bar is 25 μ m. The data are representative of experiments repeated three times with similar results. ( m ) NHEKs were treated with FICZ (100 nM) in the absence or presence of IL-4 (10 ng/ml) for 18 h. Cellular nuclear protein was extracted using a biochemical subcellular fractionation technique. The OVOL1 levels in the nuclear protein fraction of NHEKs were evaluated by western blotting. The activation of AHR by FICZ increased the nuclear OVOL1 expression; in contrast, IL-4 did not change nuclear expression of OVOL1. The IL-4-mediated blockade of the OVOL1 nuclear translocation was partially reversed by treatment with FICZ. The data are representative of experiments repeated three times with similar results
    Figure Legend Snippet: Nuclear translocation of OVOL1 was likely to be inhibited in AD skin, leading to the reduced FLG expression in AD skin. Normal skin ( a ) and AD skin ( d ) were stained with hematoxylin and eosin. The scale bar is 100 μ m. Expression of FLG and OVOL1 in the epidermis of the same skin lesion was analyzed by IHC staining for FLG (red) or OVOL1 (red). The expression of FLG was observed in normal skin ( b ) and was low in AD skin ( e ). The expression of OVOL1 was observed mainly in the nuclei of keratinocytes in normal skin ( c ); however, nuclear OVOL1 expression was lower in AD skin ( f ). For semiquantitative analysis of IHC staining, microscopic visual fields of the samples from each group were randomly chosen and examined. In a high-power field (× 400 magnification), the nuclear-OVOL1-stained cells of the epidermis were counted, as were all the cells with hematoxylin staining. Nuclear OVOL1 expression was lower in AD skin (AD) compared with normal skin (NS) ( g ). NHEKs treated with DMSO ( h ), FICZ (100 nM) ( i ), IL-4 (10 ng/ml) ( j ), or FICZ plus IL-4 ( k) for 24 h were stained with an anti-OVOL1 antibody (primary antibody) and an Alexa Fluor 488-conjugated anti-rabbit IgG antibody (secondary). The nuclei were counterstained with DAPI (blue). Confocal laser scanning images revealed that OVOL1 expression was noticeable mainly in the cytoplasm in a steady state ( h ) and that the AHR activation by FICZ induced nuclear translocation of OVOL1 ( i ). In contrast, IL-4 did not induce nuclear translocation of OVOL1, and the latter was retained in the cytoplasm ( j ). IL-4-mediated blockade of the nuclear translocation of OVOL1 was overridden by treatment with FICZ ( k ). ( l ) Isotype negative control. The scale bar is 25 μ m. The data are representative of experiments repeated three times with similar results. ( m ) NHEKs were treated with FICZ (100 nM) in the absence or presence of IL-4 (10 ng/ml) for 18 h. Cellular nuclear protein was extracted using a biochemical subcellular fractionation technique. The OVOL1 levels in the nuclear protein fraction of NHEKs were evaluated by western blotting. The activation of AHR by FICZ increased the nuclear OVOL1 expression; in contrast, IL-4 did not change nuclear expression of OVOL1. The IL-4-mediated blockade of the OVOL1 nuclear translocation was partially reversed by treatment with FICZ. The data are representative of experiments repeated three times with similar results

    Techniques Used: Translocation Assay, Expressing, Staining, Immunohistochemistry, Activation Assay, Negative Control, Fractionation, Western Blot

    3) Product Images from "Multiple Functions of Glutamate Uptake via Meningococcal GltT-GltM l-Glutamate ABC Transporter in Neisseria meningitidis Internalization into Human Brain Microvascular Endothelial Cells"

    Article Title: Multiple Functions of Glutamate Uptake via Meningococcal GltT-GltM l-Glutamate ABC Transporter in Neisseria meningitidis Internalization into Human Brain Microvascular Endothelial Cells

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00654-15

    N. meningitidis formed larger colonies on HBMEC at higher MOIs. Shown are phase-contrast and immunofluorescent staining with anti- N. meningitidis (α-Nm) rabbit serum and anti-rabbit IgG-Alexa 488, respectively. HBMEC infections were done with wild-type (HT1125) and Δ gltT Δ gltM (HT1414) strains in AM at MOIs of 5, 50, and 500. Magnification, ×1,000.
    Figure Legend Snippet: N. meningitidis formed larger colonies on HBMEC at higher MOIs. Shown are phase-contrast and immunofluorescent staining with anti- N. meningitidis (α-Nm) rabbit serum and anti-rabbit IgG-Alexa 488, respectively. HBMEC infections were done with wild-type (HT1125) and Δ gltT Δ gltM (HT1414) strains in AM at MOIs of 5, 50, and 500. Magnification, ×1,000.

    Techniques Used: Staining

    Phase-contrast and immunofluorescence microscopy showing ezrin accumulation beneath N. meningitidis -infected HBMEC. Noninfected controls are also shown. HBMEC monolayers were infected with N. meningitidis wild-type (HT1125) and Δ gltT Δ gltM (HT1414) strains in AM (A), AM(−S) (B), and AM(−S, +Glu) (C). N. meningitidis and HBMEC were observed by phase-contrast microscopy (left column). N. meningitidis strains and ezrin were immunostained with two sets of primary and secondary antibodies: anti- N. meningitidis rabbit serum and Alexa Fluor 488-conjugated anti-rabbit IgG (middle column) and anti-ezrin monoclonal antibody (MAb) and Alexa Fluor 594-conjugated anti-mouse IgG (right column). Magnification, ×400.
    Figure Legend Snippet: Phase-contrast and immunofluorescence microscopy showing ezrin accumulation beneath N. meningitidis -infected HBMEC. Noninfected controls are also shown. HBMEC monolayers were infected with N. meningitidis wild-type (HT1125) and Δ gltT Δ gltM (HT1414) strains in AM (A), AM(−S) (B), and AM(−S, +Glu) (C). N. meningitidis and HBMEC were observed by phase-contrast microscopy (left column). N. meningitidis strains and ezrin were immunostained with two sets of primary and secondary antibodies: anti- N. meningitidis rabbit serum and Alexa Fluor 488-conjugated anti-rabbit IgG (middle column) and anti-ezrin monoclonal antibody (MAb) and Alexa Fluor 594-conjugated anti-mouse IgG (right column). Magnification, ×400.

    Techniques Used: Immunofluorescence, Microscopy, Infection

    4) Product Images from "Suppression of the Insulin Receptors in Adult Schistosoma japonicum Impacts on Parasite Growth and Development: Further Evidence of Vaccine Potential"

    Article Title: Suppression of the Insulin Receptors in Adult Schistosoma japonicum Impacts on Parasite Growth and Development: Further Evidence of Vaccine Potential

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0003730

    A. Western blot analysis of adult S . japonicum worm extracts obtained on day 6 following treatment with SjIR dsRNAs. Results are shown for proteins recognised by anti-Sm-Pmy antibody (top panel), anti-SjLD1 (middle panel) and anti-SjLD2 (bottom panel) antibodies. The intensity of Sm-Pmy expression was evaluated so as to determine equal protein loading. The arrows indicate the diminished level of SjIR proteins relative to the luciferase treatment control in the first lane. The experiment was repeated twice with similar results obtained. B . Western blot analysis showing no immunological cross reactivity between recombinant HIR and the SjLDs. Commercial recombinant human insulin receptor (rHIR) and recombinant SjLD1 (rSjLD1) and SjLD2 (rSjLD2) were electrophoresed on SDS-PAGE gels, blotted to membrane and probed with rabbit anti-HIR polyclonal antibody (left panel), rabbit anti-SjLD1 (middle panel) and anti-SjLD2 (right panel) as primary antibodies with anti-rabbit IgG conjugated to horseradish peroxidise used as secondary antibody.
    Figure Legend Snippet: A. Western blot analysis of adult S . japonicum worm extracts obtained on day 6 following treatment with SjIR dsRNAs. Results are shown for proteins recognised by anti-Sm-Pmy antibody (top panel), anti-SjLD1 (middle panel) and anti-SjLD2 (bottom panel) antibodies. The intensity of Sm-Pmy expression was evaluated so as to determine equal protein loading. The arrows indicate the diminished level of SjIR proteins relative to the luciferase treatment control in the first lane. The experiment was repeated twice with similar results obtained. B . Western blot analysis showing no immunological cross reactivity between recombinant HIR and the SjLDs. Commercial recombinant human insulin receptor (rHIR) and recombinant SjLD1 (rSjLD1) and SjLD2 (rSjLD2) were electrophoresed on SDS-PAGE gels, blotted to membrane and probed with rabbit anti-HIR polyclonal antibody (left panel), rabbit anti-SjLD1 (middle panel) and anti-SjLD2 (right panel) as primary antibodies with anti-rabbit IgG conjugated to horseradish peroxidise used as secondary antibody.

    Techniques Used: Western Blot, Expressing, Luciferase, Recombinant, SDS Page

    5) Product Images from "Selective and strain-specific NFAT4 activation by the Toxoplasma gondii polymorphic dense granule protein GRA6"

    Article Title: Selective and strain-specific NFAT4 activation by the Toxoplasma gondii polymorphic dense granule protein GRA6

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20131272

    NFAT4 is selectively activated by ectopic expression of GRA6. (A) 293T cells were transfected with the NFAT-dependent luciferase reporter together with indicated expression vectors. Luciferase activities in the presence or absence of CAMLG were expressed as indicated above. Error bars represent means ± SD of triplicates. (B) 293T cells were transfected with the NFAT-dependent luciferase reporter together with indicated expression vectors. Luciferase activities in the presence or absence of PMA/ionophore were expressed as indicated above. Error bars represent means ± SD of triplicates. (C) Lysates of 293T cells transiently transfected with HA-tagged CAMLG were immunoprecipitated with the indicated Abs and subjected to Western blotting. (D) Lysates of 293T cells transiently cotransfected with Flag-tagged indicated NFAT vectors and/or HA-tagged CAMLG were immunoprecipitated with the indicated Abs and subjected to Western blot. (E) Quantitative RT-PCR analysis was performed using cDNA reversely transcribed from RNA extracted from 293T cells stably expressing shRNA for control or NFAT1-4. Relative mRNA levels of indicated genes compared with the GAPDH level were shown in the y axis. Error bars represent means ± SD of triplicates. (F) 293T cells stably expressing shRNA for human NFAT1-4 or control were transfected with the NFAT-dependent luciferase reporter together with empty or GRA6 expression vectors. Error bars represent means ± SD of triplicates. (G) MEFs stably expressing RFP-tagged NFAT4 were transfected with empty or Flag-tagged GRA6 or GRA15 expression vectors. 24 h after transfection, the cells were fixed and stained with mouse anti-Flag, and then Alexa Fluor 488–conjugated anti–mouse IgG (green) or DAPI (blue). Bars, 5 µm. *, P
    Figure Legend Snippet: NFAT4 is selectively activated by ectopic expression of GRA6. (A) 293T cells were transfected with the NFAT-dependent luciferase reporter together with indicated expression vectors. Luciferase activities in the presence or absence of CAMLG were expressed as indicated above. Error bars represent means ± SD of triplicates. (B) 293T cells were transfected with the NFAT-dependent luciferase reporter together with indicated expression vectors. Luciferase activities in the presence or absence of PMA/ionophore were expressed as indicated above. Error bars represent means ± SD of triplicates. (C) Lysates of 293T cells transiently transfected with HA-tagged CAMLG were immunoprecipitated with the indicated Abs and subjected to Western blotting. (D) Lysates of 293T cells transiently cotransfected with Flag-tagged indicated NFAT vectors and/or HA-tagged CAMLG were immunoprecipitated with the indicated Abs and subjected to Western blot. (E) Quantitative RT-PCR analysis was performed using cDNA reversely transcribed from RNA extracted from 293T cells stably expressing shRNA for control or NFAT1-4. Relative mRNA levels of indicated genes compared with the GAPDH level were shown in the y axis. Error bars represent means ± SD of triplicates. (F) 293T cells stably expressing shRNA for human NFAT1-4 or control were transfected with the NFAT-dependent luciferase reporter together with empty or GRA6 expression vectors. Error bars represent means ± SD of triplicates. (G) MEFs stably expressing RFP-tagged NFAT4 were transfected with empty or Flag-tagged GRA6 or GRA15 expression vectors. 24 h after transfection, the cells were fixed and stained with mouse anti-Flag, and then Alexa Fluor 488–conjugated anti–mouse IgG (green) or DAPI (blue). Bars, 5 µm. *, P

    Techniques Used: Expressing, Transfection, Luciferase, Immunoprecipitation, Western Blot, Quantitative RT-PCR, Stable Transfection, shRNA, Staining

    6) Product Images from "Specific Enzyme Complex of ?-1,4-Galactosyltransferase-II and Glucuronyltransferase-P Facilitates Biosynthesis of N-linked Human Natural Killer-1 (HNK-1) Carbohydrate *"

    Article Title: Specific Enzyme Complex of ?-1,4-Galactosyltransferase-II and Glucuronyltransferase-P Facilitates Biosynthesis of N-linked Human Natural Killer-1 (HNK-1) Carbohydrate *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.233353

    Pulldown assays using soluble forms of GlcAT-P-sol, prot.A-GalT-I, and prot.A-GalT-II. A , schematic diagrams of GlcAT-P-sol, prot.A-GalT-I (Ser-43), and prot.A-GalT-II (Asp-33). SS , signal sequence. B , culture medium of N2a cells transiently expressing GlcAT-P-sol and prot.A-GalT-I (Ser-43) or prot.A-GalT-II (Asp-33) was incubated with IgG-Sepharose TM6 Fast Flow (pulldown), subjected to SDS-PAGE, and Western-blotted with HRP-conjugated normal rabbit IgG or GP2 pAb. To examine the level of each protein, the culture medium was directly subjected to SDS-PAGE and then Western-blotted with HRP-conjugated normal rabbit IgG or GP2 pAb ( input ). CT , cytoplasmic tail, TM , transmembrane domain.
    Figure Legend Snippet: Pulldown assays using soluble forms of GlcAT-P-sol, prot.A-GalT-I, and prot.A-GalT-II. A , schematic diagrams of GlcAT-P-sol, prot.A-GalT-I (Ser-43), and prot.A-GalT-II (Asp-33). SS , signal sequence. B , culture medium of N2a cells transiently expressing GlcAT-P-sol and prot.A-GalT-I (Ser-43) or prot.A-GalT-II (Asp-33) was incubated with IgG-Sepharose TM6 Fast Flow (pulldown), subjected to SDS-PAGE, and Western-blotted with HRP-conjugated normal rabbit IgG or GP2 pAb. To examine the level of each protein, the culture medium was directly subjected to SDS-PAGE and then Western-blotted with HRP-conjugated normal rabbit IgG or GP2 pAb ( input ). CT , cytoplasmic tail, TM , transmembrane domain.

    Techniques Used: Sequencing, Expressing, Incubation, Flow Cytometry, SDS Page, Western Blot

    7) Product Images from "Bioplasmonic Paper as a Platform for Detection of Kidney Cancer Biomarkers"

    Article Title: Bioplasmonic Paper as a Platform for Detection of Kidney Cancer Biomarkers

    Journal: Analytical chemistry

    doi: 10.1021/ac302332g

    (A-B) SEM images of paper adsorbed with AuNR-IgG conjugates. (C) Extinction spectra of AuNR-IgG conjugates in solution and on paper (inset: shows photographs of the bare filter paper (left) and filter paper after adsorption of AuNR+IgG conjugates (right)). (D) Six extinction spectra collected from different spots on a plasmonic paper of 0.5×0.5 cm 2 area showing the remarkable spectral homogeneity of bioplasmonic paper.
    Figure Legend Snippet: (A-B) SEM images of paper adsorbed with AuNR-IgG conjugates. (C) Extinction spectra of AuNR-IgG conjugates in solution and on paper (inset: shows photographs of the bare filter paper (left) and filter paper after adsorption of AuNR+IgG conjugates (right)). (D) Six extinction spectra collected from different spots on a plasmonic paper of 0.5×0.5 cm 2 area showing the remarkable spectral homogeneity of bioplasmonic paper.

    Techniques Used: Adsorption

    8) Product Images from "Epithelial and dendritic cells in the thymic medulla promote CD4+Foxp3+ regulatory T cell development via the CD27-CD70 pathway"

    Article Title: Epithelial and dendritic cells in the thymic medulla promote CD4+Foxp3+ regulatory T cell development via the CD27-CD70 pathway

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20112061

    CD70 is expressed by Aire + and Aire − epithelial cells in the thymic medulla. (I–IV, VII, and VIII) Thymic sections of WT and Cd70 Cre/Cre mice were stained to detect CD70 on mTECs using sequential staining with ER-TR5 mAb, Alexa Fluor 488–conjugated anti-IgG, anti-CD70 mAb, and Alexa Fluor 568–conjugated anti-IgG (I, II, VII, and VIII) or sequential staining with anti–Keratin-5 (K5) mAb, Alexa Fluor 488–conjugated anti-IgG, anti-CD70 mAb, and Alexa Fluor 568–conjugated anti-IgG (III and IV). (V and VI) Thymic sections of WT and Cd70 Cre/Cre mice were stained to detect CD70 and Aire using sequential staining with anti-CD70 mAb, Alexa Fluor 568–conjugated anti-IgG, and Alexa Fluor 488–conjugated anti-Aire mAb. Sections I–VI were subsequently stained with DAPI to detect the nuclei of all cells present. The same magnification is shown for all panels. High magnification (I–VI) and lower magnification (VII–VIII) images of the thymus are shown. Data for each staining are representative of at least three to four different sections that were stained and analyzed independently.
    Figure Legend Snippet: CD70 is expressed by Aire + and Aire − epithelial cells in the thymic medulla. (I–IV, VII, and VIII) Thymic sections of WT and Cd70 Cre/Cre mice were stained to detect CD70 on mTECs using sequential staining with ER-TR5 mAb, Alexa Fluor 488–conjugated anti-IgG, anti-CD70 mAb, and Alexa Fluor 568–conjugated anti-IgG (I, II, VII, and VIII) or sequential staining with anti–Keratin-5 (K5) mAb, Alexa Fluor 488–conjugated anti-IgG, anti-CD70 mAb, and Alexa Fluor 568–conjugated anti-IgG (III and IV). (V and VI) Thymic sections of WT and Cd70 Cre/Cre mice were stained to detect CD70 and Aire using sequential staining with anti-CD70 mAb, Alexa Fluor 568–conjugated anti-IgG, and Alexa Fluor 488–conjugated anti-Aire mAb. Sections I–VI were subsequently stained with DAPI to detect the nuclei of all cells present. The same magnification is shown for all panels. High magnification (I–VI) and lower magnification (VII–VIII) images of the thymus are shown. Data for each staining are representative of at least three to four different sections that were stained and analyzed independently.

    Techniques Used: Mouse Assay, Staining

    CD70 on CD8α + thymic cDCs contributes to T reg cell development. (A) Thymic sections were stained sequentially using anti-CD70 mAb, Alexa Fluor 568–conjugated anti-IgG, FITC-conjugated anti-CD11c mAb, and APC-conjugated anti-CD8α mAb. Arrowheads indicate cells with double staining for CD70 and CD11c. (B and C) DCs were enriched from WT or Cd70 Cre/Cre thymi and analyzed by flow cytometry. (B) Shown are representative plots of CD8α versus SIRPα expression on gated CD11c + B220 lo DCs. (C) CD8α + and SIRPα + DC subsets were purified from WT and Cd70 Cre/Cre thymi, RNA was isolated, and RT-PCR was performed on cDNA. Shown are PCR products from cDNA samples amplified with primers specific for CD70 and HPRT. (D) CD8α + and SIRPα + CD11c + B220 low/− cDCs and CD11c + B220 + pDCs were purified from the pooled thymi of two WT or Cd70 Cre/Cre mice. The purified DC subsets were co-cultured for 5 d with purified CD4 + CD25 − WT thymocytes. After culture, gated CD4 + cells were analyzed for expression of Foxp3 and CD25. Shown is one representative plot of Foxp3 and CD25 expression on gated CD4 + cells in the presence of the indicated DC population. (E) Data from D, expressed as the percentage of Foxp3 + CD25 + cells among gated CD4 + cells in the presence of the indicated DC populations. Results are from four to five separate wells over two independent experiments. The Mann–Whitney U rank sum test was used to analyze results (*, P
    Figure Legend Snippet: CD70 on CD8α + thymic cDCs contributes to T reg cell development. (A) Thymic sections were stained sequentially using anti-CD70 mAb, Alexa Fluor 568–conjugated anti-IgG, FITC-conjugated anti-CD11c mAb, and APC-conjugated anti-CD8α mAb. Arrowheads indicate cells with double staining for CD70 and CD11c. (B and C) DCs were enriched from WT or Cd70 Cre/Cre thymi and analyzed by flow cytometry. (B) Shown are representative plots of CD8α versus SIRPα expression on gated CD11c + B220 lo DCs. (C) CD8α + and SIRPα + DC subsets were purified from WT and Cd70 Cre/Cre thymi, RNA was isolated, and RT-PCR was performed on cDNA. Shown are PCR products from cDNA samples amplified with primers specific for CD70 and HPRT. (D) CD8α + and SIRPα + CD11c + B220 low/− cDCs and CD11c + B220 + pDCs were purified from the pooled thymi of two WT or Cd70 Cre/Cre mice. The purified DC subsets were co-cultured for 5 d with purified CD4 + CD25 − WT thymocytes. After culture, gated CD4 + cells were analyzed for expression of Foxp3 and CD25. Shown is one representative plot of Foxp3 and CD25 expression on gated CD4 + cells in the presence of the indicated DC population. (E) Data from D, expressed as the percentage of Foxp3 + CD25 + cells among gated CD4 + cells in the presence of the indicated DC populations. Results are from four to five separate wells over two independent experiments. The Mann–Whitney U rank sum test was used to analyze results (*, P

    Techniques Used: Staining, Double Staining, Flow Cytometry, Cytometry, Expressing, Purification, Isolation, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Mouse Assay, Cell Culture, MANN-WHITNEY

    9) Product Images from "SPDL-1 functions as a kinetochore receptor for MDF-1 in Caenorhabditis elegans"

    Article Title: SPDL-1 functions as a kinetochore receptor for MDF-1 in Caenorhabditis elegans

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200805185

    SPDL-1 localizes to and targets MDF-1 to kinetochores that face away from the mono centrosome. (A) Immunofluorescence images of AB or P1 cells of ZYG-1–depleted embryos in the second mitosis. Indicated proteins (red) were stained with each specific antibody. DNA (white) and tubulin (green) were also stained with DAPI and anti-tubulin antibody, respectively. The numbers before and after the slash in parentheses are, respectively, the numbers of cells in which the indicated protein localized at both sides of kinetochores and those of cells in which the indicated protein localized only at the outer side of the chromosome arc. KNL-1, NDC-80, BUB-1, and CZW-1 localize at both sides of the chromosomes along the longitudinal axis, showing two parallel bars (white arrows). In contrast, SPDL-1 and MDF-1 localize along the side of the chromosome that faces away from the centrosome (white arrows). KNL-1 and BUB-1 also localize on the plus-end terminus region of microtubules that extends beyond chromosomes. Bar, 10 μm. (B) Embryos expressing GFP–MDF-1 and mCherry–histone H2B (RFP–H2B) were depleted of the indicated genes and subjected to time-lapse fluorescence microscopy. Images of embryos at metaphase of the second mitosis in AB cells are shown. The anterior of the embryo is at the right. Kinetochore localization of MDF-1 depends on both SPDL-1 and BUB-1. Bar, 20 μm. (C) Immunoprecipitations were performed on extracts from wild-type (WT), zyg-1 ( b1 ) homozygotes ( zyg-1ts ), and mdf-1 ( gk2 ); fzy-1 ( h1983 ) homozygotes ( mdf-1Δ ) using an anti–SPDL-1 antibody, an anti–MDF-1 antibody, or control IgG. Immunoprecipitants were separated on a gel, transferred onto a nitrocellulose membrane, and probed with antibodies to MDF-1, SPDL-1, and MDF-2. SPDL-1 was detected in MDF-1 immunoprecipitants and MDF-1 and MDF-2 in SPDL-1 immunoprecipitants. (D) Schematic model of the position of SPDL-1 in the hierarchical dependency of kinetochore assembly. SPDL-1 is downstream of CZW-1 and required for unattached kinetochore localization of MDF-1. BUB-1 is also required for unattached kinetochore localization of MDF-1 but not for that of SPDL-1. Together, MDF-1 targeting is regulated by two independent pathways, one includes SPDL-1 and the other includes BUB-1.
    Figure Legend Snippet: SPDL-1 localizes to and targets MDF-1 to kinetochores that face away from the mono centrosome. (A) Immunofluorescence images of AB or P1 cells of ZYG-1–depleted embryos in the second mitosis. Indicated proteins (red) were stained with each specific antibody. DNA (white) and tubulin (green) were also stained with DAPI and anti-tubulin antibody, respectively. The numbers before and after the slash in parentheses are, respectively, the numbers of cells in which the indicated protein localized at both sides of kinetochores and those of cells in which the indicated protein localized only at the outer side of the chromosome arc. KNL-1, NDC-80, BUB-1, and CZW-1 localize at both sides of the chromosomes along the longitudinal axis, showing two parallel bars (white arrows). In contrast, SPDL-1 and MDF-1 localize along the side of the chromosome that faces away from the centrosome (white arrows). KNL-1 and BUB-1 also localize on the plus-end terminus region of microtubules that extends beyond chromosomes. Bar, 10 μm. (B) Embryos expressing GFP–MDF-1 and mCherry–histone H2B (RFP–H2B) were depleted of the indicated genes and subjected to time-lapse fluorescence microscopy. Images of embryos at metaphase of the second mitosis in AB cells are shown. The anterior of the embryo is at the right. Kinetochore localization of MDF-1 depends on both SPDL-1 and BUB-1. Bar, 20 μm. (C) Immunoprecipitations were performed on extracts from wild-type (WT), zyg-1 ( b1 ) homozygotes ( zyg-1ts ), and mdf-1 ( gk2 ); fzy-1 ( h1983 ) homozygotes ( mdf-1Δ ) using an anti–SPDL-1 antibody, an anti–MDF-1 antibody, or control IgG. Immunoprecipitants were separated on a gel, transferred onto a nitrocellulose membrane, and probed with antibodies to MDF-1, SPDL-1, and MDF-2. SPDL-1 was detected in MDF-1 immunoprecipitants and MDF-1 and MDF-2 in SPDL-1 immunoprecipitants. (D) Schematic model of the position of SPDL-1 in the hierarchical dependency of kinetochore assembly. SPDL-1 is downstream of CZW-1 and required for unattached kinetochore localization of MDF-1. BUB-1 is also required for unattached kinetochore localization of MDF-1 but not for that of SPDL-1. Together, MDF-1 targeting is regulated by two independent pathways, one includes SPDL-1 and the other includes BUB-1.

    Techniques Used: Immunofluorescence, Staining, Expressing, Fluorescence, Microscopy

    10) Product Images from "Association of diacylglycerol kinase ? with protein kinase C ?"

    Article Title: Association of diacylglycerol kinase ? with protein kinase C ?

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200208120

    DGK ζ and PKC α associate with a signaling complex. (A) Lysates from HEK293 cells transiently transfected with PKCα and vector, FLAG-tagged DGKζ (WT or ΔATP), were immunoprecipitated using anti-FLAG or a control antibody (mouse IgG), and then the immunoprecipitates were subjected to immunoblot analysis with anti-PKCα. The blot was then stripped and reprobed to detect DGKζ. Expression of PKCα and DGKζ in the cell lysates is also shown. (B) Purified PKCα was incubated with purified DGKζ–FLAG bound to anti–FLAG-M2 agarose affinity gel or with affinity gel alone. The beads were washed, and proteins bound to the beads were immunoblotted with anti-PKCα. Input represents 15% of the initial recombinant PKCα used in this experiment. (C) Rat brain extracts were immunoprecipitated with anti-DGKζ or a control antibody (rabbit IgG), followed by immunoblotting with anti-PKCα. The blot was then stripped and reprobed to detect DGKζ. Expression of PKCα and DGKζ in the rat brain extracts is also shown. (D) Endogenous DGKζ in A172 cell lysates was immunoprecipitated using anti-DGKζ. Normal rabbit IgG was used as a control. The precipitates were subjected to immunoblot analysis with anti-PKCα. The blot was then stripped and reprobed to detect DGKζ. Expression of PKCα and DGKζ in the A172 cell lysate is shown in the bottom panel.
    Figure Legend Snippet: DGK ζ and PKC α associate with a signaling complex. (A) Lysates from HEK293 cells transiently transfected with PKCα and vector, FLAG-tagged DGKζ (WT or ΔATP), were immunoprecipitated using anti-FLAG or a control antibody (mouse IgG), and then the immunoprecipitates were subjected to immunoblot analysis with anti-PKCα. The blot was then stripped and reprobed to detect DGKζ. Expression of PKCα and DGKζ in the cell lysates is also shown. (B) Purified PKCα was incubated with purified DGKζ–FLAG bound to anti–FLAG-M2 agarose affinity gel or with affinity gel alone. The beads were washed, and proteins bound to the beads were immunoblotted with anti-PKCα. Input represents 15% of the initial recombinant PKCα used in this experiment. (C) Rat brain extracts were immunoprecipitated with anti-DGKζ or a control antibody (rabbit IgG), followed by immunoblotting with anti-PKCα. The blot was then stripped and reprobed to detect DGKζ. Expression of PKCα and DGKζ in the rat brain extracts is also shown. (D) Endogenous DGKζ in A172 cell lysates was immunoprecipitated using anti-DGKζ. Normal rabbit IgG was used as a control. The precipitates were subjected to immunoblot analysis with anti-PKCα. The blot was then stripped and reprobed to detect DGKζ. Expression of PKCα and DGKζ in the A172 cell lysate is shown in the bottom panel.

    Techniques Used: Transfection, Plasmid Preparation, Immunoprecipitation, Expressing, Purification, Incubation, Recombinant

    A portion of the catalytic domain of DGK ζ is sufficient to bind PKC α . (A) PKCα was transfected into HEK293 cells along with wild-type (WT) DGKζ or deletion mutants of DGKζ (B, H, X, L, ΔM, and Bsu) containing FLAG epitope tags at their COOH termini. DGKζ proteins in the cell lysates were immunoprecipitated with anti-FLAG or a control antibody (mouse IgG), and coimmunoprecipitation of PKCα was detected by immunoblotting. The blot was then stripped and reprobed with anti-DGKζ. Because the DGKζ antibody we used was the NH 2 -terminal anti-peptide rabbit antibody, we could not detect the NH 2 terminus deletion DGKζ mutant L (lane 6). However, we detected DGKζ L protein in the same blot using anti-FLAG antibody (not depicted). Expression of PKCα and DGKζ in the cell lysates is also shown. (B) Purified recombinant PKCα was incubated with the glutathione-sepharose–bound GST (lane 1) or GST fusion proteins that contain either full-length DGKζ (GST–DGKζ, lane 2) or a portion of the catalytic domain of DGKζ (GST–BD, lane 3). The beads were collected by centrifugation, and then the proteins bound to beads were subjected to immunoblot analysis with anti-PKCα. Input represents 5% of initial recombinant PKCα used in this experiment.
    Figure Legend Snippet: A portion of the catalytic domain of DGK ζ is sufficient to bind PKC α . (A) PKCα was transfected into HEK293 cells along with wild-type (WT) DGKζ or deletion mutants of DGKζ (B, H, X, L, ΔM, and Bsu) containing FLAG epitope tags at their COOH termini. DGKζ proteins in the cell lysates were immunoprecipitated with anti-FLAG or a control antibody (mouse IgG), and coimmunoprecipitation of PKCα was detected by immunoblotting. The blot was then stripped and reprobed with anti-DGKζ. Because the DGKζ antibody we used was the NH 2 -terminal anti-peptide rabbit antibody, we could not detect the NH 2 terminus deletion DGKζ mutant L (lane 6). However, we detected DGKζ L protein in the same blot using anti-FLAG antibody (not depicted). Expression of PKCα and DGKζ in the cell lysates is also shown. (B) Purified recombinant PKCα was incubated with the glutathione-sepharose–bound GST (lane 1) or GST fusion proteins that contain either full-length DGKζ (GST–DGKζ, lane 2) or a portion of the catalytic domain of DGKζ (GST–BD, lane 3). The beads were collected by centrifugation, and then the proteins bound to beads were subjected to immunoblot analysis with anti-PKCα. Input represents 5% of initial recombinant PKCα used in this experiment.

    Techniques Used: Transfection, FLAG-tag, Immunoprecipitation, Mutagenesis, Expressing, Purification, Recombinant, Incubation, Centrifugation

    Activation of PKC α impairs its association with DGK ζ . (A) HEK293 cells transfected with PKCα and DGKζ–FLAG were stimulated with PMA or vehicle for 30 min. DGKζ in the cell lysates was immunoprecipitated by anti-FLAG, and coimmunoprecipitation of PKCα was detected by immunoblotting. To inhibit PKC activity, cells were treated with Gö 6983 for 10 min before PMA stimulation. The blot was then stripped and reprobed to detect DGKζ. Expression of DGKζ and PKCα in the cell lysates is also shown. (B) Purified recombinant PKCα was incubated with purified DGKζ–FLAG bound to anti–FLAG-M2 agarose affinity gel or with affinity gel alone in PKC assay buffer (containing phosphatase inhibitors) in the presence or absence of PMA. After 2 h, the beads were washed, and proteins bound to beads were immunoblotted to detect PKCα. Input represents 5% of the initial recombinant PKCα. (C) A172 cells, treated with either 50 ng/ml of PDGF or vehicle for 30 min, were lysed, and then endogenous PKCα proteins were immunoprecipitated with anti-PKCα or normal rabbit IgG as a control. The precipitates were then used for DGK activity assays. To inhibit PKC activity, the cells were treated with Gö 6983 before PDGF stimulation. Data are expressed as the mean ± SEM of three independent experiments. An asterisk indicates P
    Figure Legend Snippet: Activation of PKC α impairs its association with DGK ζ . (A) HEK293 cells transfected with PKCα and DGKζ–FLAG were stimulated with PMA or vehicle for 30 min. DGKζ in the cell lysates was immunoprecipitated by anti-FLAG, and coimmunoprecipitation of PKCα was detected by immunoblotting. To inhibit PKC activity, cells were treated with Gö 6983 for 10 min before PMA stimulation. The blot was then stripped and reprobed to detect DGKζ. Expression of DGKζ and PKCα in the cell lysates is also shown. (B) Purified recombinant PKCα was incubated with purified DGKζ–FLAG bound to anti–FLAG-M2 agarose affinity gel or with affinity gel alone in PKC assay buffer (containing phosphatase inhibitors) in the presence or absence of PMA. After 2 h, the beads were washed, and proteins bound to beads were immunoblotted to detect PKCα. Input represents 5% of the initial recombinant PKCα. (C) A172 cells, treated with either 50 ng/ml of PDGF or vehicle for 30 min, were lysed, and then endogenous PKCα proteins were immunoprecipitated with anti-PKCα or normal rabbit IgG as a control. The precipitates were then used for DGK activity assays. To inhibit PKC activity, the cells were treated with Gö 6983 before PDGF stimulation. Data are expressed as the mean ± SEM of three independent experiments. An asterisk indicates P

    Techniques Used: Activation Assay, Transfection, Immunoprecipitation, Activity Assay, Expressing, Purification, Recombinant, Incubation

    Phosphorylation of the MARCKS motif induces the dissociation between DGK ζ and PKC α . (A) HEK293 cells transfected with PKCα and either wild-type (WT) DGKζ or a MARCKS deletion mutant (ΔM). After 48 h, the cells were stimulated with PMA or vehicle for 30 min. DGKζ in the cell lysates was immunoprecipitated by anti-FLAG, and coimmunoprecipitation of PKCα was detected by immunoblotting. The blot was then stripped and reprobed to detect DGKζ. Expression of PKCα and DGKζ in the cell lysates is also shown. (B) PKCα was transfected into HEK293 cells along with wild-type (WT) DGKζ, DGKζ S/D, or DGKζ S/N. DGKζ proteins in the cell lysates were immunoprecipitated with anti-FLAG or a control antibody (mouse IgG), and coimmunoprecipitation of PKCα was detected by immunoblotting. The blot was then stripped and reprobed to detect DGKζ. Expression of PKCα and DGKζ in the cell lysates is also shown. (C) HEK293 cells transfected with PKCα and either wild-type (WT) DGKζ or DGKζ S/N were stimulated with PMA or vehicle for 30 min. DGKζ in the cell lysates was immunoprecipitated by anti-FLAG, and coimmunoprecipitation of PKCα was detected by immunoblotting. To inhibit PKC activity, cells were treated with Gö 6983 for 10 min before PMA stimulation. The blot was then stripped and reprobed to detect DGKζ. Expression of DGKζ and PKCα in the cell lysates is also shown.
    Figure Legend Snippet: Phosphorylation of the MARCKS motif induces the dissociation between DGK ζ and PKC α . (A) HEK293 cells transfected with PKCα and either wild-type (WT) DGKζ or a MARCKS deletion mutant (ΔM). After 48 h, the cells were stimulated with PMA or vehicle for 30 min. DGKζ in the cell lysates was immunoprecipitated by anti-FLAG, and coimmunoprecipitation of PKCα was detected by immunoblotting. The blot was then stripped and reprobed to detect DGKζ. Expression of PKCα and DGKζ in the cell lysates is also shown. (B) PKCα was transfected into HEK293 cells along with wild-type (WT) DGKζ, DGKζ S/D, or DGKζ S/N. DGKζ proteins in the cell lysates were immunoprecipitated with anti-FLAG or a control antibody (mouse IgG), and coimmunoprecipitation of PKCα was detected by immunoblotting. The blot was then stripped and reprobed to detect DGKζ. Expression of PKCα and DGKζ in the cell lysates is also shown. (C) HEK293 cells transfected with PKCα and either wild-type (WT) DGKζ or DGKζ S/N were stimulated with PMA or vehicle for 30 min. DGKζ in the cell lysates was immunoprecipitated by anti-FLAG, and coimmunoprecipitation of PKCα was detected by immunoblotting. To inhibit PKC activity, cells were treated with Gö 6983 for 10 min before PMA stimulation. The blot was then stripped and reprobed to detect DGKζ. Expression of DGKζ and PKCα in the cell lysates is also shown.

    Techniques Used: Transfection, Mutagenesis, Immunoprecipitation, Expressing, Activity Assay

    11) Product Images from "Fam40b is required for lineage commitment of murine embryonic stem cells"

    Article Title: Fam40b is required for lineage commitment of murine embryonic stem cells

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2014.273

    Molecular weight and cellular localization of FAM40B protein. ( a ) Protein lysates were prepared from undifferentiated ESCs. After separation of 40 μ g protein by SDS polyacrylamide (10%) gel electrophoresis (SDS-PAGE), western blotting of the proteins was done on nitrocellulose membrane. Chemiluminescence detection of FAM40B has been performed using the Fam40b-433–450 polyclonal antibodies and anti-Mouse IgG alkaline phosphatase-conjugated secondary antibodies. ( b and d ) Localization of FAM40B in ESCs. ESCs were transfected with the HaloTag Flexi Vector containing the Fam40b cDNA using TurboFect. After 48 h, Fam40b was detected using the HaloTag Oregon Green ligand in the nucleoli by confocal microscopy. Normal arrows show Fam40b in the nucleoli and dashed arrows the perinuclear Fam40b. ( c ) The transparent light microscopy of ( b ). ( e ) After fixing of the ESCs (in d ), Fam40b has also been detected by immunohistochemistry using primary Anti HaloTag pAb (1 : 500 dilution) and anti-mouse IgG Alexa Fluor 594 secondary antibodies. ( f ) Immunostaining of Fam40b in WT ESCs using primary anti-Fam40b antibodies (sc-162799; 1 : 200) and donkey anti goat IgG-FITC secondary antibody (sc-2024, 1 : 200) as secondary antibody (upper scan, green pseudocolor). Cells were co-stained with the nuclear marker Hoechst 33342 (scan in the middle, blue). The overlay of nuclear and Fam40b staining ( f , bottom) reveals that the presence of Fam40b is not restricted to the nucleus but also extends to perinuclear or even cytoplasmic domains of the ESCs (scale bar: 10 μ m)
    Figure Legend Snippet: Molecular weight and cellular localization of FAM40B protein. ( a ) Protein lysates were prepared from undifferentiated ESCs. After separation of 40 μ g protein by SDS polyacrylamide (10%) gel electrophoresis (SDS-PAGE), western blotting of the proteins was done on nitrocellulose membrane. Chemiluminescence detection of FAM40B has been performed using the Fam40b-433–450 polyclonal antibodies and anti-Mouse IgG alkaline phosphatase-conjugated secondary antibodies. ( b and d ) Localization of FAM40B in ESCs. ESCs were transfected with the HaloTag Flexi Vector containing the Fam40b cDNA using TurboFect. After 48 h, Fam40b was detected using the HaloTag Oregon Green ligand in the nucleoli by confocal microscopy. Normal arrows show Fam40b in the nucleoli and dashed arrows the perinuclear Fam40b. ( c ) The transparent light microscopy of ( b ). ( e ) After fixing of the ESCs (in d ), Fam40b has also been detected by immunohistochemistry using primary Anti HaloTag pAb (1 : 500 dilution) and anti-mouse IgG Alexa Fluor 594 secondary antibodies. ( f ) Immunostaining of Fam40b in WT ESCs using primary anti-Fam40b antibodies (sc-162799; 1 : 200) and donkey anti goat IgG-FITC secondary antibody (sc-2024, 1 : 200) as secondary antibody (upper scan, green pseudocolor). Cells were co-stained with the nuclear marker Hoechst 33342 (scan in the middle, blue). The overlay of nuclear and Fam40b staining ( f , bottom) reveals that the presence of Fam40b is not restricted to the nucleus but also extends to perinuclear or even cytoplasmic domains of the ESCs (scale bar: 10 μ m)

    Techniques Used: Molecular Weight, Nucleic Acid Electrophoresis, SDS Page, Western Blot, Transfection, Plasmid Preparation, Confocal Microscopy, Light Microscopy, Immunohistochemistry, Immunostaining, Staining, Marker

    12) Product Images from "Autoantibodies to IgG/HLA class II complexes are associated with rheumatoid arthritis susceptibility"

    Article Title: Autoantibodies to IgG/HLA class II complexes are associated with rheumatoid arthritis susceptibility

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

    doi: 10.1073/pnas.1401105111

    A strong correlation between autoantibody binding to IgG complexed with each HLA-DR allele and the odds ratio for that allele’s association with RA. ( A ) The IgGH was cotransfected with Ig light chain (L), Ii, and HLA-DRα in combination
    Figure Legend Snippet: A strong correlation between autoantibody binding to IgG complexed with each HLA-DR allele and the odds ratio for that allele’s association with RA. ( A ) The IgGH was cotransfected with Ig light chain (L), Ii, and HLA-DRα in combination

    Techniques Used: Binding Assay

    IgGH is transported to the cell surface by HLA-DR. ( A ) The secreted forms of IgGHs cloned from human PBMCs that have different V regions were cotransfected with HLA-DRα, DRB1*04:04 (HLA-DR4), and GFP. Cell surface IgG on GFP-expressing cells was
    Figure Legend Snippet: IgGH is transported to the cell surface by HLA-DR. ( A ) The secreted forms of IgGHs cloned from human PBMCs that have different V regions were cotransfected with HLA-DRα, DRB1*04:04 (HLA-DR4), and GFP. Cell surface IgG on GFP-expressing cells was

    Techniques Used: Clone Assay, Expressing

    13) Product Images from "Involvement of clathrin and AP-2 in the trafficking of MHC class II molecules to antigen-processing compartments"

    Article Title: Involvement of clathrin and AP-2 in the trafficking of MHC class II molecules to antigen-processing compartments

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

    doi: 10.1073/pnas.0502206102

    Efficient knockdown of CHC and AP-μ subunits in Mel JuSo and HeLa-CIITA cells. ( A ) HeLa-CIITA and Mel JuSo cells were mock-treated or treated with siRNA oligonucleotides for μ1A, μ2, μ3A, and CHC. Immunoblot (IB) analysis was performed by using antibodies to the proteins indicated. ( B ) Immunofluorescence (IF) microscopy of mock-treated and μ4-siRNA-treated HeLa-CIITA and Mel JuSo cells with rabbit antibody to β4. ( C ) FACS analysis of TfR and MHC-I surface expression in HeLa-CIITA cells that were either mock-treated (open curves) or treated with siRNAs directed to the indicated proteins (shaded curves). Primary antibodies to TfR (DF 1513) and MHC-I (W6/32) were used, followed by incubation with phycoerythrin-conjugated anti-mouse IgG.
    Figure Legend Snippet: Efficient knockdown of CHC and AP-μ subunits in Mel JuSo and HeLa-CIITA cells. ( A ) HeLa-CIITA and Mel JuSo cells were mock-treated or treated with siRNA oligonucleotides for μ1A, μ2, μ3A, and CHC. Immunoblot (IB) analysis was performed by using antibodies to the proteins indicated. ( B ) Immunofluorescence (IF) microscopy of mock-treated and μ4-siRNA-treated HeLa-CIITA and Mel JuSo cells with rabbit antibody to β4. ( C ) FACS analysis of TfR and MHC-I surface expression in HeLa-CIITA cells that were either mock-treated (open curves) or treated with siRNAs directed to the indicated proteins (shaded curves). Primary antibodies to TfR (DF 1513) and MHC-I (W6/32) were used, followed by incubation with phycoerythrin-conjugated anti-mouse IgG.

    Techniques Used: Immunofluorescence, Microscopy, FACS, Expressing, Incubation

    14) Product Images from "Expression and localisation of Rab44 in immune-related cells change during cell differentiation and stimulation"

    Article Title: Expression and localisation of Rab44 in immune-related cells change during cell differentiation and stimulation

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-67638-7

    Immunofluorescence analysis of Rab44 and markers for haematopoietic stem cells in the bone marrow. ( a ) The fixed sections of femur containing bone marrow were blocked in PBS containing 5% normal donkey serum. The samples were incubated with rabbit polyclonal anti-Rab44 IgG (1:1,000) as the first antibody followed by fluorescent labelling with Alexa fluor 488-conjugated anti-rabbit IgG and then visualised by confocal laser microscopy. Bar: 20 μm. ( b , c ) The mouse bone was fixed, permeabilised with 0.1% Triton X-100 in PBS, and then allowed to react with antibodies against Rab44 and ( b ) CD117 or ( c ) Sca-1. After washing, the samples were incubated with a fluorescence-labelled secondary antibody and then visualised by confocal laser microscopy. Bar: 5 μm.
    Figure Legend Snippet: Immunofluorescence analysis of Rab44 and markers for haematopoietic stem cells in the bone marrow. ( a ) The fixed sections of femur containing bone marrow were blocked in PBS containing 5% normal donkey serum. The samples were incubated with rabbit polyclonal anti-Rab44 IgG (1:1,000) as the first antibody followed by fluorescent labelling with Alexa fluor 488-conjugated anti-rabbit IgG and then visualised by confocal laser microscopy. Bar: 20 μm. ( b , c ) The mouse bone was fixed, permeabilised with 0.1% Triton X-100 in PBS, and then allowed to react with antibodies against Rab44 and ( b ) CD117 or ( c ) Sca-1. After washing, the samples were incubated with a fluorescence-labelled secondary antibody and then visualised by confocal laser microscopy. Bar: 5 μm.

    Techniques Used: Immunofluorescence, Incubation, Microscopy, Fluorescence

    15) 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

    16) Product Images from "Dynamic subcellular localization of isoforms of the folate pathway enzyme serine hydroxymethyltransferase (SHMT) through the erythrocytic cycle of Plasmodium falciparum"

    Article Title: Dynamic subcellular localization of isoforms of the folate pathway enzyme serine hydroxymethyltransferase (SHMT) through the erythrocytic cycle of Plasmodium falciparum

    Journal: Malaria Journal

    doi: 10.1186/1475-2875-9-351

    Triple-labelling experiments . (A) and (B) Combined mitochondrial and apicoplast images probed with anti-PfSHMTc. These do not show nuclear morphology, therefore the erythrocytic cycle stage cannot be precisely ascertained; however, the size of the organelles and overall size of the parasites in (A) and (B) suggest that both are mid trophozoites. In (A) the parasite is probed with anti-PfSHMTc, MitoTracker and anti-ACP (plastid). The plastid is coincident with an area of marked PfSHMTc fluorescence, whereas the mitochondrion shows no evidence of coincident PfSHMTc fluorescence. In (B) the parasite is probed with anti-PfSHMTc, MitoTracker and anti-ACP (plastid). The plastid is coincident with a discrete area of PfSHMTc fluorescence, whereas the mitochondrion is located in a pocket of lower PfSHMTc fluorescence. (C) Parasite is probably a late trophozoite and (D) a mitotic schizont. Both parasites were expressing DsRED-labelled ACP and were probed with both anti-PcSHMTc (IgY) and anti-PfSHMTm (IgG). The distribution of the two SHMT fluorescence signals are similar but not identical, and both co-localize with the apicoplast (scale bars (A) and (C), 3 μm, (B) 2 μm, (D) 4 μm). The associated table shows the percentage volume (V%) and material (M%) co-localization data for PfSHMTc (Sc), PfSHMTm (Sm), MitoTracker (MIT) and acyl carrier protein (ACP) fluorescence.
    Figure Legend Snippet: Triple-labelling experiments . (A) and (B) Combined mitochondrial and apicoplast images probed with anti-PfSHMTc. These do not show nuclear morphology, therefore the erythrocytic cycle stage cannot be precisely ascertained; however, the size of the organelles and overall size of the parasites in (A) and (B) suggest that both are mid trophozoites. In (A) the parasite is probed with anti-PfSHMTc, MitoTracker and anti-ACP (plastid). The plastid is coincident with an area of marked PfSHMTc fluorescence, whereas the mitochondrion shows no evidence of coincident PfSHMTc fluorescence. In (B) the parasite is probed with anti-PfSHMTc, MitoTracker and anti-ACP (plastid). The plastid is coincident with a discrete area of PfSHMTc fluorescence, whereas the mitochondrion is located in a pocket of lower PfSHMTc fluorescence. (C) Parasite is probably a late trophozoite and (D) a mitotic schizont. Both parasites were expressing DsRED-labelled ACP and were probed with both anti-PcSHMTc (IgY) and anti-PfSHMTm (IgG). The distribution of the two SHMT fluorescence signals are similar but not identical, and both co-localize with the apicoplast (scale bars (A) and (C), 3 μm, (B) 2 μm, (D) 4 μm). The associated table shows the percentage volume (V%) and material (M%) co-localization data for PfSHMTc (Sc), PfSHMTm (Sm), MitoTracker (MIT) and acyl carrier protein (ACP) fluorescence.

    Techniques Used: Fluorescence, Expressing

    Late schizonts show a central concentration of PfSHMTc fluorescence . (A) Post-mitotic schizont showing a concentration of PfSHMTc fluorescence in the centre of the parasite, and overlapping the outer zone of haemozoin. PfSHMTc is largely excluded from the nuclei. (B) Post-mitotic schizont showing a concentration of PfSHMTc fluorescence in the centre of the parasite as well as at low intensity in the multiple small apicoplasts. Note the merozoite buds arranged in a radial pattern centred on the future residual body. (C) A post-mitotic parasite probed with both anti-PfSHMTc (IgY) and anti-PfSHMTm (IgG). Both SHMT proteins show a similar, but not identical distribution, as described for image series (A) and (B) above (scale bars 3 μm).
    Figure Legend Snippet: Late schizonts show a central concentration of PfSHMTc fluorescence . (A) Post-mitotic schizont showing a concentration of PfSHMTc fluorescence in the centre of the parasite, and overlapping the outer zone of haemozoin. PfSHMTc is largely excluded from the nuclei. (B) Post-mitotic schizont showing a concentration of PfSHMTc fluorescence in the centre of the parasite as well as at low intensity in the multiple small apicoplasts. Note the merozoite buds arranged in a radial pattern centred on the future residual body. (C) A post-mitotic parasite probed with both anti-PfSHMTc (IgY) and anti-PfSHMTm (IgG). Both SHMT proteins show a similar, but not identical distribution, as described for image series (A) and (B) above (scale bars 3 μm).

    Techniques Used: Concentration Assay, Fluorescence

    17) Product Images from "The C5a Receptor (C5aR) C5L2 Is a Modulator of C5aR-mediated Signal Transduction *"

    Article Title: The C5a Receptor (C5aR) C5L2 Is a Modulator of C5aR-mediated Signal Transduction *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.092106

    C5L2 activation is a consequence of activation of the C5aR. Human PMNs were incubated with C5a in the presence of 5 μg/ml isotype control IgG or anti-C5aR antibody 3C5, as indicated, and then permeabilized and stained with rabbit anti-C5L2 and
    Figure Legend Snippet: C5L2 activation is a consequence of activation of the C5aR. Human PMNs were incubated with C5a in the presence of 5 μg/ml isotype control IgG or anti-C5aR antibody 3C5, as indicated, and then permeabilized and stained with rabbit anti-C5L2 and

    Techniques Used: Activation Assay, Incubation, Staining

    18) Product Images from "High Mobility Group A2 protects cancer cells against telomere dysfunction"

    Article Title: High Mobility Group A2 protects cancer cells against telomere dysfunction

    Journal: Oncotarget

    doi: 10.18632/oncotarget.6938

    HMGA2 silencing reduces telomeric DNA bound to TRF2 Telomere-Chromatin immunoprecipitation was performed with TRF2 antibody in the presence of HMGA2 (−dox) and at HMGA2low conditions (+dox). The presence of telomeric DNA was quantified by real time quantitative PCR in TRF2 immunoprecipitated and purified DNA [ 73 ]. Enrichment of telomeric DNA in telomere-ChIP upon TRF2 IP was normalized to the corresponding IgG control. A. A significant reduction in fold enrichment of telomeric DNA was observed upon knockdown of HMGA2 (+dox) p
    Figure Legend Snippet: HMGA2 silencing reduces telomeric DNA bound to TRF2 Telomere-Chromatin immunoprecipitation was performed with TRF2 antibody in the presence of HMGA2 (−dox) and at HMGA2low conditions (+dox). The presence of telomeric DNA was quantified by real time quantitative PCR in TRF2 immunoprecipitated and purified DNA [ 73 ]. Enrichment of telomeric DNA in telomere-ChIP upon TRF2 IP was normalized to the corresponding IgG control. A. A significant reduction in fold enrichment of telomeric DNA was observed upon knockdown of HMGA2 (+dox) p

    Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Immunoprecipitation, Purification

    19) Product Images from "Independent Regulation of Reovirus Membrane Penetration and Apoptosis by the ?1 ? Domain"

    Article Title: Independent Regulation of Reovirus Membrane Penetration and Apoptosis by the ?1 ? Domain

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1000248

    Apoptosis-modulating ϕ mutations alter μ1 distribution in cells. (A) CV-1 cells were infected with 10 PFU/cell of rsT3D or the indicated ϕ mutant, fixed 48 h post-infection, permeabilized, and immunostained with anti-μ1 MAb 4A3 (green) and anti-μNS serum (red), followed by goat anti-mouse IgG conjugated to Alexa Fluor 488 (green) and goat anti-rabbit IgG conjugated to Alexa Fluor 594 (red). Scale bars, 10 µm. A representative image of the predominant μ1 distribution pattern following infection with each virus strain is shown. (B) The patterns of μ1 distribution in individual infected cells were scored for each time point as diffuse, associated with viral inclusions, or associated with intracellular membranes (marked by distinct ring-like distribution of μ1). Results are expressed as the mean percentage of cells showing the indicated μ1 distribution for triplicate samples. Error bars indicate SD. *, P
    Figure Legend Snippet: Apoptosis-modulating ϕ mutations alter μ1 distribution in cells. (A) CV-1 cells were infected with 10 PFU/cell of rsT3D or the indicated ϕ mutant, fixed 48 h post-infection, permeabilized, and immunostained with anti-μ1 MAb 4A3 (green) and anti-μNS serum (red), followed by goat anti-mouse IgG conjugated to Alexa Fluor 488 (green) and goat anti-rabbit IgG conjugated to Alexa Fluor 594 (red). Scale bars, 10 µm. A representative image of the predominant μ1 distribution pattern following infection with each virus strain is shown. (B) The patterns of μ1 distribution in individual infected cells were scored for each time point as diffuse, associated with viral inclusions, or associated with intracellular membranes (marked by distinct ring-like distribution of μ1). Results are expressed as the mean percentage of cells showing the indicated μ1 distribution for triplicate samples. Error bars indicate SD. *, P

    Techniques Used: Infection, Mutagenesis

    20) Product Images from "Prostacyclin Prevents Pericyte Loss and Demyelination Induced by Lysophosphatidylcholine in the Central Nervous System *"

    Article Title: Prostacyclin Prevents Pericyte Loss and Demyelination Induced by Lysophosphatidylcholine in the Central Nervous System *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.587253

    Treatment with iloprost prevents LPC-mediated vascular barrier disruption in the adult spinal cord. A , visualization of vascular leakage in the CNS. Upper panels, representative images of the thoracic spinal cord showing leakage of Alexa Fluor 555-conjugated cadaverine ( red ) into the spinal cord 1 day after LPC injection. Iloprost treatment was initiated immediately after LPC injection. The broken line represents the outline of the tissue. Scale bar, 1 mm. Middle panels, representative images of the thoracic spinal cord showing the distribution of Alexa Fluor 555-conjugated cadaverine ( red ) in the spinal cord 1 day after LPC injection. Iloprost treatment was initiated immediately after LPC injection. Sections were counterstained for CD31 (vascular endothelial cell marker, green ). Scale bar, 100 μm. Lower panels, representative images of cross-sections of the thoracic spinal cord showing leakage of endogenous IgG ( green ) into the spinal cord 1 day after LPC injection. Iloprost treatment was initiated immediately after LPC injection. Sections were vascular counterstained with DyLight 594-labeled L. esculentum lectin (vascular endothelial cell marker, red ). Scale bar, 100 μm. B , quantification of Evans Blue leakage in lesions of the spinal cord at 1 day after the operation. Values represent the mean ± S.E. of three independent experiments. **, p
    Figure Legend Snippet: Treatment with iloprost prevents LPC-mediated vascular barrier disruption in the adult spinal cord. A , visualization of vascular leakage in the CNS. Upper panels, representative images of the thoracic spinal cord showing leakage of Alexa Fluor 555-conjugated cadaverine ( red ) into the spinal cord 1 day after LPC injection. Iloprost treatment was initiated immediately after LPC injection. The broken line represents the outline of the tissue. Scale bar, 1 mm. Middle panels, representative images of the thoracic spinal cord showing the distribution of Alexa Fluor 555-conjugated cadaverine ( red ) in the spinal cord 1 day after LPC injection. Iloprost treatment was initiated immediately after LPC injection. Sections were counterstained for CD31 (vascular endothelial cell marker, green ). Scale bar, 100 μm. Lower panels, representative images of cross-sections of the thoracic spinal cord showing leakage of endogenous IgG ( green ) into the spinal cord 1 day after LPC injection. Iloprost treatment was initiated immediately after LPC injection. Sections were vascular counterstained with DyLight 594-labeled L. esculentum lectin (vascular endothelial cell marker, red ). Scale bar, 100 μm. B , quantification of Evans Blue leakage in lesions of the spinal cord at 1 day after the operation. Values represent the mean ± S.E. of three independent experiments. **, p

    Techniques Used: Injection, Marker, Labeling

    21) Product Images from "Multiple Functions of Glutamate Uptake via Meningococcal GltT-GltM l-Glutamate ABC Transporter in Neisseria meningitidis Internalization into Human Brain Microvascular Endothelial Cells"

    Article Title: Multiple Functions of Glutamate Uptake via Meningococcal GltT-GltM l-Glutamate ABC Transporter in Neisseria meningitidis Internalization into Human Brain Microvascular Endothelial Cells

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00654-15

    N. meningitidis formed larger colonies on HBMEC at higher MOIs. Shown are phase-contrast and immunofluorescent staining with anti- N. meningitidis (α-Nm) rabbit serum and anti-rabbit IgG-Alexa 488, respectively. HBMEC infections were done with wild-type (HT1125) and Δ gltT Δ gltM (HT1414) strains in AM at MOIs of 5, 50, and 500. Magnification, ×1,000.
    Figure Legend Snippet: N. meningitidis formed larger colonies on HBMEC at higher MOIs. Shown are phase-contrast and immunofluorescent staining with anti- N. meningitidis (α-Nm) rabbit serum and anti-rabbit IgG-Alexa 488, respectively. HBMEC infections were done with wild-type (HT1125) and Δ gltT Δ gltM (HT1414) strains in AM at MOIs of 5, 50, and 500. Magnification, ×1,000.

    Techniques Used: Staining

    Phase-contrast and immunofluorescence microscopy showing ezrin accumulation beneath N. meningitidis -infected HBMEC. Noninfected controls are also shown. HBMEC monolayers were infected with N. meningitidis wild-type (HT1125) and Δ gltT Δ gltM (HT1414) strains in AM (A), AM(−S) (B), and AM(−S, +Glu) (C). N. meningitidis and HBMEC were observed by phase-contrast microscopy (left column). N. meningitidis strains and ezrin were immunostained with two sets of primary and secondary antibodies: anti- N. meningitidis rabbit serum and Alexa Fluor 488-conjugated anti-rabbit IgG (middle column) and anti-ezrin monoclonal antibody (MAb) and Alexa Fluor 594-conjugated anti-mouse IgG (right column). Magnification, ×400.
    Figure Legend Snippet: Phase-contrast and immunofluorescence microscopy showing ezrin accumulation beneath N. meningitidis -infected HBMEC. Noninfected controls are also shown. HBMEC monolayers were infected with N. meningitidis wild-type (HT1125) and Δ gltT Δ gltM (HT1414) strains in AM (A), AM(−S) (B), and AM(−S, +Glu) (C). N. meningitidis and HBMEC were observed by phase-contrast microscopy (left column). N. meningitidis strains and ezrin were immunostained with two sets of primary and secondary antibodies: anti- N. meningitidis rabbit serum and Alexa Fluor 488-conjugated anti-rabbit IgG (middle column) and anti-ezrin monoclonal antibody (MAb) and Alexa Fluor 594-conjugated anti-mouse IgG (right column). Magnification, ×400.

    Techniques Used: Immunofluorescence, Microscopy, Infection

    22) Product Images from "Cell-Surface Nucleolin is a Signal Transducing P-Selectin Binding Protein for Human Colon Carcinoma Cells"

    Article Title: Cell-Surface Nucleolin is a Signal Transducing P-Selectin Binding Protein for Human Colon Carcinoma Cells

    Journal: Experimental cell research

    doi: 10.1016/j.yexcr.2008.03.016

    Cell-surface nucleolin functions as P-selectin binding protein in Colo-320 cells (A) Colo-320 cells were allowed to attach to immobilized BSA (white bars) or P-selectin (black bars) substrates for 60 min in the absence (-) or presence of 20 μg/ml anti-nucleolin mAb (D3) or non-immune mouse IgG for 60 min at 37°C. Error bars indicate standard deviation. (B) Colo-320 cells were incubated with P-selectin IgG-Fc chimera at 4°C for 45 min. After washing, cell were plated on polylysine-coated cover slips in the presence or absence of anti-human IgG-Fc (cross-linker) and incubated for 10 min at 37°C, washed, and fixed. The localization of nucleolin on non-permeabilized cells was determined by staining with antibody against nucleolin and with Alexa 488 goat anti-mouse IgG-Fc (green). P-selectin was localized with Alexa 565 goat anti-human IgG-Fc (red). The nucleus was stained with DAPI (blue). Fluorescent images were combined to determine colocalization (yellow). Bar = 10 μM.
    Figure Legend Snippet: Cell-surface nucleolin functions as P-selectin binding protein in Colo-320 cells (A) Colo-320 cells were allowed to attach to immobilized BSA (white bars) or P-selectin (black bars) substrates for 60 min in the absence (-) or presence of 20 μg/ml anti-nucleolin mAb (D3) or non-immune mouse IgG for 60 min at 37°C. Error bars indicate standard deviation. (B) Colo-320 cells were incubated with P-selectin IgG-Fc chimera at 4°C for 45 min. After washing, cell were plated on polylysine-coated cover slips in the presence or absence of anti-human IgG-Fc (cross-linker) and incubated for 10 min at 37°C, washed, and fixed. The localization of nucleolin on non-permeabilized cells was determined by staining with antibody against nucleolin and with Alexa 488 goat anti-mouse IgG-Fc (green). P-selectin was localized with Alexa 565 goat anti-human IgG-Fc (red). The nucleus was stained with DAPI (blue). Fluorescent images were combined to determine colocalization (yellow). Bar = 10 μM.

    Techniques Used: Binding Assay, Standard Deviation, Incubation, Staining

    23) Product Images from "Annexin A2 Mediates Mycoplasma pneumoniae Community-Acquired Respiratory Distress Syndrome Toxin Binding to Eukaryotic Cells"

    Article Title: Annexin A2 Mediates Mycoplasma pneumoniae Community-Acquired Respiratory Distress Syndrome Toxin Binding to Eukaryotic Cells

    Journal: mBio

    doi: 10.1128/mBio.01497-14

    Binding of CARDS toxin to recombinant AnxA2. (A) Binding of CARDS toxin to AnxA2 by ligand blotting. GST-AnxA2 or BSA (2 µg each) was separated on SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and incubated with CARDS toxin (7 µg/ml) for 2 h. CARDS toxin binding was detected by incubation with rabbit polyclonal anti-CARDS toxin antibody followed by incubation with goat anti-rabbit IgG and visualization with ECL. Lane 1, AnxA2; lane 2, BSA. (B) Dose-dependent binding of CARDS toxin to AnxA2. Microtiter wells were coated with 100 ng AnxA2, and increasing concentrations of CARDS toxin or BSA were added to individual wells for 1 h at room temperature. Bound protein was detected with rabbit polyclonal anti-CARDS toxin antibody and goat anti-rabbit HRP-conjugated polyclonal antibody, followed by development with TMB substrate. Wells with BSA alone served as negative controls, and the nonspecific bound values were subtracted from individual test scores. Values are means ± standard errors of the means (error bars) for triplicate wells from three separate experiments. No immunological cross-reactivity was observed between anti-CARDS toxin antibody and AnxA2.
    Figure Legend Snippet: Binding of CARDS toxin to recombinant AnxA2. (A) Binding of CARDS toxin to AnxA2 by ligand blotting. GST-AnxA2 or BSA (2 µg each) was separated on SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and incubated with CARDS toxin (7 µg/ml) for 2 h. CARDS toxin binding was detected by incubation with rabbit polyclonal anti-CARDS toxin antibody followed by incubation with goat anti-rabbit IgG and visualization with ECL. Lane 1, AnxA2; lane 2, BSA. (B) Dose-dependent binding of CARDS toxin to AnxA2. Microtiter wells were coated with 100 ng AnxA2, and increasing concentrations of CARDS toxin or BSA were added to individual wells for 1 h at room temperature. Bound protein was detected with rabbit polyclonal anti-CARDS toxin antibody and goat anti-rabbit HRP-conjugated polyclonal antibody, followed by development with TMB substrate. Wells with BSA alone served as negative controls, and the nonspecific bound values were subtracted from individual test scores. Values are means ± standard errors of the means (error bars) for triplicate wells from three separate experiments. No immunological cross-reactivity was observed between anti-CARDS toxin antibody and AnxA2.

    Techniques Used: Binding Assay, Recombinant, Incubation

    24) Product Images from "A Monoclonal Antibody-Based Copro-ELISA Kit for Canine Echinococcosis to Support the PAHO Effort for Hydatid Disease Control in South America"

    Article Title: A Monoclonal Antibody-Based Copro-ELISA Kit for Canine Echinococcosis to Support the PAHO Effort for Hydatid Disease Control in South America

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0001967

    Time-course stability of the copro-ELISA kit components. A) Dried or freshly-coated plates (white and gray symbols, respectively) were tested at different time points by assaying a set of 3 weak-positive and 3 negative samples (average = circles and squares, respectively). B–D) Fresh solutions of the calibrator were tested in triplicate at different time points, using fresh (grey circles) or stored dilutions of the kit reagents kept at room temperature (white) or 37°C (black symbols). All values were normalized with regard to the value obtained with the fresh reagent. B) Stability of the calibrator: triangles, PBS, 5% glycerol, 0.1% Kathon. C) MAb Eg9 stability: squares, PBS-T; triangles, PBS-T, 0.1% Kathon. D) Stability of the peroxidase anti-mouse IgG antibody: squares, PBS, 0.1% BSA; triangles, PBS, 0.1% BSA, 0.1% Kathon, 0.1 mM 3,3′,5,5′-tetramethylbenzidine (TMB).
    Figure Legend Snippet: Time-course stability of the copro-ELISA kit components. A) Dried or freshly-coated plates (white and gray symbols, respectively) were tested at different time points by assaying a set of 3 weak-positive and 3 negative samples (average = circles and squares, respectively). B–D) Fresh solutions of the calibrator were tested in triplicate at different time points, using fresh (grey circles) or stored dilutions of the kit reagents kept at room temperature (white) or 37°C (black symbols). All values were normalized with regard to the value obtained with the fresh reagent. B) Stability of the calibrator: triangles, PBS, 5% glycerol, 0.1% Kathon. C) MAb Eg9 stability: squares, PBS-T; triangles, PBS-T, 0.1% Kathon. D) Stability of the peroxidase anti-mouse IgG antibody: squares, PBS, 0.1% BSA; triangles, PBS, 0.1% BSA, 0.1% Kathon, 0.1 mM 3,3′,5,5′-tetramethylbenzidine (TMB).

    Techniques Used: Enzyme-linked Immunosorbent Assay

    25) Product Images from "Octopamine Modulates the Axons of Modulatory Projection Neurons"

    Article Title: Octopamine Modulates the Axons of Modulatory Projection Neurons

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.2078-04.2004

    Synaptic vesicle staining surrounds ion axon collaterals at the level of the stn-son junction. Axons were filled with neurobiotin, which was then visualized by streptavidin coupled to Cy5 ( B, C , green), and synaptic vesicle-containing structures were revealed by anti-synaptotagmin staining visualized with anti-rabbit IgG coupled to AlexaFluor 488 ( B, C , red). A , Schematic of the preparation showing the origin and the trajectory of the neurobiotin-filled axons. The black and white boxes represent the location of the pictures presented in B and C . B , Low magnification of the neurobiotin staining (left panel, green) and the synaptotagmin staining (middle panel, red) at the level of the stn-son junction. C , High-magnification pictures of the region surrounded in B . Note that the synaptotagmin staining is specifically located around the fine axon processes and not around the main axon branches.
    Figure Legend Snippet: Synaptic vesicle staining surrounds ion axon collaterals at the level of the stn-son junction. Axons were filled with neurobiotin, which was then visualized by streptavidin coupled to Cy5 ( B, C , green), and synaptic vesicle-containing structures were revealed by anti-synaptotagmin staining visualized with anti-rabbit IgG coupled to AlexaFluor 488 ( B, C , red). A , Schematic of the preparation showing the origin and the trajectory of the neurobiotin-filled axons. The black and white boxes represent the location of the pictures presented in B and C . B , Low magnification of the neurobiotin staining (left panel, green) and the synaptotagmin staining (middle panel, red) at the level of the stn-son junction. C , High-magnification pictures of the region surrounded in B . Note that the synaptotagmin staining is specifically located around the fine axon processes and not around the main axon branches.

    Techniques Used: Staining

    26) Product Images from "The local immune landscape determines tumor PD-L1 heterogeneity and sensitivity to therapy"

    Article Title: The local immune landscape determines tumor PD-L1 heterogeneity and sensitivity to therapy

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI127726

    PD-L1 + tumors generated differently respond to therapeutic strategies distinctly. ( A ) PD-L1 + HepG2 cells were generated by transducing with pBABE-Puro retroviral vector encoding human CD274 or incubating with tumor T cell–CM or TAM-CM. Survival of cells after 48-hour exposure to doxorubicin was determined ( n = 4). ( B and C ) Incubating with an inhibitor against NF-κB ( B ) and knockdown of P65 NF-κB subunit (si RELA , C ) in HepG2 cells attenuated TAM-CM–mediated resistance to doxorubicin (0.25 μg/mL; n = 7 for B and n = 6 for C ). ( D – G ) PD-L1 + hepatoma Hepa1-6 cells were generated by transducing with pBABE-Puro retroviral vector encoding mouse CD274 or by exposure to CM from Hepa1-6 hepatoma–derived macrophages or T cells ( D ). Thereafter, these cells were left untreated or cultured with Hepa1-6 hepatoma–derived CD8 + T cells in the presence of control IgG or an Ab against PD-L1. Survival of Hepa1-6 cells at indicated times ( E ), 24-hour production of IFN-γ and IL-2 by T cells ( F ), and 24-hour expression or CD107a on T cells ( G ) were determined. Data represent mean ± SEM. Results are representative of at least 4 separate experiments. * P
    Figure Legend Snippet: PD-L1 + tumors generated differently respond to therapeutic strategies distinctly. ( A ) PD-L1 + HepG2 cells were generated by transducing with pBABE-Puro retroviral vector encoding human CD274 or incubating with tumor T cell–CM or TAM-CM. Survival of cells after 48-hour exposure to doxorubicin was determined ( n = 4). ( B and C ) Incubating with an inhibitor against NF-κB ( B ) and knockdown of P65 NF-κB subunit (si RELA , C ) in HepG2 cells attenuated TAM-CM–mediated resistance to doxorubicin (0.25 μg/mL; n = 7 for B and n = 6 for C ). ( D – G ) PD-L1 + hepatoma Hepa1-6 cells were generated by transducing with pBABE-Puro retroviral vector encoding mouse CD274 or by exposure to CM from Hepa1-6 hepatoma–derived macrophages or T cells ( D ). Thereafter, these cells were left untreated or cultured with Hepa1-6 hepatoma–derived CD8 + T cells in the presence of control IgG or an Ab against PD-L1. Survival of Hepa1-6 cells at indicated times ( E ), 24-hour production of IFN-γ and IL-2 by T cells ( F ), and 24-hour expression or CD107a on T cells ( G ) were determined. Data represent mean ± SEM. Results are representative of at least 4 separate experiments. * P

    Techniques Used: Generated, Plasmid Preparation, Cell Culture, Expressing

    27) Product Images from "A tubular EHD1-containing compartment involved in the recycling of major histocompatibility complex class I molecules to the plasma membrane"

    Article Title: A tubular EHD1-containing compartment involved in the recycling of major histocompatibility complex class I molecules to the plasma membrane

    Journal: The EMBO Journal

    doi: 10.1093/emboj/21.11.2557

    Fig. 6. Co-localization of EHD1 and internalized MHC-I molecules. ( A – F ) Co-localization of internalized MHC-I with EHD1 tubular structures. HeLa cells were transiently transfected with a construct encoding GFP–EHD1. After 24 h, the cells were continuously pulsed with W6/32 anti-MHC-I monoclonal antibody for 30 min. After brief acid washing to remove surface-bound MHC-I antibody, fixed and permeabilized cells were incubated with Cy3-conjugated anti-mouse IgG, and examined by confocal microscopy. Internalized MHC-I is shown in (A) and (D), and GFP–EHD1 is depicted in (B) and (E). (C and F) Merged images. ( G ) HeLa cells were transiently co-transfected with Myc-EHD1 and H-2D d (mouse MHC-I) constructs. Cells were pulsed with anti-H-2D d antibody 24 h later, fixed and processed for ultrathin section electron microscopy. EHD1 is marked by 15 nm gold particles, and 10 nm gold particles mark the presence of internalized H-2D d . Arrows denote tubular structures positive for internalized MHC-I (A and D) and GFP–EHD1 (B and E). Bars: (A–C), 10 µm; (D–F), 10 µm; (G), 200 nm.
    Figure Legend Snippet: Fig. 6. Co-localization of EHD1 and internalized MHC-I molecules. ( A – F ) Co-localization of internalized MHC-I with EHD1 tubular structures. HeLa cells were transiently transfected with a construct encoding GFP–EHD1. After 24 h, the cells were continuously pulsed with W6/32 anti-MHC-I monoclonal antibody for 30 min. After brief acid washing to remove surface-bound MHC-I antibody, fixed and permeabilized cells were incubated with Cy3-conjugated anti-mouse IgG, and examined by confocal microscopy. Internalized MHC-I is shown in (A) and (D), and GFP–EHD1 is depicted in (B) and (E). (C and F) Merged images. ( G ) HeLa cells were transiently co-transfected with Myc-EHD1 and H-2D d (mouse MHC-I) constructs. Cells were pulsed with anti-H-2D d antibody 24 h later, fixed and processed for ultrathin section electron microscopy. EHD1 is marked by 15 nm gold particles, and 10 nm gold particles mark the presence of internalized H-2D d . Arrows denote tubular structures positive for internalized MHC-I (A and D) and GFP–EHD1 (B and E). Bars: (A–C), 10 µm; (D–F), 10 µm; (G), 200 nm.

    Techniques Used: Transfection, Construct, Incubation, Confocal Microscopy, Electron Microscopy

    Fig. 5. Co-localization and functional interaction of EHD1 with Arf6. HeLa cells were transiently co-transfected with constructs encoding Myc-EHD1 and wild-type Arf6 ( A – C ), GFP–EHD1 and Arf6-Q67L ( D – F ), GFP–EHD1 and Arf6-T27N ( G – I ), GFP–EHD1 and FLAG-EFA6 ( J – L ), and GFP–EHD1 and FLAG-ACAP1 ( M – O ). Cells were fixed, permeabilized and incubated with a monoclonal antibody to the Myc epitope and a rabbit polyclonal antibody to Arf6 (A–C). Bound antibodies were revealed by Alexa-488-conjugated antibody to mouse IgG (A and C), and by Cy3-conjugated anti-rabbit IgG (B and C). HeLa cells co-transfected with GFP–EHD1 and Arf6 mutant constructs (D–I) were fixed, permeabilized and incubated with a rabbit polyclonal antibody directed against Arf6 (D–I), followed by Cy3-conjugated anti-rabbit IgG (E, F, H and I). HeLa cells co-transfected with GFP–EHD1 and FLAG-EFA6 (J–L) or FLAG-ACAP1 (M–O) were fixed, permeabilized and incubated with a monoclonal antibody to the FLAG epitope, followed by a Cy3-conjugated anti-mouse IgG antibody. All images were obtained by confocal microscopy. Arrows (A and B) denote tubular structures containing both Arf6 and EHD1. Bar, 10 µm.
    Figure Legend Snippet: Fig. 5. Co-localization and functional interaction of EHD1 with Arf6. HeLa cells were transiently co-transfected with constructs encoding Myc-EHD1 and wild-type Arf6 ( A – C ), GFP–EHD1 and Arf6-Q67L ( D – F ), GFP–EHD1 and Arf6-T27N ( G – I ), GFP–EHD1 and FLAG-EFA6 ( J – L ), and GFP–EHD1 and FLAG-ACAP1 ( M – O ). Cells were fixed, permeabilized and incubated with a monoclonal antibody to the Myc epitope and a rabbit polyclonal antibody to Arf6 (A–C). Bound antibodies were revealed by Alexa-488-conjugated antibody to mouse IgG (A and C), and by Cy3-conjugated anti-rabbit IgG (B and C). HeLa cells co-transfected with GFP–EHD1 and Arf6 mutant constructs (D–I) were fixed, permeabilized and incubated with a rabbit polyclonal antibody directed against Arf6 (D–I), followed by Cy3-conjugated anti-rabbit IgG (E, F, H and I). HeLa cells co-transfected with GFP–EHD1 and FLAG-EFA6 (J–L) or FLAG-ACAP1 (M–O) were fixed, permeabilized and incubated with a monoclonal antibody to the FLAG epitope, followed by a Cy3-conjugated anti-mouse IgG antibody. All images were obtained by confocal microscopy. Arrows (A and B) denote tubular structures containing both Arf6 and EHD1. Bar, 10 µm.

    Techniques Used: Functional Assay, Transfection, Construct, Incubation, Mutagenesis, FLAG-tag, Confocal Microscopy

    Fig. 2. EHD1 localizes to an array of long tubular structures. ( A ) Detergent lysates were prepared from untransfected HeLa cells (PI and UT) or HeLa cells transfected with a GFP–EHD1 construct (T), and resolved by 4–20% SDS–PAGE. Immunoblot analysis with either pre-immune serum (PI) or rabbit polyclonal antibody directed against EHD1 (UT and T) revealed the presence of both endogenous EHD1 (55 kDa) and transgenic GFP–EHD1 (85 kDa) proteins. Following transient transfection, ∼60% of the cells expressed detectable levels of EHD1, and the relative levels of transfected and endogenous proteins were estimated by densitometric analysis of multiple film exposures. ( B ) HeLa cell extracts were subjected to sedimentation velocity analysis on a 4–20% sucrose gradient. Fractions were collected, resolved by 4–20% SDS–PAGE, and proteins were visualized by immunoblot analysis using the polyclonal antibody prepared against EHD1. Size markers indicate the positions of albumin, AP-2 complex and catalase on the sucrose gradients. ( C ) HeLa cells were transfected with a plasmid encoding Myc-EHD1. Cells were fixed and permeabilized 24 h later, and incubated with a mouse monoclonal antibody to the Myc epitope. Bound antibodies were revealed by incubation with Cy3-conjugated donkey anti-mouse IgG, demonstrating the presence of a dense network of Myc-EHD1 tubular organelles. ( D ) HeLa cells were transfected with a plasmid encoding GFP–EHD1, and were fixed and permeabilized after 24 h. ( E – G ) Untransfected HeLa cells were fixed, permeabilized and incubated with a rabbit polyclonal antibody to endogenous EHD1. Bound antibodies were revealed by incubation with Cy3-conjugated donkey anti-rabbit IgG. Images show the presence of long, tubular structures containing endogenous EHD proteins. All images were obtained by confocal microscopy. Bars: (C and D), 10 µm; (E–G), 10 µm.
    Figure Legend Snippet: Fig. 2. EHD1 localizes to an array of long tubular structures. ( A ) Detergent lysates were prepared from untransfected HeLa cells (PI and UT) or HeLa cells transfected with a GFP–EHD1 construct (T), and resolved by 4–20% SDS–PAGE. Immunoblot analysis with either pre-immune serum (PI) or rabbit polyclonal antibody directed against EHD1 (UT and T) revealed the presence of both endogenous EHD1 (55 kDa) and transgenic GFP–EHD1 (85 kDa) proteins. Following transient transfection, ∼60% of the cells expressed detectable levels of EHD1, and the relative levels of transfected and endogenous proteins were estimated by densitometric analysis of multiple film exposures. ( B ) HeLa cell extracts were subjected to sedimentation velocity analysis on a 4–20% sucrose gradient. Fractions were collected, resolved by 4–20% SDS–PAGE, and proteins were visualized by immunoblot analysis using the polyclonal antibody prepared against EHD1. Size markers indicate the positions of albumin, AP-2 complex and catalase on the sucrose gradients. ( C ) HeLa cells were transfected with a plasmid encoding Myc-EHD1. Cells were fixed and permeabilized 24 h later, and incubated with a mouse monoclonal antibody to the Myc epitope. Bound antibodies were revealed by incubation with Cy3-conjugated donkey anti-mouse IgG, demonstrating the presence of a dense network of Myc-EHD1 tubular organelles. ( D ) HeLa cells were transfected with a plasmid encoding GFP–EHD1, and were fixed and permeabilized after 24 h. ( E – G ) Untransfected HeLa cells were fixed, permeabilized and incubated with a rabbit polyclonal antibody to endogenous EHD1. Bound antibodies were revealed by incubation with Cy3-conjugated donkey anti-rabbit IgG. Images show the presence of long, tubular structures containing endogenous EHD proteins. All images were obtained by confocal microscopy. Bars: (C and D), 10 µm; (E–G), 10 µm.

    Techniques Used: Transfection, Construct, SDS Page, Transgenic Assay, Sedimentation, Plasmid Preparation, Incubation, Confocal Microscopy

    Fig. 7. EHD1 tubular structures promote recycling of MHC-I to the cell surface. ( A ) Time-dependent co-localization of internalized MHC-I with EHD1 tubules by live image analysis. MHC-I monoclonal antibodies were coupled to Alexa Fluor 568 F(ab′)2 fragment of goat anti-mouse IgG. The coupled antibodies were then used to continuously pulse HeLa cells that were transfected 24 h earlier with a GFP–EHD1 construct. Images of MHC-I uptake (left panels) and GFP–EHD1 tubules (right panels) are depicted. Arrows (white) mark MHC-I tubular structures that appear at 15–20 min of internalization and co-localize with pre-existing GFP–EHD1 tubules (black arrows). Images are shown inverted to facilitate analysis (see Supplementary time-lapse video). Bar, 10 µm. ( B ) Quantification of EHD1-enhanced MHC-I recycling by a CELISA assay. HeLa cells were transfected with cDNA coding for H-2D d (mouse MHC-I), H-2D d and GFP–EHD1, H-2D d and Myc-EHD1, H-2D d and GFP–EHD1-G65R, or H-2D d and GFP–EHD1-K220N. Internalization of MHC-I over time was monitored 24 h after transfection by CELISA utilizing a biotinylated anti-MHC-I antibody (see Materials and methods), and the fraction of MHC-I antibody on the surface at each time point was recorded. A representative experiment from four independent CELISA assays is depicted, with triplicates at each time point.
    Figure Legend Snippet: Fig. 7. EHD1 tubular structures promote recycling of MHC-I to the cell surface. ( A ) Time-dependent co-localization of internalized MHC-I with EHD1 tubules by live image analysis. MHC-I monoclonal antibodies were coupled to Alexa Fluor 568 F(ab′)2 fragment of goat anti-mouse IgG. The coupled antibodies were then used to continuously pulse HeLa cells that were transfected 24 h earlier with a GFP–EHD1 construct. Images of MHC-I uptake (left panels) and GFP–EHD1 tubules (right panels) are depicted. Arrows (white) mark MHC-I tubular structures that appear at 15–20 min of internalization and co-localize with pre-existing GFP–EHD1 tubules (black arrows). Images are shown inverted to facilitate analysis (see Supplementary time-lapse video). Bar, 10 µm. ( B ) Quantification of EHD1-enhanced MHC-I recycling by a CELISA assay. HeLa cells were transfected with cDNA coding for H-2D d (mouse MHC-I), H-2D d and GFP–EHD1, H-2D d and Myc-EHD1, H-2D d and GFP–EHD1-G65R, or H-2D d and GFP–EHD1-K220N. Internalization of MHC-I over time was monitored 24 h after transfection by CELISA utilizing a biotinylated anti-MHC-I antibody (see Materials and methods), and the fraction of MHC-I antibody on the surface at each time point was recorded. A representative experiment from four independent CELISA assays is depicted, with triplicates at each time point.

    Techniques Used: Transfection, Construct

    28) Product Images from "Receptor Activator of NF-?B Ligand Regulates the Proliferation of Mammary Epithelial Cells via Id2"

    Article Title: Receptor Activator of NF-?B Ligand Regulates the Proliferation of Mammary Epithelial Cells via Id2

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.26.3.1002-1013.2006

    RANKL induces the nuclear translocation of Id2 in HC11 and primary mammary epithelial cells. (A) Primary MECs from P14.5 mice were treated with 1 μg of RANKL/ml for the indicated times. Cytoplasmic (C) and nuclear (N) proteins were analyzed by Western blotting for the presence of Id2. Protein disulfide isomerase (PDI) and poly(ADP-ribose) polymerase (PARP) are shown as loading controls for cytoplasmic and nuclear proteins, respectively. One result, representative of four independent experiments, is shown. (B) Primary MECs from P14.5 mice were placed in DMEM containing 1% FBS for 24 h. Serum-starved cells were untreated (left panels) or stimulated (right panels) with 1 μg of RANKL/ml for 3 h. Id2 was detected with an anti-Id2 antibody, followed by Alexa 594-labeled anti-rabbit IgG antibody (red; upper panel). Nuclear DNA was stained with Hoechst (blue; middle panel). Lower panels indicate merged images. At the bottom, the numbers indicate the localization of Id2 in the cytoplasm (C) and nucleus (N) of each cell. The values shown are means ± the standard error of the mean of three separate experiments. One result, representative of three independent experiments, is shown. (C) HC11 cells transiently transfected with HA-tagged Id2 were placed in RPMI containing 0.5% FBS for 24 h. Serum-starved cells were untreated (left panels) or stimulated (right panels) with 1 μg of RANKL/ml for 3 h. HA epitopes were detected with an anti-HA antibody, followed by Alexa 594-labeled anti-mouse IgG antibody (red; upper panels). Nuclear DNA was stained with Hoechst (blue; middle panels). Lower panels indicate merged images. One result, representative of four independent experiments, is shown.
    Figure Legend Snippet: RANKL induces the nuclear translocation of Id2 in HC11 and primary mammary epithelial cells. (A) Primary MECs from P14.5 mice were treated with 1 μg of RANKL/ml for the indicated times. Cytoplasmic (C) and nuclear (N) proteins were analyzed by Western blotting for the presence of Id2. Protein disulfide isomerase (PDI) and poly(ADP-ribose) polymerase (PARP) are shown as loading controls for cytoplasmic and nuclear proteins, respectively. One result, representative of four independent experiments, is shown. (B) Primary MECs from P14.5 mice were placed in DMEM containing 1% FBS for 24 h. Serum-starved cells were untreated (left panels) or stimulated (right panels) with 1 μg of RANKL/ml for 3 h. Id2 was detected with an anti-Id2 antibody, followed by Alexa 594-labeled anti-rabbit IgG antibody (red; upper panel). Nuclear DNA was stained with Hoechst (blue; middle panel). Lower panels indicate merged images. At the bottom, the numbers indicate the localization of Id2 in the cytoplasm (C) and nucleus (N) of each cell. The values shown are means ± the standard error of the mean of three separate experiments. One result, representative of three independent experiments, is shown. (C) HC11 cells transiently transfected with HA-tagged Id2 were placed in RPMI containing 0.5% FBS for 24 h. Serum-starved cells were untreated (left panels) or stimulated (right panels) with 1 μg of RANKL/ml for 3 h. HA epitopes were detected with an anti-HA antibody, followed by Alexa 594-labeled anti-mouse IgG antibody (red; upper panels). Nuclear DNA was stained with Hoechst (blue; middle panels). Lower panels indicate merged images. One result, representative of four independent experiments, is shown.

    Techniques Used: Translocation Assay, Mouse Assay, Western Blot, Labeling, Staining, Transfection

    Nuclear localization of Id2 in mammary epithelial cells. (A and B) Localization and p21 promoter luciferase assays of Id2 and NLS-tagged Id2. MCF7 cells (A) and primary mammary epithelial cells from rankl −/− P14.5 mice (B) were transiently transfected with HA-Id2 (Id2) and NLS-HA-Id2 (NLS-Id2) constructs and placed in DMEM containing 10% FBS for 48 h. HA epitopes were detected with an anti-HA antibody, followed by Alexa 488-labeled anti-mouse IgG antibody (A, green) and Alexa 594-labeled anti-mouse IgG antibody (B, red). Nuclear DNA was stained with PI (A, red) and Hoechst (B, blue). One result, representative of three independent experiments, is shown. (C and D) p21 promoter luciferase assays in MCF7 cells (C) and primary mammary epithelial cells from rankl −/− P14.5 mice (D). Cells were cotransfected with p21-luciferase and Id2 constructs and, after 48 h, the luciferase reporter activity was measured and normalized to the Renilla luciferase activity. The results are shown as mean values ± the standard error of the mean of three separate transfection experiments. ✽, significant difference ( P
    Figure Legend Snippet: Nuclear localization of Id2 in mammary epithelial cells. (A and B) Localization and p21 promoter luciferase assays of Id2 and NLS-tagged Id2. MCF7 cells (A) and primary mammary epithelial cells from rankl −/− P14.5 mice (B) were transiently transfected with HA-Id2 (Id2) and NLS-HA-Id2 (NLS-Id2) constructs and placed in DMEM containing 10% FBS for 48 h. HA epitopes were detected with an anti-HA antibody, followed by Alexa 488-labeled anti-mouse IgG antibody (A, green) and Alexa 594-labeled anti-mouse IgG antibody (B, red). Nuclear DNA was stained with PI (A, red) and Hoechst (B, blue). One result, representative of three independent experiments, is shown. (C and D) p21 promoter luciferase assays in MCF7 cells (C) and primary mammary epithelial cells from rankl −/− P14.5 mice (D). Cells were cotransfected with p21-luciferase and Id2 constructs and, after 48 h, the luciferase reporter activity was measured and normalized to the Renilla luciferase activity. The results are shown as mean values ± the standard error of the mean of three separate transfection experiments. ✽, significant difference ( P

    Techniques Used: Luciferase, Mouse Assay, Transfection, Construct, Labeling, Staining, Activity Assay

    29) Product Images from "Functional Kir7.1 channels localized at the root of apical processes in rat retinal pigment epithelium"

    Article Title: Functional Kir7.1 channels localized at the root of apical processes in rat retinal pigment epithelium

    Journal: The Journal of Physiology

    doi: 10.1111/j.1469-7793.2001.0027j.x

    Immunohistochemical and immunoelectron microscopy analyses of Kir4.1 and Kir7.1 in the retina A , a 12 μm sagittal section from a paraformaldehyde-fixed rat retina was stained with affinity-purified anti-Kir4.1 antibody (left) and affinity-purified anti-Kir7.1 antibody (middle) followed by FITC-conjugated anti-rabbit IgG (green). Kir4.1 is expressed mainly in the apical processes of RPE cells. In contrast, Kir7.1 is expressed mainly in the apical membrane, especially at the root of apical processes of RPE cells. The right panel shows double staining of a sagittal section of methanol-fixed rat retina with anti-Kir7.1 antibody followed by anti-rabbit IgG (Alexa Fluor 568; red) and monoclonal anti-Na + ,K + -ATPase α1-subunit antibody followed by FITC-labelled anti-mouse IgG (green). ONL, outer nuclear layer; OS, outer segment layer; RPE, retinal pigment epithelial cell layer. Scale bars = 10 μm. B , ultra-thin sections were stained with anti-Kir4.1 antibody or anti-Kir7.1 antibody, and anti-rabbit IgG coupled to colloidal gold particles. The portions from which electron microscope images were obtained are indicated as a, b and c in the schematic drawing on the right. Positive gold particles (arrows) coupled to Kir4.1 antibodies were detected on the membranes of middle-distal portions of apical processes ( a , Kir4.1). In contrast, positive gold particles (arrows) coupled to Kir7.1 antibodies were not detected at all on the middle of the processes ( a , Kir7.1). They were abundantly detected on the root of apical processes ( b, Kir7.1). Gold particles coupled to either Kir4.1 or Kir7.1 antibodies were not detected on the basolateral membrane ( c, Kir7.1). OS, photoreceptor outer segment; AP (in a ), apical process; P (in b ), apical process; F, basal infoldings; B, basement membrane of RPE. Scale bars = 200 nm.
    Figure Legend Snippet: Immunohistochemical and immunoelectron microscopy analyses of Kir4.1 and Kir7.1 in the retina A , a 12 μm sagittal section from a paraformaldehyde-fixed rat retina was stained with affinity-purified anti-Kir4.1 antibody (left) and affinity-purified anti-Kir7.1 antibody (middle) followed by FITC-conjugated anti-rabbit IgG (green). Kir4.1 is expressed mainly in the apical processes of RPE cells. In contrast, Kir7.1 is expressed mainly in the apical membrane, especially at the root of apical processes of RPE cells. The right panel shows double staining of a sagittal section of methanol-fixed rat retina with anti-Kir7.1 antibody followed by anti-rabbit IgG (Alexa Fluor 568; red) and monoclonal anti-Na + ,K + -ATPase α1-subunit antibody followed by FITC-labelled anti-mouse IgG (green). ONL, outer nuclear layer; OS, outer segment layer; RPE, retinal pigment epithelial cell layer. Scale bars = 10 μm. B , ultra-thin sections were stained with anti-Kir4.1 antibody or anti-Kir7.1 antibody, and anti-rabbit IgG coupled to colloidal gold particles. The portions from which electron microscope images were obtained are indicated as a, b and c in the schematic drawing on the right. Positive gold particles (arrows) coupled to Kir4.1 antibodies were detected on the membranes of middle-distal portions of apical processes ( a , Kir4.1). In contrast, positive gold particles (arrows) coupled to Kir7.1 antibodies were not detected at all on the middle of the processes ( a , Kir7.1). They were abundantly detected on the root of apical processes ( b, Kir7.1). Gold particles coupled to either Kir4.1 or Kir7.1 antibodies were not detected on the basolateral membrane ( c, Kir7.1). OS, photoreceptor outer segment; AP (in a ), apical process; P (in b ), apical process; F, basal infoldings; B, basement membrane of RPE. Scale bars = 200 nm.

    Techniques Used: Immunohistochemistry, Immuno-Electron Microscopy, Staining, Affinity Purification, Double Staining, Microscopy

    Expression of Kir7.1 mRNA in RPE cells and immunological analysis of Kir7.1 A , RT-PCR amplification of Kir7.1 cDNA from a sheet of RPE cells. Kir7.1 fragments (682 bp) were amplified from cDNA/mRNA from the RPE cells (RPE). Retina, rat sensory retina; -, negative control (distilled water instead of cDNA); +, positive control (rat Kir7.1 cDNA). Numbers on the right indicate the positions of molecular weight markers in base pairs. Ba , immunoblot analysis of an affinity-purified, polyclonal rabbit antibody against the C-terminal region of rat Kir7.1. Membrane fractions obtained from HEK293T cells with (lanes 1 and 3) or without (lane 2) transfection of Kir7.1 were separated by SDS-PAGE, transferred to PVDF membranes, and then immunoblotted with the anti-Kir7.1 antibody. In lane 3 the antibody was preincubated with antigenic peptide. Numbers on the left indicate the positions of molecular mass markers in kilodaltons. Immunolabelling was blocked by preincubation with the antigenic peptides. Bb , immunofluorescence images of HEK293T cells transfected with Kir7.1 obtained with anti-Kir7.1 antibody followed by FITC-conjugated anti-rabbit IgG (green) and analysed with confocal microscopy. Nuclei staining was obtained with propidium iodide (red). Scale bar = 10 μm. Ca , membrane fractions obtained from rat RPE cells (RPE), the sensory retina (Retina), choroid plexus (CP), cerebellum and cerebrum were separated by SDS-PAGE, transferred to PVDF membranes, and then immunoblotted with the anti-Kir7.1 antibody. Numbers on the left indicate the positions of molecular mass markers in kilodaltons. Cb , double staining of a sagittal section of rat brain with anti-Kir7.1 antibody followed by FITC-conjugated anti-rabbit IgG (green) and monoclonal anti-pan cytokeratin antibody followed by Texas Red-labelled anti-mouse IgG (red). Cc , a Nomarski image of the same sagittal section as in Cb . Scale bar = 50 μm.
    Figure Legend Snippet: Expression of Kir7.1 mRNA in RPE cells and immunological analysis of Kir7.1 A , RT-PCR amplification of Kir7.1 cDNA from a sheet of RPE cells. Kir7.1 fragments (682 bp) were amplified from cDNA/mRNA from the RPE cells (RPE). Retina, rat sensory retina; -, negative control (distilled water instead of cDNA); +, positive control (rat Kir7.1 cDNA). Numbers on the right indicate the positions of molecular weight markers in base pairs. Ba , immunoblot analysis of an affinity-purified, polyclonal rabbit antibody against the C-terminal region of rat Kir7.1. Membrane fractions obtained from HEK293T cells with (lanes 1 and 3) or without (lane 2) transfection of Kir7.1 were separated by SDS-PAGE, transferred to PVDF membranes, and then immunoblotted with the anti-Kir7.1 antibody. In lane 3 the antibody was preincubated with antigenic peptide. Numbers on the left indicate the positions of molecular mass markers in kilodaltons. Immunolabelling was blocked by preincubation with the antigenic peptides. Bb , immunofluorescence images of HEK293T cells transfected with Kir7.1 obtained with anti-Kir7.1 antibody followed by FITC-conjugated anti-rabbit IgG (green) and analysed with confocal microscopy. Nuclei staining was obtained with propidium iodide (red). Scale bar = 10 μm. Ca , membrane fractions obtained from rat RPE cells (RPE), the sensory retina (Retina), choroid plexus (CP), cerebellum and cerebrum were separated by SDS-PAGE, transferred to PVDF membranes, and then immunoblotted with the anti-Kir7.1 antibody. Numbers on the left indicate the positions of molecular mass markers in kilodaltons. Cb , double staining of a sagittal section of rat brain with anti-Kir7.1 antibody followed by FITC-conjugated anti-rabbit IgG (green) and monoclonal anti-pan cytokeratin antibody followed by Texas Red-labelled anti-mouse IgG (red). Cc , a Nomarski image of the same sagittal section as in Cb . Scale bar = 50 μm.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification, Negative Control, Positive Control, Molecular Weight, Affinity Purification, Transfection, SDS Page, Immunofluorescence, Confocal Microscopy, Staining, Double Staining

    Immunocytochemical analyses of Kir4.1 and Kir7.1 in enzymatically isolated RPE cells Enzymatically isolated rat RPE cells were stained with affinity-purified anti-Kir7.1 ( A , green) or Kir4.1 ( C and D , green) antibody followed by FITC-conjugated anti-rabbit IgG and monoclonal anti-α1-subunit of Na + ,K + -ATPase antibody followed by Texas Red-labelled anti-mouse IgG ( B , red). Immunoreactivity of Kir7.1 and Na + ,K + -ATPase was observed in the apical membrane of RPE cells. In contrast, immunoreactivity of Kir4.1 was remarkably weak in isolated RPE cells that seemed to have lost most of their apical processes. AP, apical membrane; BA, basolateral membrane. Scale bar = 10 μm.
    Figure Legend Snippet: Immunocytochemical analyses of Kir4.1 and Kir7.1 in enzymatically isolated RPE cells Enzymatically isolated rat RPE cells were stained with affinity-purified anti-Kir7.1 ( A , green) or Kir4.1 ( C and D , green) antibody followed by FITC-conjugated anti-rabbit IgG and monoclonal anti-α1-subunit of Na + ,K + -ATPase antibody followed by Texas Red-labelled anti-mouse IgG ( B , red). Immunoreactivity of Kir7.1 and Na + ,K + -ATPase was observed in the apical membrane of RPE cells. In contrast, immunoreactivity of Kir4.1 was remarkably weak in isolated RPE cells that seemed to have lost most of their apical processes. AP, apical membrane; BA, basolateral membrane. Scale bar = 10 μm.

    Techniques Used: Isolation, Staining, Affinity Purification

    30) Product Images from "Cdc42-interacting protein-4 functionally links actin and microtubule networks at the cytolytic NK cell immunological synapse"

    Article Title: Cdc42-interacting protein-4 functionally links actin and microtubule networks at the cytolytic NK cell immunological synapse

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20061893

    Alteration in association of endogenous CIP4 after NK cell activation. YTS cells were incubated on slides coated with IgG (A) or anti-CD28 (B) and ex vivo NK cells on slides coated with IgG (C) or anti-NKp30 (D) for 30 min and evaluated via confocal microscopy in the x-z plane. The arrowheads show the plane of the slide. Fluorescence demonstrating F-actin (cyan), α-tubulin (blue), and CIP4 (red) is shown. (E) 10 6 YTS cells were incubated in wells containing IgG, anti-CD28 mAb, or media for 30 min and then lysed. CIP4 was immunoprecipitated using mAb anti-CIP4 and probed for WASp and CIP4 by Western blotting. Immunoprecipitation with nonspecific mIgG was performed as a control. (F) 10 7 ex vivo NK cells were lysed after a 30-min incubation in wells coated with IgG or anti-NKp30 mAb, and CIP4 was immunoprecipitated using mAb anti-CIP4. WASp and CIP4 were detected in immunoprecipitates by Western blotting. Immunoprecipitation with nonspecific mIgG was performed in parallel as a control. Blots represent at least three independent results.
    Figure Legend Snippet: Alteration in association of endogenous CIP4 after NK cell activation. YTS cells were incubated on slides coated with IgG (A) or anti-CD28 (B) and ex vivo NK cells on slides coated with IgG (C) or anti-NKp30 (D) for 30 min and evaluated via confocal microscopy in the x-z plane. The arrowheads show the plane of the slide. Fluorescence demonstrating F-actin (cyan), α-tubulin (blue), and CIP4 (red) is shown. (E) 10 6 YTS cells were incubated in wells containing IgG, anti-CD28 mAb, or media for 30 min and then lysed. CIP4 was immunoprecipitated using mAb anti-CIP4 and probed for WASp and CIP4 by Western blotting. Immunoprecipitation with nonspecific mIgG was performed as a control. (F) 10 7 ex vivo NK cells were lysed after a 30-min incubation in wells coated with IgG or anti-NKp30 mAb, and CIP4 was immunoprecipitated using mAb anti-CIP4. WASp and CIP4 were detected in immunoprecipitates by Western blotting. Immunoprecipitation with nonspecific mIgG was performed in parallel as a control. Blots represent at least three independent results.

    Techniques Used: Activation Assay, Incubation, Ex Vivo, Confocal Microscopy, Fluorescence, Immunoprecipitation, Western Blot

    Associations of endogenous and overexpressed CIP4. (A) CIP4 was precipitated from lysates of 2 ×10 6 YTS CIP4 cells, 2 ×10 6 parental YTS cells, or 2 × 10 7 ex vivo NK cells using mAb anti-CIP4. Immunoprecipitation with nonspecific mouse isotype-matched mAb IgG (mIgG) is shown as a control. CIP4 was identified in immunoprecipitates by Western blotting using anti-CIP4 mAb (top). Blots were stripped and reprobed for α-tubulin (middle) and WASp (bottom; blots represent three to six independent results). (B) YTS cell lysates cleared of nuclei and debris were incubated with or without stabilized microtubules, after which microtubules and associated proteins were precipitated. CIP4 and α-tubulin were identified by Western blotting in the supernatant (Supt) and precipitate (PPT).
    Figure Legend Snippet: Associations of endogenous and overexpressed CIP4. (A) CIP4 was precipitated from lysates of 2 ×10 6 YTS CIP4 cells, 2 ×10 6 parental YTS cells, or 2 × 10 7 ex vivo NK cells using mAb anti-CIP4. Immunoprecipitation with nonspecific mouse isotype-matched mAb IgG (mIgG) is shown as a control. CIP4 was identified in immunoprecipitates by Western blotting using anti-CIP4 mAb (top). Blots were stripped and reprobed for α-tubulin (middle) and WASp (bottom; blots represent three to six independent results). (B) YTS cell lysates cleared of nuclei and debris were incubated with or without stabilized microtubules, after which microtubules and associated proteins were precipitated. CIP4 and α-tubulin were identified by Western blotting in the supernatant (Supt) and precipitate (PPT).

    Techniques Used: Ex Vivo, Immunoprecipitation, Western Blot, Incubation

    CIP4 expression in NK cells. (A) RT-PCR for CIP4 message in NK cell lines and ex vivo NK cells. (B) Western blot (10 μg of protein per lane) for CIP4 in NK92, YTS, and ex vivo NK cells, as well as WASp and α-tubulin after stripping and reprobing membranes. (C) Intracellular CIP4 FACS using CIP4 mAb or IgG clone MOPC21 (in YTS cells as a specificity control, which was comparable with IgG control for the other cell types). Ex vivo NK cells were identified by FACS in total PBMCs by costaining for CD3 and CD56 and gating on CD3 − , CD56 + lymphocytes (NK). (D) The increase in CIP4 mean fluorescence intensity (MFI) over control IgG detected by FACS for YTS, NK92, ex vivo NK, and CIP4 YTS cells in three experiments and with six different donors of ex vivo cells is shown. IgG MFI was determined in parallel with each repeated assessment of CIP4. Error bars represent the SD.
    Figure Legend Snippet: CIP4 expression in NK cells. (A) RT-PCR for CIP4 message in NK cell lines and ex vivo NK cells. (B) Western blot (10 μg of protein per lane) for CIP4 in NK92, YTS, and ex vivo NK cells, as well as WASp and α-tubulin after stripping and reprobing membranes. (C) Intracellular CIP4 FACS using CIP4 mAb or IgG clone MOPC21 (in YTS cells as a specificity control, which was comparable with IgG control for the other cell types). Ex vivo NK cells were identified by FACS in total PBMCs by costaining for CD3 and CD56 and gating on CD3 − , CD56 + lymphocytes (NK). (D) The increase in CIP4 mean fluorescence intensity (MFI) over control IgG detected by FACS for YTS, NK92, ex vivo NK, and CIP4 YTS cells in three experiments and with six different donors of ex vivo cells is shown. IgG MFI was determined in parallel with each repeated assessment of CIP4. Error bars represent the SD.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Ex Vivo, Western Blot, Stripping Membranes, FACS, Fluorescence

    31) Product Images from "ER stress-induced mediator C/EBP homologous protein thwarts effector T cell activity in tumors through T-bet repression"

    Article Title: ER stress-induced mediator C/EBP homologous protein thwarts effector T cell activity in tumors through T-bet repression

    Journal: Nature Communications

    doi: 10.1038/s41467-019-09263-1

    Chop negatively regulates T-bet expression. a Time-dependent expression (upper panel) and corresponding densitometry quantitation (lower panel) of T-bet in primed wild-type and Ddit3 −/− CD8 + T cells. Left: protein level (0–72 h); right: Tbx21 mRNA levels 48 h post-activation. CD8 + T cells were stimulated with plate-bound anti-CD3/CD28 ( n = 3). b Tbx21 and Ifng mRNA expression in activated CD8 + T cells infected with control retrovirus (Retro-Ctrl) or Ddit3 -expressing retrovirus (Retro-Chop). Cells were primed for 48 h and then infected for additional 48 h in the presence of the stimulating anti-CD3/CD28 antibodies ( n = 4). c Ifng , Il12b2 , Cbfa3 , and Cxcr3 mRNA levels in control vs. Ddit3 −/− CD8 + T cells primed as in a ( n = 5). d Predicted Chop-binding site in the Tbx21 promoter region (GGGTGCAATCTTC). e Chromatin immunoprecipitation assay for the endogenous binding of Chop to Tbx21 promoter in primed wild-type or Ddit3 −/− CD8 + T cells. Chop-binding activity was measured by real-time quantitative PCR, compared with IgG binding activity after normalizing to the activity of anti-H3 ( n = 4). f A dual luciferase system composed of 2x-CRE containing Firefly luciferase reporter and the control Renilla luciferase reporter was transfected into 293T cells in combination with Ddit3 -expressing or control vectors. n = 4 experimental repeats. g Expression of Chop (left) and T-bet (right) by fluorescence-activated cell sorter (FACS) upon transduction of primed CD8 + T cells with green fluorescent protein (GFP)-coding retroviruses containing control or 8x-CRE sequences. Cells were primed for 48 h and then infected for another 48 h in the presence of the stimulating anti-CD3/CD28 antibodies plus interleukin (IL)-2 (50 U/ml). n = 3 independent repeats. h Interferon-γ (IFNγ) levels in primed CD8 + T cells transduced with: (1) GFP/CD90.1-expressing control virus (Ctrl); (2) Chop/CD90.1-expressing virus and GFP-expressing control virus (Chop); (3) CD90.1-expressing control virus and T-bet/GFP-expressing virus (T-bet); or (4) Chop/CD90.1-expressing virus and T-bet/GFP-expressing virus (Chop/T-bet). Cells were primed for 24 h and then transduced for additional 72 h in the presence of stimulating antibodies plus IL-2 (50 U/ml). Then IFNγ levels were detected by FACS in gated CD90.1 + GFP + cells. Right: Representative FACS result; left: Merged results from three independent experiments. In the bar graphs showing mean ± s.e.m., * p
    Figure Legend Snippet: Chop negatively regulates T-bet expression. a Time-dependent expression (upper panel) and corresponding densitometry quantitation (lower panel) of T-bet in primed wild-type and Ddit3 −/− CD8 + T cells. Left: protein level (0–72 h); right: Tbx21 mRNA levels 48 h post-activation. CD8 + T cells were stimulated with plate-bound anti-CD3/CD28 ( n = 3). b Tbx21 and Ifng mRNA expression in activated CD8 + T cells infected with control retrovirus (Retro-Ctrl) or Ddit3 -expressing retrovirus (Retro-Chop). Cells were primed for 48 h and then infected for additional 48 h in the presence of the stimulating anti-CD3/CD28 antibodies ( n = 4). c Ifng , Il12b2 , Cbfa3 , and Cxcr3 mRNA levels in control vs. Ddit3 −/− CD8 + T cells primed as in a ( n = 5). d Predicted Chop-binding site in the Tbx21 promoter region (GGGTGCAATCTTC). e Chromatin immunoprecipitation assay for the endogenous binding of Chop to Tbx21 promoter in primed wild-type or Ddit3 −/− CD8 + T cells. Chop-binding activity was measured by real-time quantitative PCR, compared with IgG binding activity after normalizing to the activity of anti-H3 ( n = 4). f A dual luciferase system composed of 2x-CRE containing Firefly luciferase reporter and the control Renilla luciferase reporter was transfected into 293T cells in combination with Ddit3 -expressing or control vectors. n = 4 experimental repeats. g Expression of Chop (left) and T-bet (right) by fluorescence-activated cell sorter (FACS) upon transduction of primed CD8 + T cells with green fluorescent protein (GFP)-coding retroviruses containing control or 8x-CRE sequences. Cells were primed for 48 h and then infected for another 48 h in the presence of the stimulating anti-CD3/CD28 antibodies plus interleukin (IL)-2 (50 U/ml). n = 3 independent repeats. h Interferon-γ (IFNγ) levels in primed CD8 + T cells transduced with: (1) GFP/CD90.1-expressing control virus (Ctrl); (2) Chop/CD90.1-expressing virus and GFP-expressing control virus (Chop); (3) CD90.1-expressing control virus and T-bet/GFP-expressing virus (T-bet); or (4) Chop/CD90.1-expressing virus and T-bet/GFP-expressing virus (Chop/T-bet). Cells were primed for 24 h and then transduced for additional 72 h in the presence of stimulating antibodies plus IL-2 (50 U/ml). Then IFNγ levels were detected by FACS in gated CD90.1 + GFP + cells. Right: Representative FACS result; left: Merged results from three independent experiments. In the bar graphs showing mean ± s.e.m., * p

    Techniques Used: Expressing, Quantitation Assay, Activation Assay, Infection, Binding Assay, Chromatin Immunoprecipitation, Activity Assay, Real-time Polymerase Chain Reaction, Luciferase, Transfection, Fluorescence, FACS, Transduction

    32) Product Images from "USP18 recruits USP20 to promote innate antiviral response through deubiquitinating STING/MITA"

    Article Title: USP18 recruits USP20 to promote innate antiviral response through deubiquitinating STING/MITA

    Journal: Cell Research

    doi: 10.1038/cr.2016.125

    USP18 recruits USP20 to deubiquitinate STING. (A) Immunoblot of HEK293 cells that were transfected to express FLAG-STING, HA-Ubiquitin, along with vector, USP18 or USP18 (C64S), lysed and immunoprecipitated with control IgG or anti-FLAG. Cell lysate was analyzed by immunoblot with anti-FLAG or anti-USP18. (B) Immunoblot of HEK293 cells that were transfected to express FLAG-STING, HA-Ubiquitin, along with vector, USP20 or USP20 (C560/563S), lysed and immunoprecipitated with control IgG or anti-FLAG. Cell lysate was analyzed by immunoblot with anti-FLAG or anti-USP20. (C) Immunoblot of HEK293 cells transfected with a control siRNA, siUSP18 (left panels) or siUSP20 (right panels) for 12 h followed by transfection of HA-STING and Myc-Ubiquitin, along with FLAG-USP20 (left panels) or pCMV-USP18 (right panels) for 24 h, lysed and immunoprecipitated with anti-HA (left) or anti-FLAG (right). Cell lysate was analyzed by immunoblot with anti-FLAG, anti-HA, anti-USP20, anti-USP18 or anti-β-Actin. The intensities of ubiquitin-modified STING were normalized to β-Actin (graphs below). (D) Immunoblot of Usp18 +/+ and Usp18 −/− BMDCs infected with HSV-1 for 4-8 h, lysed and immunoprecipitated with anti-STING followed by immunoblot analysis with antibodies to the indicated proteins. (E) In vitro deubiquitination analysis of ubiquitin-modified STING eluted from the denature immunoprecipitates (anti-FLAG) from HEK293 cells transfected with FLAG-STING and HA-ubiquitin with FLAG peptide followed by incubation with in vitro generated USP18, USP18(C64S), USP20, USP20(C560/563S) by an in vitro transcription and translation kit. The mixtures were analyzed by immunoblot analysis with antibodies against HA, FLAG, USP18 or USP20. Data are three (A - C) or two (D - E) independent experiments.
    Figure Legend Snippet: USP18 recruits USP20 to deubiquitinate STING. (A) Immunoblot of HEK293 cells that were transfected to express FLAG-STING, HA-Ubiquitin, along with vector, USP18 or USP18 (C64S), lysed and immunoprecipitated with control IgG or anti-FLAG. Cell lysate was analyzed by immunoblot with anti-FLAG or anti-USP18. (B) Immunoblot of HEK293 cells that were transfected to express FLAG-STING, HA-Ubiquitin, along with vector, USP20 or USP20 (C560/563S), lysed and immunoprecipitated with control IgG or anti-FLAG. Cell lysate was analyzed by immunoblot with anti-FLAG or anti-USP20. (C) Immunoblot of HEK293 cells transfected with a control siRNA, siUSP18 (left panels) or siUSP20 (right panels) for 12 h followed by transfection of HA-STING and Myc-Ubiquitin, along with FLAG-USP20 (left panels) or pCMV-USP18 (right panels) for 24 h, lysed and immunoprecipitated with anti-HA (left) or anti-FLAG (right). Cell lysate was analyzed by immunoblot with anti-FLAG, anti-HA, anti-USP20, anti-USP18 or anti-β-Actin. The intensities of ubiquitin-modified STING were normalized to β-Actin (graphs below). (D) Immunoblot of Usp18 +/+ and Usp18 −/− BMDCs infected with HSV-1 for 4-8 h, lysed and immunoprecipitated with anti-STING followed by immunoblot analysis with antibodies to the indicated proteins. (E) In vitro deubiquitination analysis of ubiquitin-modified STING eluted from the denature immunoprecipitates (anti-FLAG) from HEK293 cells transfected with FLAG-STING and HA-ubiquitin with FLAG peptide followed by incubation with in vitro generated USP18, USP18(C64S), USP20, USP20(C560/563S) by an in vitro transcription and translation kit. The mixtures were analyzed by immunoblot analysis with antibodies against HA, FLAG, USP18 or USP20. Data are three (A - C) or two (D - E) independent experiments.

    Techniques Used: Transfection, Plasmid Preparation, Immunoprecipitation, Modification, Infection, In Vitro, Incubation, Generated

    Identification of USP18 as a STING-interacting protein. (A) Immunoblot of HEK293 cells that were transfected to express HA-STING and FLAG-tagged DUBs, lysed and immunoprecipitated (IP) with anti-FLAG. Cell lysate was analyzed by immunoblot with anti-FLAG or anti-HA. (B) Immunoblot of MEFs (upper panels) and BMDCs (lower panels) that were left uninfected or infected with HSV-1 or SeV for 6-12 h, lysed and immunoprecipitated with a control immunoglobulin G (IgG), or anti-STING followed by immunoblot analysis with antibodies to the indicated proteins. (C , D) Immunoblot of HEK293 cells that were transfected to express HA-STING and FLAG-tagged WT or mutant USP18 (C) or FLAG-USP18 and HA-tagged WT or mutant STING (D) , lysed and immunoprecipitated with anti-FLAG. Cell lysate was analyzed by immunoblot with anti-FLAG or anti-HA. Data are representative of three (A , B) or two (C , D) independent experiments.
    Figure Legend Snippet: Identification of USP18 as a STING-interacting protein. (A) Immunoblot of HEK293 cells that were transfected to express HA-STING and FLAG-tagged DUBs, lysed and immunoprecipitated (IP) with anti-FLAG. Cell lysate was analyzed by immunoblot with anti-FLAG or anti-HA. (B) Immunoblot of MEFs (upper panels) and BMDCs (lower panels) that were left uninfected or infected with HSV-1 or SeV for 6-12 h, lysed and immunoprecipitated with a control immunoglobulin G (IgG), or anti-STING followed by immunoblot analysis with antibodies to the indicated proteins. (C , D) Immunoblot of HEK293 cells that were transfected to express HA-STING and FLAG-tagged WT or mutant USP18 (C) or FLAG-USP18 and HA-tagged WT or mutant STING (D) , lysed and immunoprecipitated with anti-FLAG. Cell lysate was analyzed by immunoblot with anti-FLAG or anti-HA. Data are representative of three (A , B) or two (C , D) independent experiments.

    Techniques Used: Transfection, Immunoprecipitation, Infection, Mutagenesis

    33) Product Images from "Stability of proICA512/IA-2 and Its Targeting to Insulin Secretory Granules Require β4-Sheet-Mediated Dimerization of Its Ectodomain in the Endoplasmic Reticulum"

    Article Title: Stability of proICA512/IA-2 and Its Targeting to Insulin Secretory Granules Require β4-Sheet-Mediated Dimerization of Its Ectodomain in the Endoplasmic Reticulum

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00994-14

    NTF targets ICA512 to SGs. (A to C) Confocal microscopy images of resting (A and B) or HGHK-stimulated (C) INS-1 cells transfected with ICA512-GFP (top panels; green) or ICA512-GFP ΔNTF (bottom panels; green). Live cells were incubated at 4°C with the mouse anti-ME ICA512 antibody (A and C), together with the guinea pig anti-insulin antibody in panel A, or were incubated with the mouse anti-ICA512 CT antibody (B). After fixation, the binding of the primary antibodies to the cell surface was detected by incubation with Alexa Fluor-conjugated anti-mouse (A to C) (red) or anti-guinea pig (A) (white) IgG. Merged images are additionally shown for panels A and C. Bars = 10 μm ( n ≥ 3). (D and E) Immunoblotting for GFP (D) and ME ICA512 (E) in lysates of resting (R) or HGHK-stimulated (S) INS-1 cells transfected with ICA512-GFP or ICA512-GFP ΔNTF ( n ≥ 3). For normalization, the same lysates were also immunoblotted for γ-tubulin.
    Figure Legend Snippet: NTF targets ICA512 to SGs. (A to C) Confocal microscopy images of resting (A and B) or HGHK-stimulated (C) INS-1 cells transfected with ICA512-GFP (top panels; green) or ICA512-GFP ΔNTF (bottom panels; green). Live cells were incubated at 4°C with the mouse anti-ME ICA512 antibody (A and C), together with the guinea pig anti-insulin antibody in panel A, or were incubated with the mouse anti-ICA512 CT antibody (B). After fixation, the binding of the primary antibodies to the cell surface was detected by incubation with Alexa Fluor-conjugated anti-mouse (A to C) (red) or anti-guinea pig (A) (white) IgG. Merged images are additionally shown for panels A and C. Bars = 10 μm ( n ≥ 3). (D and E) Immunoblotting for GFP (D) and ME ICA512 (E) in lysates of resting (R) or HGHK-stimulated (S) INS-1 cells transfected with ICA512-GFP or ICA512-GFP ΔNTF ( n ≥ 3). For normalization, the same lysates were also immunoblotted for γ-tubulin.

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

    34) 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

    35) Product Images from "Methyllysine Reader Plant Homeodomain (PHD) Finger Protein 20-like 1 (PHF20L1) Antagonizes DNA (Cytosine-5) Methyltransferase 1 (DNMT1) Proteasomal Degradation *"

    Article Title: Methyllysine Reader Plant Homeodomain (PHD) Finger Protein 20-like 1 (PHF20L1) Antagonizes DNA (Cytosine-5) Methyltransferase 1 (DNMT1) Proteasomal Degradation *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.525279

    PHF20L1 binds to DNMT1 in vivo . A , schematic illustrating the three different known isoforms of PHF20L1. All three isoforms contain an MBT and Tudor domain, but only isoform a, the largest, contains a PHD finger domain. B , co-immunoprecipitation ( IP ) of DNMT1 and PHF20L1 reveals in vivo binding of these two proteins. Molecular weight marker and immunoprecipitation antibodies, anti-DNMT1, anti-PHF20L1, and anti-IgG, are indicated at the top of the Western blots. Antibodies used for protein detection, anti-PHF20L1 and anti-DNMT1, are indicated to the bottom of the Western blots. Immunoprecipitations revealed that DNMT1 binds all three isoforms of PHF20L1 (indicated by an asterisk ), but the largest PHF20L1a isoform is the predominant species bound to DNMT1 on the chromatin. Densitometry measurements of PHF20L1 immunoprecipitated with DNMT1 antibody were compared with its corresponding chromatin ( chr .) input sample to determine the percentage of PHF20L1 bound to DNMT1. C , PHF20L1a binds DNMT1 in human HEK293 and HCT116 cells. On the left is a Western blot revealing that PHF20L1a (indicated by an arrow ) is the predominant chromatin binder in both HEK293 and HCT116 cells. To the right are Western blots of DNMT1 and PHF20L1a co-immunoprecipitations in HEK293 and HCT116 cells, revealing in vivo interaction of these two proteins. D , size exclusion chromatography fraction of nuclear extract, demonstrating co-elution of DNMT1K142me1 and PHF20L1a with molecular weight markers indicated at the top . Western blot antibodies for DNMT1, DNMT1K142me1, and PHF20L1a are shown to the left .
    Figure Legend Snippet: PHF20L1 binds to DNMT1 in vivo . A , schematic illustrating the three different known isoforms of PHF20L1. All three isoforms contain an MBT and Tudor domain, but only isoform a, the largest, contains a PHD finger domain. B , co-immunoprecipitation ( IP ) of DNMT1 and PHF20L1 reveals in vivo binding of these two proteins. Molecular weight marker and immunoprecipitation antibodies, anti-DNMT1, anti-PHF20L1, and anti-IgG, are indicated at the top of the Western blots. Antibodies used for protein detection, anti-PHF20L1 and anti-DNMT1, are indicated to the bottom of the Western blots. Immunoprecipitations revealed that DNMT1 binds all three isoforms of PHF20L1 (indicated by an asterisk ), but the largest PHF20L1a isoform is the predominant species bound to DNMT1 on the chromatin. Densitometry measurements of PHF20L1 immunoprecipitated with DNMT1 antibody were compared with its corresponding chromatin ( chr .) input sample to determine the percentage of PHF20L1 bound to DNMT1. C , PHF20L1a binds DNMT1 in human HEK293 and HCT116 cells. On the left is a Western blot revealing that PHF20L1a (indicated by an arrow ) is the predominant chromatin binder in both HEK293 and HCT116 cells. To the right are Western blots of DNMT1 and PHF20L1a co-immunoprecipitations in HEK293 and HCT116 cells, revealing in vivo interaction of these two proteins. D , size exclusion chromatography fraction of nuclear extract, demonstrating co-elution of DNMT1K142me1 and PHF20L1a with molecular weight markers indicated at the top . Western blot antibodies for DNMT1, DNMT1K142me1, and PHF20L1a are shown to the left .

    Techniques Used: In Vivo, Immunoprecipitation, Binding Assay, Molecular Weight, Marker, Western Blot, Size-exclusion Chromatography, Co-Elution Assay

    36) Product Images from "Overexpression of Trypanosoma cruzi High Mobility Group B protein (TcHMGB) alters the nuclear structure, impairs cytokinesis and reduces the parasite infectivity"

    Article Title: Overexpression of Trypanosoma cruzi High Mobility Group B protein (TcHMGB) alters the nuclear structure, impairs cytokinesis and reduces the parasite infectivity

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-36718-0

    Tc HMGB overexpression affects nuclear structure and the protein localization. Confocal Immunofluorescence microscopy of non-induced (Tet−) and induced (Tet+) (0.5 μg/ml tetracycline, 12 h) T . cruzi Dm28c/p Tc INDEX-GW- Tc HMGB(HA) 2 epimastigotes, trypomastigotes and amastigotes. The nucleus and kinetoplast were labeled with DAPI (blue); rabbit anti- Tc HMGB (a- Tc HMGB) was revealed with Cy3-conjugated anti-rabbit IgG antibodies (red). Arrows indicate the nucleolar region and open arrows the kinetoplast. Scale bar: 1 μm.
    Figure Legend Snippet: Tc HMGB overexpression affects nuclear structure and the protein localization. Confocal Immunofluorescence microscopy of non-induced (Tet−) and induced (Tet+) (0.5 μg/ml tetracycline, 12 h) T . cruzi Dm28c/p Tc INDEX-GW- Tc HMGB(HA) 2 epimastigotes, trypomastigotes and amastigotes. The nucleus and kinetoplast were labeled with DAPI (blue); rabbit anti- Tc HMGB (a- Tc HMGB) was revealed with Cy3-conjugated anti-rabbit IgG antibodies (red). Arrows indicate the nucleolar region and open arrows the kinetoplast. Scale bar: 1 μm.

    Techniques Used: Over Expression, Immunofluorescence, Microscopy, Labeling

    37) Product Images from "Overexpression of Trypanosoma cruzi High Mobility Group B protein (TcHMGB) alters the nuclear structure, impairs cytokinesis and reduces the parasite infectivity"

    Article Title: Overexpression of Trypanosoma cruzi High Mobility Group B protein (TcHMGB) alters the nuclear structure, impairs cytokinesis and reduces the parasite infectivity

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-36718-0

    Tc HMGB overexpression affects nuclear structure and the protein localization. Confocal Immunofluorescence microscopy of non-induced (Tet−) and induced (Tet+) (0.5 μg/ml tetracycline, 12 h) T . cruzi Dm28c/p Tc INDEX-GW- Tc HMGB(HA) 2 epimastigotes, trypomastigotes and amastigotes. The nucleus and kinetoplast were labeled with DAPI (blue); rabbit anti- Tc HMGB (a- Tc HMGB) was revealed with Cy3-conjugated anti-rabbit IgG antibodies (red). Arrows indicate the nucleolar region and open arrows the kinetoplast. Scale bar: 1 μm.
    Figure Legend Snippet: Tc HMGB overexpression affects nuclear structure and the protein localization. Confocal Immunofluorescence microscopy of non-induced (Tet−) and induced (Tet+) (0.5 μg/ml tetracycline, 12 h) T . cruzi Dm28c/p Tc INDEX-GW- Tc HMGB(HA) 2 epimastigotes, trypomastigotes and amastigotes. The nucleus and kinetoplast were labeled with DAPI (blue); rabbit anti- Tc HMGB (a- Tc HMGB) was revealed with Cy3-conjugated anti-rabbit IgG antibodies (red). Arrows indicate the nucleolar region and open arrows the kinetoplast. Scale bar: 1 μm.

    Techniques Used: Over Expression, Immunofluorescence, Microscopy, Labeling

    38) Product Images from "High Expression of the Newly Found Long Noncoding RNA Z38 Promotes Cell Proliferation and Oncogenic Activity in Breast Cancer"

    Article Title: High Expression of the Newly Found Long Noncoding RNA Z38 Promotes Cell Proliferation and Oncogenic Activity in Breast Cancer

    Journal: Journal of Cancer

    doi: 10.7150/jca.13117

    Non-protein-coding features of Z38. (A) The expression of EGFP in MCF-7 cells after transfection with the following plasmids: pEGFP-C1, pEGFP-C1-Z38, pEGFP-C1-Z38(S), pEGFP-N3, pEGFP-N3-Z38, pEGFP-N3-Z38(S). (B) The transcription level of Z38 in the in vitro translation experiments, assayed by semi-quantitative PCR. PBSIIKS-Z38-P represents the circular plasmid, and PBSIIKS-Z38-F represents the linearized plasmid. +RT represents reverse transcription, and -RT represents non-reverse transcription. (C) The results of in vitro translation assays. Arrow 1 indicates the positive control provided in reagent kit, arrow 2 indicates the nonspecific bands detailed by manufacturer, and arrow 3 indicates the target protein. PstI represents a kind of endonuclease. (D) The expression of fusion proteins of FLAG and CLDND1, assayed by western blotting. (E) The expression of CLDND1 proteins in MDA-MB-231 cells, assayed by immunofluorescence. The normal rabbit IgG was used as the negative control. (F) The expression of CLDND1 proteins in cancer cells, assayed by western blotting.
    Figure Legend Snippet: Non-protein-coding features of Z38. (A) The expression of EGFP in MCF-7 cells after transfection with the following plasmids: pEGFP-C1, pEGFP-C1-Z38, pEGFP-C1-Z38(S), pEGFP-N3, pEGFP-N3-Z38, pEGFP-N3-Z38(S). (B) The transcription level of Z38 in the in vitro translation experiments, assayed by semi-quantitative PCR. PBSIIKS-Z38-P represents the circular plasmid, and PBSIIKS-Z38-F represents the linearized plasmid. +RT represents reverse transcription, and -RT represents non-reverse transcription. (C) The results of in vitro translation assays. Arrow 1 indicates the positive control provided in reagent kit, arrow 2 indicates the nonspecific bands detailed by manufacturer, and arrow 3 indicates the target protein. PstI represents a kind of endonuclease. (D) The expression of fusion proteins of FLAG and CLDND1, assayed by western blotting. (E) The expression of CLDND1 proteins in MDA-MB-231 cells, assayed by immunofluorescence. The normal rabbit IgG was used as the negative control. (F) The expression of CLDND1 proteins in cancer cells, assayed by western blotting.

    Techniques Used: Expressing, Transfection, In Vitro, Real-time Polymerase Chain Reaction, Plasmid Preparation, Positive Control, Western Blot, Multiple Displacement Amplification, Immunofluorescence, Negative Control

    39) Product Images from "Reovirus ?NS Protein Localizes to Inclusions through an Association Requiring the ?NS Amino Terminus"

    Article Title: Reovirus ?NS Protein Localizes to Inclusions through an Association Requiring the ?NS Amino Terminus

    Journal: Journal of Virology

    doi: 10.1128/JVI.77.8.4566-4576.2003

    ) followed by HRP-conjugated anti-rabbit IgG. Bound HRP conjugates were detected by chemiluminescence.
    Figure Legend Snippet: ) followed by HRP-conjugated anti-rabbit IgG. Bound HRP conjugates were detected by chemiluminescence.

    Techniques Used:

    ) followed by HRP-conjugated anti-rabbit IgG. Bound HRP conjugates were detected by chemiluminescence.
    Figure Legend Snippet: ) followed by HRP-conjugated anti-rabbit IgG. Bound HRP conjugates were detected by chemiluminescence.

    Techniques Used:

    ) followed by HRP-conjugated anti-mouse IgG (left and middle) or μNS antiserum followed by HRP-conjugated anti-rabbit IgG (right). Bound HRP conjugates were detected by chemiluminescence.
    Figure Legend Snippet: ) followed by HRP-conjugated anti-mouse IgG (left and middle) or μNS antiserum followed by HRP-conjugated anti-rabbit IgG (right). Bound HRP conjugates were detected by chemiluminescence.

    Techniques Used:

    Colocalization and co-IP of σNS and μNS in T1L- and T3D N -infected CV-1 cells. (A) IF microscopy of CV-1 cells infected with reovirus T1L (top two rows) or T3D N ) (right column). Insets show higher-magnification views of the σNS and μNS staining patterns at 6 h p.i. Scale bars, 10 μm. (B) Co-IP of σNS and μNS from T1L- and T3D N -infected CV-1 cells. At 18 h p.i., T1L-, T3D N ) followed by HRP-conjugated anti-rabbit IgG (left) or with μNS antiserum followed by HRP-conjugated anti-rabbit IgG (right). Bound HRP conjugates were detected by chemiluminescence. The background levels of σNS and μNS in the BA and PI lanes represent nonspecific binding of these proteins to the beads.
    Figure Legend Snippet: Colocalization and co-IP of σNS and μNS in T1L- and T3D N -infected CV-1 cells. (A) IF microscopy of CV-1 cells infected with reovirus T1L (top two rows) or T3D N ) (right column). Insets show higher-magnification views of the σNS and μNS staining patterns at 6 h p.i. Scale bars, 10 μm. (B) Co-IP of σNS and μNS from T1L- and T3D N -infected CV-1 cells. At 18 h p.i., T1L-, T3D N ) followed by HRP-conjugated anti-rabbit IgG (left) or with μNS antiserum followed by HRP-conjugated anti-rabbit IgG (right). Bound HRP conjugates were detected by chemiluminescence. The background levels of σNS and μNS in the BA and PI lanes represent nonspecific binding of these proteins to the beads.

    Techniques Used: Co-Immunoprecipitation Assay, Infection, Microscopy, Staining, Binding Assay

    ) (left). In parallel, CV-1 cells transfected with both pCI-M3 and pCI-S3 or both pCI-M3(41-721) and pCI-S3 were lysed in nondenaturing buffer and immunoprecipitated with σNS MAb (right). Immunoprecipitated proteins were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted using μNS antiserum followed by HRP-conjugated anti-rabbit IgG. Bound HRP conjugates were detected by chemiluminescence.
    Figure Legend Snippet: ) (left). In parallel, CV-1 cells transfected with both pCI-M3 and pCI-S3 or both pCI-M3(41-721) and pCI-S3 were lysed in nondenaturing buffer and immunoprecipitated with σNS MAb (right). Immunoprecipitated proteins were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted using μNS antiserum followed by HRP-conjugated anti-rabbit IgG. Bound HRP conjugates were detected by chemiluminescence.

    Techniques Used: Transfection, Immunoprecipitation, SDS Page

    ) followed by HRP-conjugated anti-rabbit IgG (left and middle) or using μNS antiserum followed by HRP-conjugated anti-rabbit IgG (right). Bound HRP conjugates were detected by chemiluminescence.
    Figure Legend Snippet: ) followed by HRP-conjugated anti-rabbit IgG (left and middle) or using μNS antiserum followed by HRP-conjugated anti-rabbit IgG (right). Bound HRP conjugates were detected by chemiluminescence.

    Techniques Used:

    40) Product Images from "Identification of the PDZ3 Domain of the Adaptor Protein PDZK1 as a Second, Physiologically Functional Binding Site for the C Terminus of the High Density Lipoprotein Receptor Scavenger Receptor Class B Type I *"

    Article Title: Identification of the PDZ3 Domain of the Adaptor Protein PDZK1 as a Second, Physiologically Functional Binding Site for the C Terminus of the High Density Lipoprotein Receptor Scavenger Receptor Class B Type I *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.242362

    Immunohistochemical analysis of hepatic SR-BI in WT and PDZK1 KO nontransgenic ( A and B ), PDZK1 [Phe 145 → Ala]transgenic ( C and D ), PDZK1 [Tyr 253 → Ala]transgenic ( E and F ), PDZK1 [Tyr 388 → Ala]transgenic ( G and H ), and PDZK1 [Tyr 20 → Ala + Tyr 253 → Ala]transgenic ( I and J ) mice. Livers from mice of the indicated genotypes and Tg were fixed, frozen, and sectioned. The sections were then stained with a polyclonal anti-SR-BI antibody and a biotinylated anti-rabbit IgG secondary antibody and visualized by immunoperoxidase staining (magnification ×600).
    Figure Legend Snippet: Immunohistochemical analysis of hepatic SR-BI in WT and PDZK1 KO nontransgenic ( A and B ), PDZK1 [Phe 145 → Ala]transgenic ( C and D ), PDZK1 [Tyr 253 → Ala]transgenic ( E and F ), PDZK1 [Tyr 388 → Ala]transgenic ( G and H ), and PDZK1 [Tyr 20 → Ala + Tyr 253 → Ala]transgenic ( I and J ) mice. Livers from mice of the indicated genotypes and Tg were fixed, frozen, and sectioned. The sections were then stained with a polyclonal anti-SR-BI antibody and a biotinylated anti-rabbit IgG secondary antibody and visualized by immunoperoxidase staining (magnification ×600).

    Techniques Used: Immunohistochemistry, Transgenic Assay, Mouse Assay, Staining, Immunoperoxidase Staining

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    FLAG-tag:

    Article Title: A tubular EHD1-containing compartment involved in the recycling of major histocompatibility complex class I molecules to the plasma membrane
    Article Snippet: .. The following monoclonal antibodies were used: 9E10 and HA.11 antibodies to the Myc and HA epitopes, respectively (Covance); W6/32 antibody to human MHC-I (peptide-bound) (American Type Culture Collection); 34-5-8 antibody to H-2Dd (peptide-bound) (Pharmingen); 34-5-8 antibody conjugated to biotin (Cedarlane Laboratories Limited); M5 antibody to the FLAG epitope (Sigma-Aldrich); Cy3-conjugated anti-mouse and anti-rabbit IgG (Molecular Probes, Inc.); Alexa-488-conjugated antibody to mouse IgG (Molecular Probes, Inc.); and Alexa Fluor 568 F(ab′)2 fragment of goat anti-mouse IgG (Molecular Probes, Inc.). ..

    Incubation:

    Article Title: Native low density lipoprotein induces pancreatic ? cell apoptosis through generating excess reactive oxygen species
    Article Snippet: .. In brief, after fixation and blocking, the cells were incubated with the first antibody against rabbit active caspase-3 (Sigma-Aldrich, St. Louis, Missouri, USA) followed by incubation with anti-rabbit IgG antibody conjugated with Alexa Fluor 488 (Molecular Probes, Eugene, OR). .. The densitometric analysis was performed using a Multi Gauge software in LAS-1000 (Fuji Film).

    Article Title: Prostacyclin Prevents Pericyte Loss and Demyelination Induced by Lysophosphatidylcholine in the Central Nervous System *
    Article Snippet: .. Sections were incubated with antibodies against rabbit anti-mouse cleaved caspase-3 and biotinylated anti-mouse PDGDRβ overnight at 4 °C and then incubated with Alexa Fluor 568 conjugated to anti-rabbit IgG (1:500, Invitrogen) and streptavidin-conjugated Alexa Fluor 488 (1:500, Invitrogen). .. To visualize vascular endothelial cells using lectin, sections were incubated with DyLight 594-labeled Lycopersicon esculentum (tomato) lectin (vascular endothelial cell marker, 1:100; Vector Laboratories, catalog no. DL-1177) according to the manufacturer's instructions.

    other:

    Article Title: Trimeric Tn Antigen on Syndecan 1 Produced by ppGalNAc-T13 Enhances Cancer Metastasis via a Complex Formation with Integrin α5β1 and Matrix Metalloproteinase 9 *
    Article Snippet: Anti-rat IgG antibody conjugated with Alexa Fluor 405, anti-rabbit IgG antibody conjugated with Alexa Fluor 488, and anti-mouse IgG antibody conjugated with Alexa Fluor 564 were purchased from Invitrogen.

    Blocking Assay:

    Article Title: Native low density lipoprotein induces pancreatic ? cell apoptosis through generating excess reactive oxygen species
    Article Snippet: .. In brief, after fixation and blocking, the cells were incubated with the first antibody against rabbit active caspase-3 (Sigma-Aldrich, St. Louis, Missouri, USA) followed by incubation with anti-rabbit IgG antibody conjugated with Alexa Fluor 488 (Molecular Probes, Eugene, OR). .. The densitometric analysis was performed using a Multi Gauge software in LAS-1000 (Fuji Film).

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    Thermo Fisher rabbit anti goat igg hrp conjugated antibody
    The immunized mouse serum <t>IgG</t> (H+L) titer and IgG subtypes were detected using the indirect ELISA assay. (A) IgG titers of BALB/c mice immunized with rEF-Tu, rHSP70, Mo extracts and PBS sera of each groups were collected on days 3, 7, 14, 21, 28, 35, 42, 49, 56, 63 and 70 DAI. (B) Determination of IgG subtypes in sera of the immunized mice. The sera of each group were collected at 35 DAI. ELISA plates were coated with purified rEF-Tu proteins or rHSP70 proteins or Mo extracts of M . ovipneumoniae wild strain Mo-1 at a concentration of 100 ng per well. Anti-mouse IgG (H+L) or IgG1 or IgG2a <t>HRP-conjugated</t> antibodies were used as secondary antibodies. Asterisks indicates the results of the One-Way ANOVA using the Tukey test, compared with the PBS negative control group, with P
    Rabbit Anti Goat Igg Hrp Conjugated Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Thermo Fisher alexa fluor 488 conjugated goat anti rabbit igg secondary antibody
    Determination of AvBD8 protein expression in immune cell lines by performing immunocytochemical analysis. Cultured chicken CU91 T-, DT40 B-, HD11 macrophage-, and OU2 fibroblast-cell lines were incubated with the rabbit anti-AvBD8 primary antibody, followed by incubation with the <t>Alexa</t> Fluor 488-conjugated goat anti-rabbit <t>IgG</t> secondary antibody, and were counterstained with 4′,6-diamidino-2-phenylindole. Control cells were incubated with secondary antibody only. AvBD8, avian beta-defensin 8; IgG, immunoglobulin G. Scale bar: 20 μm.
    Alexa Fluor 488 Conjugated Goat Anti Rabbit Igg Secondary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher fitc conjugated goat anti rabbit igg
    Pf MSP8 is expressed during P. falciparum gametocyte development but is absent on the surface of activated macrogametes. (A) Detection of Pf MSP8 expression in fixed and permeabilized P. falciparum gametocytes (stages II to V) by an immunofluorescence assay with rabbit anti-r Pf MSP8 <t>IgG</t> or control IgG followed by <t>FITC-conjugated</t> secondary antibodies. DAPI was used to stain parasite DNA. (B) Analysis of Pf MSP8 expression on activated, live P. falciparum macrogametes by an immunofluorescence assay, as described above, with rabbit anti-r Pf MSP8 IgG. Samples were costained with MAb 4B7, which is specific for Pf s25 (MAb 4B7), followed by TRITC-conjugated secondary IgG. DIC, differential interference contrast.
    Fitc Conjugated Goat Anti Rabbit Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher rabbit anti goat igg abs
    CD82 and its cholesterol-binding differentially regulate cellular release of EVs. (a) Extracellular staining by filipin and <t>Alexa488-conjugated</t> CTxb in Du145 transfectants. Equal number of the cells were cultured on glass coverslips for 2 days, then fixed and labelled with filipin or Alexa488-conjugated CTxb. For filipin staining, intercellular regions were imaged. For CTxb staining, pericellular regions were imaged. Scale bar: 10 µm. (b) Distributions of Annexin V and Annexin A2 in Du145 transfectant cells. Alexa488-conjugated recombinant Annexin V was used for phosphatidylserine labelling, while Annexin-A2 Ab was used for Annexin-A2 staining. Scale bar: 10 μm. (c) Colocalization of Ezrin with GM1 or Annexin A2 in EVs. For Ezrin and GM1 co-staining, the cells were labelled with the Abs, Alexa488-conjugated CTxB and DAPI. For Ezrin and Annexin A2 co-staining, the cells were incubated sequentially with the primary Abs, Cy3-conjugated donkey anti-goat <t>IgG,</t> normal goat IgG and Alexa594-conjugated goat anti-mouse IgG. Images were obtained by confocal microscopy. Scale bar: 10 µm. (d) The cells were seeded in six-well plate at 50% confluence and cultured in DMEM containing 1% exosome-depleted FBS for 2 – 3 days. The culture supernatants were collected, spun at 2000 × g for 10 min to remove cell debris, and then analysed with NanoSight instrument for EV number and size. Data are presented as mean ± SD (n = 3 individual experiments). *: p
    Rabbit Anti Goat Igg Abs, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The immunized mouse serum IgG (H+L) titer and IgG subtypes were detected using the indirect ELISA assay. (A) IgG titers of BALB/c mice immunized with rEF-Tu, rHSP70, Mo extracts and PBS sera of each groups were collected on days 3, 7, 14, 21, 28, 35, 42, 49, 56, 63 and 70 DAI. (B) Determination of IgG subtypes in sera of the immunized mice. The sera of each group were collected at 35 DAI. ELISA plates were coated with purified rEF-Tu proteins or rHSP70 proteins or Mo extracts of M . ovipneumoniae wild strain Mo-1 at a concentration of 100 ng per well. Anti-mouse IgG (H+L) or IgG1 or IgG2a HRP-conjugated antibodies were used as secondary antibodies. Asterisks indicates the results of the One-Way ANOVA using the Tukey test, compared with the PBS negative control group, with P

    Journal: PLoS ONE

    Article Title: Elongation Factor Tu and Heat Shock Protein 70 Are Membrane-Associated Proteins from Mycoplasma ovipneumoniae Capable of Inducing Strong Immune Response in Mice

    doi: 10.1371/journal.pone.0161170

    Figure Lengend Snippet: The immunized mouse serum IgG (H+L) titer and IgG subtypes were detected using the indirect ELISA assay. (A) IgG titers of BALB/c mice immunized with rEF-Tu, rHSP70, Mo extracts and PBS sera of each groups were collected on days 3, 7, 14, 21, 28, 35, 42, 49, 56, 63 and 70 DAI. (B) Determination of IgG subtypes in sera of the immunized mice. The sera of each group were collected at 35 DAI. ELISA plates were coated with purified rEF-Tu proteins or rHSP70 proteins or Mo extracts of M . ovipneumoniae wild strain Mo-1 at a concentration of 100 ng per well. Anti-mouse IgG (H+L) or IgG1 or IgG2a HRP-conjugated antibodies were used as secondary antibodies. Asterisks indicates the results of the One-Way ANOVA using the Tukey test, compared with the PBS negative control group, with P

    Article Snippet: Following washing with PBST three times, membranes were incubated with rabbit anti-goat IgG HRP- conjugated antibody at 37°C for 1 h and exposed to SuperSignal® West Pico Chemiluminescent Substrate reagent for detection (Thermo Scientific, USA).

    Techniques: Indirect ELISA, Mouse Assay, Enzyme-linked Immunosorbent Assay, Purification, Concentration Assay, Negative Control

    Determination of AvBD8 protein expression in immune cell lines by performing immunocytochemical analysis. Cultured chicken CU91 T-, DT40 B-, HD11 macrophage-, and OU2 fibroblast-cell lines were incubated with the rabbit anti-AvBD8 primary antibody, followed by incubation with the Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody, and were counterstained with 4′,6-diamidino-2-phenylindole. Control cells were incubated with secondary antibody only. AvBD8, avian beta-defensin 8; IgG, immunoglobulin G. Scale bar: 20 μm.

    Journal: Asian-Australasian Journal of Animal Sciences

    Article Title: Expression and regulation of avian beta-defensin 8 protein in immune tissues and cell lines of chickens

    doi: 10.5713/ajas.17.0836

    Figure Lengend Snippet: Determination of AvBD8 protein expression in immune cell lines by performing immunocytochemical analysis. Cultured chicken CU91 T-, DT40 B-, HD11 macrophage-, and OU2 fibroblast-cell lines were incubated with the rabbit anti-AvBD8 primary antibody, followed by incubation with the Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody, and were counterstained with 4′,6-diamidino-2-phenylindole. Control cells were incubated with secondary antibody only. AvBD8, avian beta-defensin 8; IgG, immunoglobulin G. Scale bar: 20 μm.

    Article Snippet: After blocking, the cells were incubated overnight with the rabbit anti-AvBD8 primary antibody (dilution, 1:100) at 4°C, followed by incubation with the Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody (dilution, 1:500) for 1 h. Control cells received secondary antibody only.

    Techniques: Expressing, Cell Culture, Incubation

    Determination of AvBD8 protein expression in immune tissues of male WL chickens by performing immunohistochemical analysis. Immunohistochemical analysis was performed to assess AvBD8 protein expression in the thymus, spleen, liver, small intestine, and ceca of male WL chickens aged 25 weeks. Frozen sections were incubated with the rabbit anti-AvBD8 primary antibody, followed by incubation with the Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody, and were counterstained with 4′,6-diamidino-2-phenylindole. Control sections were incubated with secondary antibody only. Boxed region in the middle column is enlarged in the right column. AvBD8, avian beta-defensin 8; WL, White Leghorn; IgG, immunoglobulin G. Scale bar: 200 μm (left and middle columns) and 50 μm (right column). Arrowheads indicate strong AvBD8 signal in the intestinal mucosal layer.

    Journal: Asian-Australasian Journal of Animal Sciences

    Article Title: Expression and regulation of avian beta-defensin 8 protein in immune tissues and cell lines of chickens

    doi: 10.5713/ajas.17.0836

    Figure Lengend Snippet: Determination of AvBD8 protein expression in immune tissues of male WL chickens by performing immunohistochemical analysis. Immunohistochemical analysis was performed to assess AvBD8 protein expression in the thymus, spleen, liver, small intestine, and ceca of male WL chickens aged 25 weeks. Frozen sections were incubated with the rabbit anti-AvBD8 primary antibody, followed by incubation with the Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody, and were counterstained with 4′,6-diamidino-2-phenylindole. Control sections were incubated with secondary antibody only. Boxed region in the middle column is enlarged in the right column. AvBD8, avian beta-defensin 8; WL, White Leghorn; IgG, immunoglobulin G. Scale bar: 200 μm (left and middle columns) and 50 μm (right column). Arrowheads indicate strong AvBD8 signal in the intestinal mucosal layer.

    Article Snippet: After blocking, the cells were incubated overnight with the rabbit anti-AvBD8 primary antibody (dilution, 1:100) at 4°C, followed by incubation with the Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody (dilution, 1:500) for 1 h. Control cells received secondary antibody only.

    Techniques: Expressing, Immunohistochemistry, Incubation

    Pf MSP8 is expressed during P. falciparum gametocyte development but is absent on the surface of activated macrogametes. (A) Detection of Pf MSP8 expression in fixed and permeabilized P. falciparum gametocytes (stages II to V) by an immunofluorescence assay with rabbit anti-r Pf MSP8 IgG or control IgG followed by FITC-conjugated secondary antibodies. DAPI was used to stain parasite DNA. (B) Analysis of Pf MSP8 expression on activated, live P. falciparum macrogametes by an immunofluorescence assay, as described above, with rabbit anti-r Pf MSP8 IgG. Samples were costained with MAb 4B7, which is specific for Pf s25 (MAb 4B7), followed by TRITC-conjugated secondary IgG. DIC, differential interference contrast.

    Journal: Infection and Immunity

    Article Title: Evaluation of a Plasmodium-Specific Carrier Protein To Enhance Production of Recombinant Pfs25, a Leading Transmission-Blocking Vaccine Candidate

    doi: 10.1128/IAI.00486-17

    Figure Lengend Snippet: Pf MSP8 is expressed during P. falciparum gametocyte development but is absent on the surface of activated macrogametes. (A) Detection of Pf MSP8 expression in fixed and permeabilized P. falciparum gametocytes (stages II to V) by an immunofluorescence assay with rabbit anti-r Pf MSP8 IgG or control IgG followed by FITC-conjugated secondary antibodies. DAPI was used to stain parasite DNA. (B) Analysis of Pf MSP8 expression on activated, live P. falciparum macrogametes by an immunofluorescence assay, as described above, with rabbit anti-r Pf MSP8 IgG. Samples were costained with MAb 4B7, which is specific for Pf s25 (MAb 4B7), followed by TRITC-conjugated secondary IgG. DIC, differential interference contrast.

    Article Snippet: Bound IgG was detected with FITC-conjugated goat anti-rabbit IgG, FITC-conjugated goat anti-mouse IgG, or tetramethylrhodamine isothiocyanate (TRITC)-labeled goat anti-mouse IgG as needed and then analyzed by fluorescence microscopy as described above.

    Techniques: Expressing, Immunofluorescence, Staining

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

    Journal: Journal of Extracellular Vesicles

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

    doi: 10.1080/20013078.2019.1692417

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

    Article Snippet: Secondary Abs were Alexa488- or Alexa594-conjugated goat anti-mouse IgG, goat anti-rabbit IgG, and rabbit anti-goat IgG Abs (ThermoFisher, MA), Cy3-conjugated donkey anti-goat IgG Ab (Jackson ImmunoResearch Laboratories, PA), Fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG Ab (Sigma-Aldrich, MO), and Horseradish Peroxidase (HRP)-conjugated goat anti-mouse and anti-rabbit IgG Abs (Sigma-Aldrich).

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