polyclonal rabbit antibodies  (Millipore)


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    Name:
    LH Rabbit Polyclonal Antibody
    Description:
    Anti LH is a useful marker in classification of pituitary tumors and the study of pituitary disease It reacts with LH producing cells gonadotrophs
    Catalog Number:
    209a-1
    Price:
    None
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    Structured Review

    Millipore polyclonal rabbit antibodies
    LH Rabbit Polyclonal Antibody
    Anti LH is a useful marker in classification of pituitary tumors and the study of pituitary disease It reacts with LH producing cells gonadotrophs
    https://www.bioz.com/result/polyclonal rabbit antibodies/product/Millipore
    Average 99 stars, based on 31 article reviews
    Price from $9.99 to $1999.99
    polyclonal rabbit antibodies - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Spatiotemporal Expression Patterns of Metalloproteinases and Their Inhibitors in the Postnatal Developing Rat Cerebellum"

    Article Title: Spatiotemporal Expression Patterns of Metalloproteinases and Their Inhibitors in the Postnatal Developing Rat Cerebellum

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.19-12-04994.1999

    Immunohistochemical distribution pattern of MMP 2 ( A ), MMP 3 ( B ), MMP 9 ( C ), TIMP 1 ( D ), TIMP 2 ( E ), and TIMP 3 ( F ) in the cerebellar cortex of 10-d-old rats. Immunohistochemical labeling was performed on 10-μm-thick frontal rat cerebellar sections showing the EGL, the ML, the PCL, and the IGL. The labeling with polyclonal antibodies was enhanced using the fluorescein tyramide signal amplification system (New England Nuclear). In the EGL, MMP 3 ( B ), MMP 9 ( C ), TIMP 1 ( D ), TIMP 2 ( E ), and to less extents for TIMP 3 ( F ), were seen in granular precursor cells. In addition, MMP 9 was detected along the Bergmann glial fibers in this layer ( C ). Arrows point to strips of stained granular precursor cells. In the ML, MMP 3, MMP 9, and TIMP 1 were diffusely present over PK cells dendrites, whereas TIMP 3 was restricted to the cytoplasm of the PK cell dendrites (the small arrow in F points to the labeled PK cell dendritic arborization). In the PCL, the PK cell somata showed labeling for each marker, and MMP 9 expression was seen in the Bergmann glial cell somata. Some granular cells in the IGL were immunopositive for MMPs 3 and 9 and TIMPs 1, 2, and 3. Scale bar, 50 μm.
    Figure Legend Snippet: Immunohistochemical distribution pattern of MMP 2 ( A ), MMP 3 ( B ), MMP 9 ( C ), TIMP 1 ( D ), TIMP 2 ( E ), and TIMP 3 ( F ) in the cerebellar cortex of 10-d-old rats. Immunohistochemical labeling was performed on 10-μm-thick frontal rat cerebellar sections showing the EGL, the ML, the PCL, and the IGL. The labeling with polyclonal antibodies was enhanced using the fluorescein tyramide signal amplification system (New England Nuclear). In the EGL, MMP 3 ( B ), MMP 9 ( C ), TIMP 1 ( D ), TIMP 2 ( E ), and to less extents for TIMP 3 ( F ), were seen in granular precursor cells. In addition, MMP 9 was detected along the Bergmann glial fibers in this layer ( C ). Arrows point to strips of stained granular precursor cells. In the ML, MMP 3, MMP 9, and TIMP 1 were diffusely present over PK cells dendrites, whereas TIMP 3 was restricted to the cytoplasm of the PK cell dendrites (the small arrow in F points to the labeled PK cell dendritic arborization). In the PCL, the PK cell somata showed labeling for each marker, and MMP 9 expression was seen in the Bergmann glial cell somata. Some granular cells in the IGL were immunopositive for MMPs 3 and 9 and TIMPs 1, 2, and 3. Scale bar, 50 μm.

    Techniques Used: Immunohistochemistry, Labeling, Amplification, Staining, Marker, Expressing

    Immunohistochemical distribution pattern of MMP 2 ( A ), MMP 3 ( B ), MMP 9 ( C ), TIMP 1 ( D ), TIMP 2 ( E ), and TIMP 3 ( F ) in adult rat cerebellar cortex. Ten micrometer frontal sections of adult rat cerebellum showed the EGL, the ML, the PCL, and the IGL. Labeling with polyclonal antibodies was amplified by using tyramide signal amplification (New England Nuclear). Note, for each marker, the cytoplasmic staining at the adult age compared with the diffuse staining seen on the developmental stages P10 and P15. In the ML, MMPs 3 and 9 and TIMPs 1, 2, and 3 protein expression was lost in PK cell arborization. However, MMP 9 expression persisted in the Bergmann glial fibers. Double arrows point to one fiber in C . Only the anti-TIMP 3 antibodies labeled PK cells dendrites. Arrow indicates TIMP 3 dendritic immunolabeling in F . Interneurons of the ML were labeled for MMPs 3 and 9 and TIMPs 1 and 2. In the PCL, all markers were detected in PK cell somata. In addition, MMP 9 and TIMP 1 were seen in the Bergmann glial cell bodies surrounding the PK cell somata. In the IGL, MMPs 3 and 9 and TIMPs 2 and 3 were strongly expressed in many granular cells, whereas TIMP 1 was poorly expressed. Magnification of the left of each panel, 100×. Magnification of the right of each panel, 400×. Scale bars, 100 μm.
    Figure Legend Snippet: Immunohistochemical distribution pattern of MMP 2 ( A ), MMP 3 ( B ), MMP 9 ( C ), TIMP 1 ( D ), TIMP 2 ( E ), and TIMP 3 ( F ) in adult rat cerebellar cortex. Ten micrometer frontal sections of adult rat cerebellum showed the EGL, the ML, the PCL, and the IGL. Labeling with polyclonal antibodies was amplified by using tyramide signal amplification (New England Nuclear). Note, for each marker, the cytoplasmic staining at the adult age compared with the diffuse staining seen on the developmental stages P10 and P15. In the ML, MMPs 3 and 9 and TIMPs 1, 2, and 3 protein expression was lost in PK cell arborization. However, MMP 9 expression persisted in the Bergmann glial fibers. Double arrows point to one fiber in C . Only the anti-TIMP 3 antibodies labeled PK cells dendrites. Arrow indicates TIMP 3 dendritic immunolabeling in F . Interneurons of the ML were labeled for MMPs 3 and 9 and TIMPs 1 and 2. In the PCL, all markers were detected in PK cell somata. In addition, MMP 9 and TIMP 1 were seen in the Bergmann glial cell bodies surrounding the PK cell somata. In the IGL, MMPs 3 and 9 and TIMPs 2 and 3 were strongly expressed in many granular cells, whereas TIMP 1 was poorly expressed. Magnification of the left of each panel, 100×. Magnification of the right of each panel, 400×. Scale bars, 100 μm.

    Techniques Used: Immunohistochemistry, Labeling, Amplification, Marker, Staining, Expressing, Immunolabeling

    Immunohistochemical distribution pattern of MMP 2 ( A ), MMP 3 ( B ), MMP 9 ( C ), TIMP 1 ( D ), TIMP 2 ( E ), and TIMP 3 ( F ) in the cerebellar cortex of 15-d-old rats. Ten-micrometer-thick frontal sections of rat cerebellum showed the EGL, the ML, the PCL, and the IGL. Immunohistochemical staining was performed using polyclonal antibodies and amplified by tyramide signal amplification (New England Nuclear). EGL immunostaining for MMPs 3 and 9 and TIMPs 1, 2, and 3 was lost. The PK cell dendrites were still stained for MMP 3 and TIMP 3. Note the diffuse extracellular distribution of MMP 3 as opposed to the cytoplasmic distribution of TIMP 3. ML labeling for MMP 9 and TIMP 1 in the PK cell dendrites decreased, but MMP 9 labeling of Bergmann glial fibers was maintained. MMP 9 labeling in ML cells, probably corresponding to stellate and basket cells, was detected. The large double arrows point to MMP 9-positive Bergmann glial fibers, and the small arrows point to MMP 9-positive ML cells ( C ). In the PCL, expression of MMPs 2, 3, and 9 and TIMPs 1, 2, and 3 was noted in the PK cell somata. TIMP 1 was differentially expressed in clusters of PK cell somata (stained and unstained PK cell somata are respectively indicated by large arrows at the left and right of D ). TIMP 1 was also present in Bergmann glial somata around labeled and unlabeled PK cell bodies ( D ). Granular cells in the IGL were positive for MMPs 3 and 9 and TIMPs 1 and 3. Scale bar, 25 μm.
    Figure Legend Snippet: Immunohistochemical distribution pattern of MMP 2 ( A ), MMP 3 ( B ), MMP 9 ( C ), TIMP 1 ( D ), TIMP 2 ( E ), and TIMP 3 ( F ) in the cerebellar cortex of 15-d-old rats. Ten-micrometer-thick frontal sections of rat cerebellum showed the EGL, the ML, the PCL, and the IGL. Immunohistochemical staining was performed using polyclonal antibodies and amplified by tyramide signal amplification (New England Nuclear). EGL immunostaining for MMPs 3 and 9 and TIMPs 1, 2, and 3 was lost. The PK cell dendrites were still stained for MMP 3 and TIMP 3. Note the diffuse extracellular distribution of MMP 3 as opposed to the cytoplasmic distribution of TIMP 3. ML labeling for MMP 9 and TIMP 1 in the PK cell dendrites decreased, but MMP 9 labeling of Bergmann glial fibers was maintained. MMP 9 labeling in ML cells, probably corresponding to stellate and basket cells, was detected. The large double arrows point to MMP 9-positive Bergmann glial fibers, and the small arrows point to MMP 9-positive ML cells ( C ). In the PCL, expression of MMPs 2, 3, and 9 and TIMPs 1, 2, and 3 was noted in the PK cell somata. TIMP 1 was differentially expressed in clusters of PK cell somata (stained and unstained PK cell somata are respectively indicated by large arrows at the left and right of D ). TIMP 1 was also present in Bergmann glial somata around labeled and unlabeled PK cell bodies ( D ). Granular cells in the IGL were positive for MMPs 3 and 9 and TIMPs 1 and 3. Scale bar, 25 μm.

    Techniques Used: Immunohistochemistry, Staining, Amplification, Immunostaining, Labeling, Expressing

    2) Product Images from "Analysis of close associations of uropod-associated proteins in human T-cells using the proximity ligation assay"

    Article Title: Analysis of close associations of uropod-associated proteins in human T-cells using the proximity ligation assay

    Journal: PeerJ

    doi: 10.7717/peerj.186

    Interaction of flotillin-1 and -2 in human T-cells studied with PLA. (A, B) T-cells were preincubated for 30 min at 37°C, followed by a further incubation for 15 min without or with 40 ng/ml SDF-1, fixation with TCA and staining for endogenous flotillin-1 (flo1) (rabbit polyclonal antibody) and flotillin-2 (flo2) (monoclonal murine antibody), followed by (A) fluorescently labeled anti-murine and anti-rabbit IgG second antibodies (IF) or (B) PLA probes minus and plus, ligation and amplification. (C) For negative controls, T-cells were treated as described for (B) except that the anti-flotillin-2 antibody was omitted. For (B) and (C), the top panels are overviews at lower magnification whereas in the lower panels single cells are shown at higher magnification. The pictures are representative of 3 experiments. The percentage of cells with one or more red fluorescent dots per cell was determined for 100 cells per sample and experiment (mean ± sem of 3 experiments). Note that the majority of the cells incubated with both flo1 and flo2 antibodies exhibited several dots per cell, whereas for controls only incubated with flo1 antibody, maximally 1 dot per cell occurred. Scale bars, 10 µm.
    Figure Legend Snippet: Interaction of flotillin-1 and -2 in human T-cells studied with PLA. (A, B) T-cells were preincubated for 30 min at 37°C, followed by a further incubation for 15 min without or with 40 ng/ml SDF-1, fixation with TCA and staining for endogenous flotillin-1 (flo1) (rabbit polyclonal antibody) and flotillin-2 (flo2) (monoclonal murine antibody), followed by (A) fluorescently labeled anti-murine and anti-rabbit IgG second antibodies (IF) or (B) PLA probes minus and plus, ligation and amplification. (C) For negative controls, T-cells were treated as described for (B) except that the anti-flotillin-2 antibody was omitted. For (B) and (C), the top panels are overviews at lower magnification whereas in the lower panels single cells are shown at higher magnification. The pictures are representative of 3 experiments. The percentage of cells with one or more red fluorescent dots per cell was determined for 100 cells per sample and experiment (mean ± sem of 3 experiments). Note that the majority of the cells incubated with both flo1 and flo2 antibodies exhibited several dots per cell, whereas for controls only incubated with flo1 antibody, maximally 1 dot per cell occurred. Scale bars, 10 µm.

    Techniques Used: Proximity Ligation Assay, Incubation, Staining, Labeling, Ligation, Amplification

    3) Product Images from "Elevated tumor tissue protein expression levels of kallikrein-related peptidases KLK10 and KLK11 are associated with a better prognosis in advanced high-grade serous ovarian cancer patients"

    Article Title: Elevated tumor tissue protein expression levels of kallikrein-related peptidases KLK10 and KLK11 are associated with a better prognosis in advanced high-grade serous ovarian cancer patients

    Journal: American Journal of Cancer Research

    doi:

    KLK10 and KLK11 immunoexpression in tumor tissue of advanced high-grade serous ovarian cancer patients (FIGO III/IV) specimens. Tissue sections were stained with polyclonal rabbit antibodies directed to KLK10 (Sigma HPA017195) and KLK11 (Abcam ab131038), respectively, applying the polymer one step system based on a horseradish peroxidase-linked reporter assay. Micrographs (A-F) illustrate representative core punches corresponding to low, moderate, and high KLK10 and KLK11 immunoexpression in tumor cells, respectively.
    Figure Legend Snippet: KLK10 and KLK11 immunoexpression in tumor tissue of advanced high-grade serous ovarian cancer patients (FIGO III/IV) specimens. Tissue sections were stained with polyclonal rabbit antibodies directed to KLK10 (Sigma HPA017195) and KLK11 (Abcam ab131038), respectively, applying the polymer one step system based on a horseradish peroxidase-linked reporter assay. Micrographs (A-F) illustrate representative core punches corresponding to low, moderate, and high KLK10 and KLK11 immunoexpression in tumor cells, respectively.

    Techniques Used: Staining, Reporter Assay

    4) Product Images from "ADAM10 Is the Major Sheddase Responsible for the Release of Membrane-associated Meprin A *"

    Article Title: ADAM10 Is the Major Sheddase Responsible for the Release of Membrane-associated Meprin A *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.559088

    Meprin A distribution in kidneys of mice subjected to ischemia-reperfusion or cisplatin injury. A–D , double-immunofluorescence of meprin A ( red ) and Na/K-ATPase ( green ) in kidney sections of control mice ( A and C ) and mice subjected to IR (24 h of reperfusion after 45 min of ischemia) ( B ) or cisplatin injury ( D ). Kidney tissue was fixed in phosphate-buffered 4% formalin and paraffin-embedded. Tissue sections (8 μm) were incubated with goat polyclonal anti-meprin-β and rabbit polyclonal anti-Na + /K + -ATPase antibodies overnight. After washing with PBS, slices were incubated with secondary antibodies from Molecular Probes (donkey anti-goat Alexa Fluor 594 ( red ) and donkey anti-rabbit Alexa Fluor 488 ( green ). Nuclei were stained with DAPI. Epifluorescence pictures were recorded on an Olympus (IX51, IX71, or BX51) microscope.
    Figure Legend Snippet: Meprin A distribution in kidneys of mice subjected to ischemia-reperfusion or cisplatin injury. A–D , double-immunofluorescence of meprin A ( red ) and Na/K-ATPase ( green ) in kidney sections of control mice ( A and C ) and mice subjected to IR (24 h of reperfusion after 45 min of ischemia) ( B ) or cisplatin injury ( D ). Kidney tissue was fixed in phosphate-buffered 4% formalin and paraffin-embedded. Tissue sections (8 μm) were incubated with goat polyclonal anti-meprin-β and rabbit polyclonal anti-Na + /K + -ATPase antibodies overnight. After washing with PBS, slices were incubated with secondary antibodies from Molecular Probes (donkey anti-goat Alexa Fluor 594 ( red ) and donkey anti-rabbit Alexa Fluor 488 ( green ). Nuclei were stained with DAPI. Epifluorescence pictures were recorded on an Olympus (IX51, IX71, or BX51) microscope.

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

    5) Product Images from "Kinesin-4 KIF21B limits microtubule growth to allow rapid centrosome polarization in T cells"

    Article Title: Kinesin-4 KIF21B limits microtubule growth to allow rapid centrosome polarization in T cells

    Journal: bioRxiv

    doi: 10.1101/2020.08.28.271643

    Immunological synapse formation is impaired in KIF21B-KO Jurkat T cells A-B) Schematic representation of T cells forming an immunological synapse upon target recognition. In vivo, T cells recognize an antigen-presenting cell (APC) via the T cell receptor (CD3)-complex which triggers T cell activation. Upon activation, several organelles including lysosomes are transported to the centrosome by dynein. Simultaneously, the centrosome and its associated organelle cluster polarize to the synapse through MT pulling by membrane-anchored dynein at the synapse. B) To achieve spatiotemporal control on T cell activation we used anti-CD3 coated glass surfaces to mimic the APC and induce T cell activation and centrosome polarization. C) Western blot analysis of the indicated Jurkat knockout (KO) cell lines with indicated antibodies. KIF21B-KO clone #1 and #2 were transduced with a KIF21B-GFP construct to generate two polyclonal cell lines re-expressing full-length KIF21B. D) Phalloidin staining to show F-actin structures in indicated Jurkat T cell lines imaged on a widefield microscope. E) Quantification of synapse size expressed as surface area based on a F-actin staining of indicated Jurkat T cells. n = 134, 218, 241, 185 and 194 cells from three independent experiments. ** p
    Figure Legend Snippet: Immunological synapse formation is impaired in KIF21B-KO Jurkat T cells A-B) Schematic representation of T cells forming an immunological synapse upon target recognition. In vivo, T cells recognize an antigen-presenting cell (APC) via the T cell receptor (CD3)-complex which triggers T cell activation. Upon activation, several organelles including lysosomes are transported to the centrosome by dynein. Simultaneously, the centrosome and its associated organelle cluster polarize to the synapse through MT pulling by membrane-anchored dynein at the synapse. B) To achieve spatiotemporal control on T cell activation we used anti-CD3 coated glass surfaces to mimic the APC and induce T cell activation and centrosome polarization. C) Western blot analysis of the indicated Jurkat knockout (KO) cell lines with indicated antibodies. KIF21B-KO clone #1 and #2 were transduced with a KIF21B-GFP construct to generate two polyclonal cell lines re-expressing full-length KIF21B. D) Phalloidin staining to show F-actin structures in indicated Jurkat T cell lines imaged on a widefield microscope. E) Quantification of synapse size expressed as surface area based on a F-actin staining of indicated Jurkat T cells. n = 134, 218, 241, 185 and 194 cells from three independent experiments. ** p

    Techniques Used: In Vivo, Activation Assay, Western Blot, Knock-Out, Transduction, Construct, Expressing, Staining, Microscopy

    6) Product Images from "Membrane alterations induced by nonstructural proteins of human norovirus"

    Article Title: Membrane alterations induced by nonstructural proteins of human norovirus

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006705

    Subcellular localization of NS1-2 analyzed by IF and CLEM. A plasmid encoding eGFP-NS1-2 (green) was transfected into Huh7-T7 cells. Twenty hours post transfection cells were fixed and analyzed by confocal microscopy (A, B) or CLEM (D). Cellular markers (red) were stained using monoclonal or polyclonal antibodies, LDs were stained with LipidTox and Mitochondria were stained with Mitotracker. A white and a yellow asterisk mark a cell with a focal and an intermediate focal/filamentous distribution of NS1-2, respectively. Mito, mitochondria. (B) Pearson correlation of the eGFP-NS1-2 signal with different cellular markers shown in (A). Each dot represents a single cell. (C) HA-NS1-2 was expressed in Huh7-T7 cells and detected by immunofluorescence using a HA-specific antibody (red). Nuclei were counterstained with DAPI (right panel, blue). (D) For CLEM, cells were seeded onto gridded coverslips, fixed and subjected to optical sectioning using a confocal microscope. Maximum-intensity Z-projection of a selected cell is shown on the top. eGFP-NS1-2 signal is depicted in green, LDs in red and the nucleus (DAPI) in blue. Samples were subsequently processed for electron microscopy by using the coordinates etched onto the surface of the gridded coverslips to record the position of the selected cells. The correlated panel was obtained by re-orientation and superimposition of light and electron micrographs as described in M M. Boxed and numbered areas are shown in higher magnification in subsequent panels. White scale bars, 5 μm. Yellow scale bar, 1 μm. Red scale bars, 200 nm.
    Figure Legend Snippet: Subcellular localization of NS1-2 analyzed by IF and CLEM. A plasmid encoding eGFP-NS1-2 (green) was transfected into Huh7-T7 cells. Twenty hours post transfection cells were fixed and analyzed by confocal microscopy (A, B) or CLEM (D). Cellular markers (red) were stained using monoclonal or polyclonal antibodies, LDs were stained with LipidTox and Mitochondria were stained with Mitotracker. A white and a yellow asterisk mark a cell with a focal and an intermediate focal/filamentous distribution of NS1-2, respectively. Mito, mitochondria. (B) Pearson correlation of the eGFP-NS1-2 signal with different cellular markers shown in (A). Each dot represents a single cell. (C) HA-NS1-2 was expressed in Huh7-T7 cells and detected by immunofluorescence using a HA-specific antibody (red). Nuclei were counterstained with DAPI (right panel, blue). (D) For CLEM, cells were seeded onto gridded coverslips, fixed and subjected to optical sectioning using a confocal microscope. Maximum-intensity Z-projection of a selected cell is shown on the top. eGFP-NS1-2 signal is depicted in green, LDs in red and the nucleus (DAPI) in blue. Samples were subsequently processed for electron microscopy by using the coordinates etched onto the surface of the gridded coverslips to record the position of the selected cells. The correlated panel was obtained by re-orientation and superimposition of light and electron micrographs as described in M M. Boxed and numbered areas are shown in higher magnification in subsequent panels. White scale bars, 5 μm. Yellow scale bar, 1 μm. Red scale bars, 200 nm.

    Techniques Used: Plasmid Preparation, Transfection, Confocal Microscopy, Staining, Immunofluorescence, Microscopy, Electron Microscopy

    7) Product Images from "Pertussis toxin-sensitive Gi-proteins and intracellular calcium sensitivity of vasoconstriction in the intact rat tail artery"

    Article Title: Pertussis toxin-sensitive Gi-proteins and intracellular calcium sensitivity of vasoconstriction in the intact rat tail artery

    Journal: British Journal of Pharmacology

    doi: 10.1038/sj.bjp.0703703

    Membrane preparations (10 μg) of rat tail artery labelled with G iα - (upper) and G oα (lower)-antibodies. Control was bovine brain purified G-proteins subunits. Lanes: 1, bovine brain purified G-proteins subunits with polyclonal rabbit antibodies directed against G iα1-2 +G iα3 (upper) or monoclonal mouse antibody directed against G oα (lower), 2, membrane preparation of the rat tail artery with polyclonal rabbit antibodies directed against G iα1-2 +G iα3 (upper) or monoclonal mouse antibody directed against G oα (lower), 3, polyclonal rabbit antibodies directed against G iα1-2 +G iα3 (upper) or monoclonal mouse antibody directed against G oα (lower), 4, bovine brain purified G-proteins subunits, 5, membrane preparation of the rat tail artery. The arrows indicate the positions of the molecular markers ovalbumin (45 kDa) and carbonic anhydrase (31 kDa).
    Figure Legend Snippet: Membrane preparations (10 μg) of rat tail artery labelled with G iα - (upper) and G oα (lower)-antibodies. Control was bovine brain purified G-proteins subunits. Lanes: 1, bovine brain purified G-proteins subunits with polyclonal rabbit antibodies directed against G iα1-2 +G iα3 (upper) or monoclonal mouse antibody directed against G oα (lower), 2, membrane preparation of the rat tail artery with polyclonal rabbit antibodies directed against G iα1-2 +G iα3 (upper) or monoclonal mouse antibody directed against G oα (lower), 3, polyclonal rabbit antibodies directed against G iα1-2 +G iα3 (upper) or monoclonal mouse antibody directed against G oα (lower), 4, bovine brain purified G-proteins subunits, 5, membrane preparation of the rat tail artery. The arrows indicate the positions of the molecular markers ovalbumin (45 kDa) and carbonic anhydrase (31 kDa).

    Techniques Used: Purification

    8) Product Images from "A phase II dose-escalation trial of perioperative desmopressin (1-desamino-8-d-arginine vasopressin) in breast cancer patients"

    Article Title: A phase II dose-escalation trial of perioperative desmopressin (1-desamino-8-d-arginine vasopressin) in breast cancer patients

    Journal: SpringerPlus

    doi: 10.1186/s40064-015-1217-y

    Immunohistochemical staining of vasopressin receptors. V2R expression was detected using polyclonal antibodies against the human receptor, as described in detail in “ Patients and methods ”. Representative pictures of tumor sections from patients enrolled in the trial and positive control tissue are depicted. a Breast carcinoma expressing V2R b V2R-negative breast carcinoma c Kidney tubules d MCF-7 human breast carcinoma xenograft. Arrowhead denotes positive staining of small vessels. Original magnification: a , b , d ×400; c ×100.
    Figure Legend Snippet: Immunohistochemical staining of vasopressin receptors. V2R expression was detected using polyclonal antibodies against the human receptor, as described in detail in “ Patients and methods ”. Representative pictures of tumor sections from patients enrolled in the trial and positive control tissue are depicted. a Breast carcinoma expressing V2R b V2R-negative breast carcinoma c Kidney tubules d MCF-7 human breast carcinoma xenograft. Arrowhead denotes positive staining of small vessels. Original magnification: a , b , d ×400; c ×100.

    Techniques Used: Immunohistochemistry, Staining, Expressing, Positive Control

    9) Product Images from "Leptin at gender-specific concentrations does not affect glucose transport, expression of glucose transporters and leptin receptors in human lymphocytes"

    Article Title: Leptin at gender-specific concentrations does not affect glucose transport, expression of glucose transporters and leptin receptors in human lymphocytes

    Journal: Endocrine

    doi: 10.1007/s12020-014-0435-3

    Representative histograms of GLUT1 from flow cytometry analysis of females samples incubated in different leptin concentration. The first antibody used was polyclonal rabbit antibody against GLUT1. The secondary antibody was swine anti-rabbit IgG-FITC. a control without leptin; b normal leptin concentration; c elevated leptin concentration; d control sample (negative) without antibody against GLUT1
    Figure Legend Snippet: Representative histograms of GLUT1 from flow cytometry analysis of females samples incubated in different leptin concentration. The first antibody used was polyclonal rabbit antibody against GLUT1. The secondary antibody was swine anti-rabbit IgG-FITC. a control without leptin; b normal leptin concentration; c elevated leptin concentration; d control sample (negative) without antibody against GLUT1

    Techniques Used: Flow Cytometry, Cytometry, Incubation, Concentration Assay

    10) Product Images from "Clumping Factor B Promotes Adherence of Staphylococcus aureus to Corneocytes in Atopic Dermatitis"

    Article Title: Clumping Factor B Promotes Adherence of Staphylococcus aureus to Corneocytes in Atopic Dermatitis

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00994-16

    Characterization of a ClfB-deficient mutant of S. aureus AD08 CC1 . (A) S. aureus strain AD08 CC1 (lane 1), its isogenic ClfB-deficient mutant AD08 CC1 Δ clfB (lane 2), and complemented mutant AD08 CC1 Δ clfB (pCU1:: clfB ) (lane 3) were grown to exponential phase. Cell wall extracts were separated on 7.5% acrylamide gels and blotted onto PVDF membranes, and ClfB was detected using polyclonal rabbit antibodies. Bound antibody was detected using horseradish peroxidase-conjugated protein A. Size markers (in kilodaltons) are indicated on the left. (B) S. aureus strain AD08 CC1 (•) and AD08 CC1 Δ clfB (■) were grown to exponential phase, washed, and incubated in microtiter plates coated with L2v. Adherent cells were stained with crystal violet, and the absorbance was read at 570 nm. The graph shown is representative of graphs from three independent experiments. (C) S. aureus strain AD08 CC1 (red) or mutants AD08 CC1 Δ clfB (blue), AD08 CC1 Δ clfB (pCU1) (white), and AD08 CC1 Δ clfB (pCU1:: clfB ) (green) were grown to exponential phase, washed, and incubated in microtiter plates coated with L2v (0.625 μg/ml). Adherent cells were stained with crystal violet, and the absorbance was read at 570 nm. Error bars represent the standard error of the mean values obtained from three independent experiments. Statistical significance was determined by Student's unpaired t test. **, P = 0.0059; ***, P
    Figure Legend Snippet: Characterization of a ClfB-deficient mutant of S. aureus AD08 CC1 . (A) S. aureus strain AD08 CC1 (lane 1), its isogenic ClfB-deficient mutant AD08 CC1 Δ clfB (lane 2), and complemented mutant AD08 CC1 Δ clfB (pCU1:: clfB ) (lane 3) were grown to exponential phase. Cell wall extracts were separated on 7.5% acrylamide gels and blotted onto PVDF membranes, and ClfB was detected using polyclonal rabbit antibodies. Bound antibody was detected using horseradish peroxidase-conjugated protein A. Size markers (in kilodaltons) are indicated on the left. (B) S. aureus strain AD08 CC1 (•) and AD08 CC1 Δ clfB (■) were grown to exponential phase, washed, and incubated in microtiter plates coated with L2v. Adherent cells were stained with crystal violet, and the absorbance was read at 570 nm. The graph shown is representative of graphs from three independent experiments. (C) S. aureus strain AD08 CC1 (red) or mutants AD08 CC1 Δ clfB (blue), AD08 CC1 Δ clfB (pCU1) (white), and AD08 CC1 Δ clfB (pCU1:: clfB ) (green) were grown to exponential phase, washed, and incubated in microtiter plates coated with L2v (0.625 μg/ml). Adherent cells were stained with crystal violet, and the absorbance was read at 570 nm. Error bars represent the standard error of the mean values obtained from three independent experiments. Statistical significance was determined by Student's unpaired t test. **, P = 0.0059; ***, P

    Techniques Used: Mutagenesis, Incubation, Staining

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    Article Snippet: .. The membranes were then incubated with a mouse monoclonal anti-Kv4.2 or anti-KChIP3 antibody or with a rabbit polyclonal anti-GFP (Millipore, Billerica, MA) or anti-DPP6 ( ) antibody at 4 °C overnight. .. The anti-Kv4.2 and anti-KChIP3 antibodies were developed by and obtained from the UC Davis/National Institutes of Health NeuroMab facility, supported by National Institutes of Health Grant U24NS050606 and maintained by the University of California at Davis; the anti-DPP6 antibody was obtained from Dr. Bernardo Rudy.

    Generated:

    Article Title: Evidence for coordinated interaction of cyclin D3 with p21 and cdk6 in directing the development of uterine stromal cell decidualization and polyploidy during implantation
    Article Snippet: .. Rabbit polyclonal antibody to p21 (Cat# PC55), generated against a recombinant protein consisting amino acids 15–61 of mouse p21, was purchased from Oncogene Research Products (Cambridge, MA). .. A monoclonal antibody for p27, obtained from mouse ascites by epitope-affinity chromatography using full-length human recombinant p27 protein, was obtained from Zymed Laboratories Inc. (San Francisco, CA).

    Sequencing:

    Article Title: Reversible pathologic and cognitive phenotypes in an inducible model of Alzheimer-amyloidosis
    Article Snippet: .. In immunohistochemistry, we used monoclonal antibodies 6E10 and rabbit polyclonal antibody OC (EMD Millipore, Billerica, MA) ( )], which recognize the human Aβ sequence and fibrillar Aβ, respectively. .. For ELISA analysis, we used human anti-Aβ42 (Rabbit Monoclonal Clone 1-11-3, Covance, Princeton, NJ) and mAb 4G8.

    Sonication:

    Article Title: Deacetylation of p53 induces autophagy by suppressing Bmf expression
    Article Snippet: .. Sonicated nuclear fractions were incubated with mouse IgG1 monoclonal antibody to p53 (sc-98 Pab1801; Santa Cruz Biotechnology, Inc.), rabbit polyclonal antibody to HDAC1 (06–720; EMD Millipore), rabbit polyclonal antibody to acetyl-H4 (06–598; EMD Millipore), rabbit polyclonal antibody to acetyl-H3 (06-599B; EMD Millipore), mouse IgG1, or rabbit as a control (CBL600; EMD Millipore). .. DNA identification was confirmed with PCR using the following primers specific for the Bmf promoter: region −560, 5′-ACCTAAGGGCTCCCCTGGA-3′ and 5′-GCAGGTCGGAAGAAAACTGCAGC-3′, and region −97, 5′-TTGGCGCTTCACTCGCCATT-3′ and 5′-ATCCCGCAAACAGCTGAT-3′.

    Recombinant:

    Article Title: Evidence for coordinated interaction of cyclin D3 with p21 and cdk6 in directing the development of uterine stromal cell decidualization and polyploidy during implantation
    Article Snippet: .. Rabbit polyclonal antibody to p21 (Cat# PC55), generated against a recombinant protein consisting amino acids 15–61 of mouse p21, was purchased from Oncogene Research Products (Cambridge, MA). .. A monoclonal antibody for p27, obtained from mouse ascites by epitope-affinity chromatography using full-length human recombinant p27 protein, was obtained from Zymed Laboratories Inc. (San Francisco, CA).

    Article Title: TRPV-5 Mediates a Receptor Activator of NF-?B (RANK) Ligand-induced Increase in Cytosolic Ca2+ in Human Osteoclasts and Down-regulates Bone Resorption *
    Article Snippet: .. Recombinant human macrophage-CSF and recombinant human granulocyte-macrophage-CSF were purchased from R & D Systems (Minneapolis, MN); goat polyclonal antibodies against calcitonin receptor (CTR) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), mouse monoclonal antibodies against TRPV-5 were from Abcam (Cambridge, MA), and rabbit polyclonal antibodies were from Upstate Biotech Millipore (Billerica, MA); rabbit polyclonal antibodies against L-type and T-type Ca2+ channels were purchased from Sigma-Aldrich; those against R-type channels were from Novus Biologicals (Littleton, CO); and rabbit polyclonal antibodies against V-ATPase were purchased from Chemicon (Temecula, CA). .. Alexa Fluor antibodies and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Invitrogen.

    Chromatin Immunoprecipitation:

    Article Title: BEX2 has a functional interplay with c-Jun/JNK and p65/RelA in breast cancer
    Article Snippet: .. ChIP-grade rabbit polyclonal p65 (AbCam) and rabbit polyclonal c-Jun (Millipore) antibodies were applied for these assays at 1:100 and 1:50 dilutions, respectively. .. This process generated chromatin fragments with an average size of 200-500 bp assessed using Agarose gel electrophoresis.

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  • 85
    Millipore rabbit polyclonal anti e1b 19k
    Formation of tumor giant cells in tumors defective for apoptosis. ( A ) Hematoxylin and eosin-stained sections reveal numerous tumor giant cells in tumors from transformed BMK cells expressing BCL-2 or <t>E1B</t> <t>19K.</t> Typical sections of carcinomas as described in the text are shown at a magnification of 200× and highlight areas enriched for tumor giant cells in tumors from animals injected with transformed BMK cells expressing BCL-2 or E1B 19K, with insets of grossly polyploid cells in mitosis (at 1000× magnification). Several tumor giant cells in each image are indicated by white arrows. Note the absence of tumor giant cells in tumors formed by the W2.3.1–5 cells. A typical section of a tumor area enriched for tumor giant cells was also immunostained for adenovirus E1A to demonstrate that the tumor giant cells are derived from the input-transformed BMK cells. Note the numerous tumor giant cells that stain brown in the E1A immunohistochemistry, including several examples indicated by arrows. A tumor section (20 μm) enriched for tumor giant cells (arrows) was stained with YOYO-1 to reveal DNA content as described in the text. This image is shown at 630×. ( B ) Aberrant metaphases and polyploid cells accumulate during tumor progression. Tumor sections were developed by immunohistochemistry using antibodies specific for phospho-histone H3 and are shown at 200×. Black arrows indicate aberrant polyploid mitotic arrays, and mitotic arrays presented at 600× in the insets are boxed. Top panels represent images of sections from mature tumors. Phosphohistone H3 immunohistochemistry of sections of transformed BMK cell masses excised from mice on days 2 and 9 after injection are shown in the bottom two rows (200×). Insets in the phospho-histone H3 panels were photographed at 600×, and areas present in the insets are boxed. Grossly aberrant mitotic arrays stained for phospho-histone H3 that are evident in the W2.Bcl2–3 and D3.zeo-2, but not W2.3.1–5, cells on day 9 are indicated in the insets by white arrows. Necrotic centers are indicated (N).
    Rabbit Polyclonal Anti E1b 19k, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Millipore rabbit polyclonal anti py39 α syn
    c-Abl induces <t>α-syn</t> phosphorylation at Y39 and Y125 residues in primary cortical neurons. ( A ) WT primary cortical neurons were infected with lentivirus encoding for c-Abl or with an empty lentivirus. Five days post-infection, neurons were lysed and the proteins were separated using SDS–PAGE and detected by WB analysis (left-hand-side panel). The α-syn phosphorylation status was assessed using the phosphorylation site-specific antibodies for Y39 or Y125 residues. α-syn and c-Abl expressions were confirmed in an additional protein blot using specific antibodies. Actin was used as a loading control. The phosphorylation of α-syn at residues Y39 and Y125 was evaluated by densitometry quantification (right-hand-side panel). The band intensities were normalized in the following manner: <t>pY39/[α-syn/actin]</t> or pY125/[α-syn/actin]. Bars represent the mean ± SD of three independent experiments. * P
    Rabbit Polyclonal Anti Py39 α Syn, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Millipore srif
    In vitro DPV-µCFE scans obtained in <t>PBS,</t> 0.1 M; pH 7.4 (LEFT) or in a PBS solution containing a mixture of ascorbic acid (AA) 5 mM; DOPAC, 50 µM; 5HIAA, 25 µM and <t>SRIF</t> 1 mM; in PBS, 0.1 M; pH 7.4. Peak 1: AA at −50 mV; Peak 2: DOPAC at +100 mV; Peak 3: 5HIAA at +300 mV; and Peak 5: SRIF at +800 mV (modified from [ 18 ]).
    Srif, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Millipore sly1 ab
    B6 <t>SLy1−</t> NK cells demonstrate multiple functional defects. (A) Clearance of RMA lymphoma expressing the NKG2D ligand Rae-1 from lungs of B6 wt or B6 SLy1− mice 18 h after i.v. injection. Comparison performed by unpaired t-test. (B) Growth of LLC injected into the flank of B6 SLy1− (red line) and B6 wt mice (black line) in untreated mice (left), those depleted of CD4 + and CD8 + lymphocytes (middle) or NK1.1 + cells (right). Comparison performed by unpaired t-test at each time point. Isotype control-treated animals demonstrated tumor growth identical to unmanipulated mice (data not shown). (C) In vitro lysis of LLC at a 50:1 effector:target ratio. Data representative of four separate experiments with comparison performed by unpaired t-test. (D) Average number of durable contacts between B6 SLy1− (top) and B6 wt (bottom) NK (crossed to express tomato cherry red on NK1.1 promoter 56 ) cells with GFP-expressing LLC injected into the lung. Yellow arrow points to 4 B6 wt NK cells establishing contact with one tumor cell. Data representative of three separate mice per group with comparison performed by unpaired t-test. (E) In vivo clearance of CFSE labeled B6 CD45.1+ wild-type or B6 CD45.2+ m157-expressing splenocytes injected into B6 SLy1− (top) and B6 wt mice (bottom) recipient mice. Data derived from flow cytometric analysis of the spleen, demonstrating one representative experiment on the left and summary of five animals on the right compared by unpaired t-test. (F) Degranulation of B6 SLy1− (red) and B6 wt (black) NK cells after plate bound stimulation with NKp46 as measured by surface CD107a expression. Left panel representative of one experiment with heavy black line representative of Cd107a in B6 wt NK cells after co-culture with plate bound NKp46 and heavy red line representative of B6 SLy1− NK cells after co-culture with plate bound NKp46. Shaded black line representative of plate-bound isotype control. Comparison performed by unpaired t-test. Summary of five separate experiments. * p
    Sly1 Ab, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Formation of tumor giant cells in tumors defective for apoptosis. ( A ) Hematoxylin and eosin-stained sections reveal numerous tumor giant cells in tumors from transformed BMK cells expressing BCL-2 or E1B 19K. Typical sections of carcinomas as described in the text are shown at a magnification of 200× and highlight areas enriched for tumor giant cells in tumors from animals injected with transformed BMK cells expressing BCL-2 or E1B 19K, with insets of grossly polyploid cells in mitosis (at 1000× magnification). Several tumor giant cells in each image are indicated by white arrows. Note the absence of tumor giant cells in tumors formed by the W2.3.1–5 cells. A typical section of a tumor area enriched for tumor giant cells was also immunostained for adenovirus E1A to demonstrate that the tumor giant cells are derived from the input-transformed BMK cells. Note the numerous tumor giant cells that stain brown in the E1A immunohistochemistry, including several examples indicated by arrows. A tumor section (20 μm) enriched for tumor giant cells (arrows) was stained with YOYO-1 to reveal DNA content as described in the text. This image is shown at 630×. ( B ) Aberrant metaphases and polyploid cells accumulate during tumor progression. Tumor sections were developed by immunohistochemistry using antibodies specific for phospho-histone H3 and are shown at 200×. Black arrows indicate aberrant polyploid mitotic arrays, and mitotic arrays presented at 600× in the insets are boxed. Top panels represent images of sections from mature tumors. Phosphohistone H3 immunohistochemistry of sections of transformed BMK cell masses excised from mice on days 2 and 9 after injection are shown in the bottom two rows (200×). Insets in the phospho-histone H3 panels were photographed at 600×, and areas present in the insets are boxed. Grossly aberrant mitotic arrays stained for phospho-histone H3 that are evident in the W2.Bcl2–3 and D3.zeo-2, but not W2.3.1–5, cells on day 9 are indicated in the insets by white arrows. Necrotic centers are indicated (N).

    Journal: Genes & Development

    Article Title: Hypoxia and defective apoptosis drive genomic instability and tumorigenesis

    doi: 10.1101/gad.1204904

    Figure Lengend Snippet: Formation of tumor giant cells in tumors defective for apoptosis. ( A ) Hematoxylin and eosin-stained sections reveal numerous tumor giant cells in tumors from transformed BMK cells expressing BCL-2 or E1B 19K. Typical sections of carcinomas as described in the text are shown at a magnification of 200× and highlight areas enriched for tumor giant cells in tumors from animals injected with transformed BMK cells expressing BCL-2 or E1B 19K, with insets of grossly polyploid cells in mitosis (at 1000× magnification). Several tumor giant cells in each image are indicated by white arrows. Note the absence of tumor giant cells in tumors formed by the W2.3.1–5 cells. A typical section of a tumor area enriched for tumor giant cells was also immunostained for adenovirus E1A to demonstrate that the tumor giant cells are derived from the input-transformed BMK cells. Note the numerous tumor giant cells that stain brown in the E1A immunohistochemistry, including several examples indicated by arrows. A tumor section (20 μm) enriched for tumor giant cells (arrows) was stained with YOYO-1 to reveal DNA content as described in the text. This image is shown at 630×. ( B ) Aberrant metaphases and polyploid cells accumulate during tumor progression. Tumor sections were developed by immunohistochemistry using antibodies specific for phospho-histone H3 and are shown at 200×. Black arrows indicate aberrant polyploid mitotic arrays, and mitotic arrays presented at 600× in the insets are boxed. Top panels represent images of sections from mature tumors. Phosphohistone H3 immunohistochemistry of sections of transformed BMK cell masses excised from mice on days 2 and 9 after injection are shown in the bottom two rows (200×). Insets in the phospho-histone H3 panels were photographed at 600×, and areas present in the insets are boxed. Grossly aberrant mitotic arrays stained for phospho-histone H3 that are evident in the W2.Bcl2–3 and D3.zeo-2, but not W2.3.1–5, cells on day 9 are indicated in the insets by white arrows. Necrotic centers are indicated (N).

    Article Snippet: Primary antibodies used were hamster anti-human BCL-2 (BD-Pharmingen); rabbit polyclonal anti-E1B 19K , mouse anti-actin (Oncogene Research Products), rabbit anti-mouse HIF-1α (Cayman), rabbit anti-BIM (Axxora), or rabbit polyclonal antisera raised in our lab against a GST-human PUMA fusion protein encoding a 102-amino acid region common to PUMA-α and PUMA-β.

    Techniques: Staining, Transformation Assay, Expressing, Injection, Derivative Assay, Immunohistochemistry, Mouse Assay

    Antiapoptotic BCL-2 family proteins block apoptosis and promote tumor formation. ( A ) Generation of stable cell lines. Cell extracts made from stable BMK cells that express both BAX and BAK (W2), or that are deficient for BAX and BAK (D3), were subjected to Western blotting with antibodies specific for BCL-2 ( left top panel) or E1B 19K ( right top panel). Note the similar expression levels of each exogenous protein in three independent clones (depicted numerically) and undetectable levels of each exogenous protein in the vector-only control cell lines (W2.3.1–2,5,6 or D3.zeo-1,2,3). Blots were then reprobed with an antibody to actin to verify nearly equivalent levels of protein in all lanes, shown below the BCL-2 and E1B 19K panels. ( B ) BCL-2 and E1B 19K block apoptosis in response to staurosporine. Stable BMK cell lines expressing BCL-2, E1B 19K, and controls were treated with media alone (open bars) or media containing 0.4 μM staurosporine (filled bars) for 24 h, and the viable cell number was determined by trypan blue exclusion. Results are presented as the percent of viable cells in each condition, which in each case represents the average of three independent plates. ( C ) BCL-2 and E1B 19K antagonize BAX and BAK to promote tumor formation. Three independent stable BMK cell lines (circles, squares, and diamonds) expressing BCL-2 (green symbols), E1B 19K (blue symbols), or controls (red symbols) were injected subcutaneously into nude mice, and tumor formation was monitored over time. Each point represents the average tumor volume for five injected animals. W2 cells, which express both BAX and BAK, are shown in the left panel. D3 cells, which are deficient for both BAX and BAK, are shown in the right panel. Note that BCL-2 or E1B 19K expression caused a profound acceleration of tumor formation in the W2 cells, whereas the kinetics of tumor formation in the D3 cells, which are deficient for both BAX and BAK, were unchanged by BCL-2 or E1B 19K expression.

    Journal: Genes & Development

    Article Title: Hypoxia and defective apoptosis drive genomic instability and tumorigenesis

    doi: 10.1101/gad.1204904

    Figure Lengend Snippet: Antiapoptotic BCL-2 family proteins block apoptosis and promote tumor formation. ( A ) Generation of stable cell lines. Cell extracts made from stable BMK cells that express both BAX and BAK (W2), or that are deficient for BAX and BAK (D3), were subjected to Western blotting with antibodies specific for BCL-2 ( left top panel) or E1B 19K ( right top panel). Note the similar expression levels of each exogenous protein in three independent clones (depicted numerically) and undetectable levels of each exogenous protein in the vector-only control cell lines (W2.3.1–2,5,6 or D3.zeo-1,2,3). Blots were then reprobed with an antibody to actin to verify nearly equivalent levels of protein in all lanes, shown below the BCL-2 and E1B 19K panels. ( B ) BCL-2 and E1B 19K block apoptosis in response to staurosporine. Stable BMK cell lines expressing BCL-2, E1B 19K, and controls were treated with media alone (open bars) or media containing 0.4 μM staurosporine (filled bars) for 24 h, and the viable cell number was determined by trypan blue exclusion. Results are presented as the percent of viable cells in each condition, which in each case represents the average of three independent plates. ( C ) BCL-2 and E1B 19K antagonize BAX and BAK to promote tumor formation. Three independent stable BMK cell lines (circles, squares, and diamonds) expressing BCL-2 (green symbols), E1B 19K (blue symbols), or controls (red symbols) were injected subcutaneously into nude mice, and tumor formation was monitored over time. Each point represents the average tumor volume for five injected animals. W2 cells, which express both BAX and BAK, are shown in the left panel. D3 cells, which are deficient for both BAX and BAK, are shown in the right panel. Note that BCL-2 or E1B 19K expression caused a profound acceleration of tumor formation in the W2 cells, whereas the kinetics of tumor formation in the D3 cells, which are deficient for both BAX and BAK, were unchanged by BCL-2 or E1B 19K expression.

    Article Snippet: Primary antibodies used were hamster anti-human BCL-2 (BD-Pharmingen); rabbit polyclonal anti-E1B 19K , mouse anti-actin (Oncogene Research Products), rabbit anti-mouse HIF-1α (Cayman), rabbit anti-BIM (Axxora), or rabbit polyclonal antisera raised in our lab against a GST-human PUMA fusion protein encoding a 102-amino acid region common to PUMA-α and PUMA-β.

    Techniques: Blocking Assay, Stable Transfection, Western Blot, Expressing, Clone Assay, Plasmid Preparation, Injection, Mouse Assay

    c-Abl induces α-syn phosphorylation at Y39 and Y125 residues in primary cortical neurons. ( A ) WT primary cortical neurons were infected with lentivirus encoding for c-Abl or with an empty lentivirus. Five days post-infection, neurons were lysed and the proteins were separated using SDS–PAGE and detected by WB analysis (left-hand-side panel). The α-syn phosphorylation status was assessed using the phosphorylation site-specific antibodies for Y39 or Y125 residues. α-syn and c-Abl expressions were confirmed in an additional protein blot using specific antibodies. Actin was used as a loading control. The phosphorylation of α-syn at residues Y39 and Y125 was evaluated by densitometry quantification (right-hand-side panel). The band intensities were normalized in the following manner: pY39/[α-syn/actin] or pY125/[α-syn/actin]. Bars represent the mean ± SD of three independent experiments. * P

    Journal: Human Molecular Genetics

    Article Title: c-Abl phosphorylates α-synuclein and regulates its degradation: implication for α-synuclein clearance and contribution to the pathogenesis of Parkinson's disease

    doi: 10.1093/hmg/ddt674

    Figure Lengend Snippet: c-Abl induces α-syn phosphorylation at Y39 and Y125 residues in primary cortical neurons. ( A ) WT primary cortical neurons were infected with lentivirus encoding for c-Abl or with an empty lentivirus. Five days post-infection, neurons were lysed and the proteins were separated using SDS–PAGE and detected by WB analysis (left-hand-side panel). The α-syn phosphorylation status was assessed using the phosphorylation site-specific antibodies for Y39 or Y125 residues. α-syn and c-Abl expressions were confirmed in an additional protein blot using specific antibodies. Actin was used as a loading control. The phosphorylation of α-syn at residues Y39 and Y125 was evaluated by densitometry quantification (right-hand-side panel). The band intensities were normalized in the following manner: pY39/[α-syn/actin] or pY125/[α-syn/actin]. Bars represent the mean ± SD of three independent experiments. * P

    Article Snippet: The sections were incubated overnight at 4°C ( ) with the following antibodies: rabbit polyclonal anti-c-Abl (K-12), rabbit polyclonal anti-pY39-α-syn, rabbit anti-pY125-α-syn, FL α-syn rabbit polyclonal (Millipore) followed by tagged secondary antibody, avidin biotin HRP and diaminobenzidine reactivity.

    Techniques: Infection, SDS Page, Western Blot

    c-Abl protein levels increase transiently upon α-syn overexpression in rat midbrains. ( A and B ) Effect of α-syn overexpression on c-Abl protein levels in vivo , low magnification (scale bar: 500 µm) (A) and high-power magnification photomicrographs (scale bar: 100 µm) (B) that illustrate the endogenous protein level of c-Abl after the overexpression of human α-syn or FPmax (*, injected side; n = 3 per condition). The use of three different antibodies raised against different epitopes of c-Abl [(Sigma), (K12, Santa Cruz), (24-11, Santa Cruz)] revealed a dramatic increase of c-Abl protein level only in the side injected with human α-syn. Overexpression of α-syn was confirmed by IHC staining on serial section (B, bottom line). No c-Abl signal was detected in FPmax-injected or in the non-injected sides. The enhancement of c-Abl protein expression induced by α-syn overexpression was primarily observed at 1 month post-injection and disappeared after 3 months post-injection, suggesting a transient interplay between c-Abl and α-syn. ( C ) Protein levels of pY39 α-syn and activated c-Abl (pY412, a marker of high kinase activity) increased only in the injected side overexpressing α-syn 1 month post-injection (scale bar: 50 µm).

    Journal: Human Molecular Genetics

    Article Title: c-Abl phosphorylates α-synuclein and regulates its degradation: implication for α-synuclein clearance and contribution to the pathogenesis of Parkinson's disease

    doi: 10.1093/hmg/ddt674

    Figure Lengend Snippet: c-Abl protein levels increase transiently upon α-syn overexpression in rat midbrains. ( A and B ) Effect of α-syn overexpression on c-Abl protein levels in vivo , low magnification (scale bar: 500 µm) (A) and high-power magnification photomicrographs (scale bar: 100 µm) (B) that illustrate the endogenous protein level of c-Abl after the overexpression of human α-syn or FPmax (*, injected side; n = 3 per condition). The use of three different antibodies raised against different epitopes of c-Abl [(Sigma), (K12, Santa Cruz), (24-11, Santa Cruz)] revealed a dramatic increase of c-Abl protein level only in the side injected with human α-syn. Overexpression of α-syn was confirmed by IHC staining on serial section (B, bottom line). No c-Abl signal was detected in FPmax-injected or in the non-injected sides. The enhancement of c-Abl protein expression induced by α-syn overexpression was primarily observed at 1 month post-injection and disappeared after 3 months post-injection, suggesting a transient interplay between c-Abl and α-syn. ( C ) Protein levels of pY39 α-syn and activated c-Abl (pY412, a marker of high kinase activity) increased only in the injected side overexpressing α-syn 1 month post-injection (scale bar: 50 µm).

    Article Snippet: The sections were incubated overnight at 4°C ( ) with the following antibodies: rabbit polyclonal anti-c-Abl (K-12), rabbit polyclonal anti-pY39-α-syn, rabbit anti-pY125-α-syn, FL α-syn rabbit polyclonal (Millipore) followed by tagged secondary antibody, avidin biotin HRP and diaminobenzidine reactivity.

    Techniques: Over Expression, In Vivo, Injection, Immunohistochemistry, Staining, Expressing, Marker, Activity Assay

    Phosphorylation of α-syn at Y39 and Y125 and an increase in c-Abl protein level can be detected in vivo in human PD brain tissue. ( A ) Representative WB of the relative phosphorylation status of α-syn observed in PD cases versus controls. Protein extracts from samples of the anterior cingulate cortex from Parkinson's disease patients ( n = 17) and age- and post-mortem delay-matched neurological and neuropathological controls ( n = 17) were resolved by SDS–PAGE and analyzed by WB (shown, n = 7 from PD cases and n = 7 from age- and post-mortem delay-matched neurological and neuropathological controls) using α-syn antibodies against pS129, pY125, pY39 and total α-syn (left-hand-side panel). Phosphorylation levels (pS129, pY125, pY39) of α-syn were evaluated by densitometry quantification (right-hand-side panel). The band intensities were normalized in the following manner: [(pY39 or pY125 or pS129/actin)/α-syn]. The bars represent the mean ± SD of PD cases ( n = 17) and control cases ( n = 17). ** P

    Journal: Human Molecular Genetics

    Article Title: c-Abl phosphorylates α-synuclein and regulates its degradation: implication for α-synuclein clearance and contribution to the pathogenesis of Parkinson's disease

    doi: 10.1093/hmg/ddt674

    Figure Lengend Snippet: Phosphorylation of α-syn at Y39 and Y125 and an increase in c-Abl protein level can be detected in vivo in human PD brain tissue. ( A ) Representative WB of the relative phosphorylation status of α-syn observed in PD cases versus controls. Protein extracts from samples of the anterior cingulate cortex from Parkinson's disease patients ( n = 17) and age- and post-mortem delay-matched neurological and neuropathological controls ( n = 17) were resolved by SDS–PAGE and analyzed by WB (shown, n = 7 from PD cases and n = 7 from age- and post-mortem delay-matched neurological and neuropathological controls) using α-syn antibodies against pS129, pY125, pY39 and total α-syn (left-hand-side panel). Phosphorylation levels (pS129, pY125, pY39) of α-syn were evaluated by densitometry quantification (right-hand-side panel). The band intensities were normalized in the following manner: [(pY39 or pY125 or pS129/actin)/α-syn]. The bars represent the mean ± SD of PD cases ( n = 17) and control cases ( n = 17). ** P

    Article Snippet: The sections were incubated overnight at 4°C ( ) with the following antibodies: rabbit polyclonal anti-c-Abl (K-12), rabbit polyclonal anti-pY39-α-syn, rabbit anti-pY125-α-syn, FL α-syn rabbit polyclonal (Millipore) followed by tagged secondary antibody, avidin biotin HRP and diaminobenzidine reactivity.

    Techniques: In Vivo, Western Blot, SDS Page

    c-Abl induces α-syn phosphorylation at Y39 and Y125 residues in HEK293T cells. ( A ) HEK293T cells were transfected with WT α-syn or its mutants (Y39F, Y125F or Y39FY125F) together with plasmids encoding for LUC (negative control) or WT c-Abl. Twenty-four hours post-transfection, cells were lysed and the proteins separated using SDS–PAGE and detected by WB analysis (top panel). α-syn phosphorylation status was assessed using phosphorylation site-specific antibodies for Y39 or Y125 residues. α-syn and c-Abl expression were confirmed in an additional protein blot using specific antibodies. Actin was used as a loading control. The phosphorylation of α-syn at residues Y39 and Y125 was evaluated by densitometry quantification (bottom panel). The band intensities were in the following manner: pY39/[α-syn/actin] or pY125/[α-syn/actin]. The bars represent the mean ± SD of three independent experiments. ** P

    Journal: Human Molecular Genetics

    Article Title: c-Abl phosphorylates α-synuclein and regulates its degradation: implication for α-synuclein clearance and contribution to the pathogenesis of Parkinson's disease

    doi: 10.1093/hmg/ddt674

    Figure Lengend Snippet: c-Abl induces α-syn phosphorylation at Y39 and Y125 residues in HEK293T cells. ( A ) HEK293T cells were transfected with WT α-syn or its mutants (Y39F, Y125F or Y39FY125F) together with plasmids encoding for LUC (negative control) or WT c-Abl. Twenty-four hours post-transfection, cells were lysed and the proteins separated using SDS–PAGE and detected by WB analysis (top panel). α-syn phosphorylation status was assessed using phosphorylation site-specific antibodies for Y39 or Y125 residues. α-syn and c-Abl expression were confirmed in an additional protein blot using specific antibodies. Actin was used as a loading control. The phosphorylation of α-syn at residues Y39 and Y125 was evaluated by densitometry quantification (bottom panel). The band intensities were in the following manner: pY39/[α-syn/actin] or pY125/[α-syn/actin]. The bars represent the mean ± SD of three independent experiments. ** P

    Article Snippet: The sections were incubated overnight at 4°C ( ) with the following antibodies: rabbit polyclonal anti-c-Abl (K-12), rabbit polyclonal anti-pY39-α-syn, rabbit anti-pY125-α-syn, FL α-syn rabbit polyclonal (Millipore) followed by tagged secondary antibody, avidin biotin HRP and diaminobenzidine reactivity.

    Techniques: Transfection, Negative Control, SDS Page, Western Blot, Expressing

    α-syn protein level does not increase in cortical neurons infected by c-Abl. ( A ) Cortical neurons were infected with lentiviruses for GFP or α-syn (top panel). After 5 days, neurons were lysed in Laemmli buffer 2× and the proteins were separated using SDS–PAGE. The expression levels of α-syn and c-Abl were assessed by WB. Actin was used as a loading control. c-Abl protein level was evaluated by densitometry quantification (bottom panel). The band intensities were normalized in the following manner: (c-Abl/actin). The bars represent the mean ± SD of three independent experiments. P > 0.05 (Student's t -test: GFP versus α-syn infected cells). ( B ) Cortical neurons were infected with lentiviruses for GFP, WT, KD or PP c-Abl. After 5 days, cells were lysed in Laemmli buffer 2× and the proteins were separated by SDS–PAGE. α-syn and c-Abl expression levels were assessed by WB (left hand panel). α-syn phosphorylation status was also confirmed using anti-pY39 or pY125 antibodies in a separate protein blot. Actin was used as a loading control. The α-syn protein levels were evaluated by densitometry quantification (right-hand-side panels). The band intensities were normalized in the following manner: (α-syn/actin). The bars represent the mean ± SD of three independent experiments. P > 0.05 (Student's t -test:GFP versus c-Abl infected cells); * P

    Journal: Human Molecular Genetics

    Article Title: c-Abl phosphorylates α-synuclein and regulates its degradation: implication for α-synuclein clearance and contribution to the pathogenesis of Parkinson's disease

    doi: 10.1093/hmg/ddt674

    Figure Lengend Snippet: α-syn protein level does not increase in cortical neurons infected by c-Abl. ( A ) Cortical neurons were infected with lentiviruses for GFP or α-syn (top panel). After 5 days, neurons were lysed in Laemmli buffer 2× and the proteins were separated using SDS–PAGE. The expression levels of α-syn and c-Abl were assessed by WB. Actin was used as a loading control. c-Abl protein level was evaluated by densitometry quantification (bottom panel). The band intensities were normalized in the following manner: (c-Abl/actin). The bars represent the mean ± SD of three independent experiments. P > 0.05 (Student's t -test: GFP versus α-syn infected cells). ( B ) Cortical neurons were infected with lentiviruses for GFP, WT, KD or PP c-Abl. After 5 days, cells were lysed in Laemmli buffer 2× and the proteins were separated by SDS–PAGE. α-syn and c-Abl expression levels were assessed by WB (left hand panel). α-syn phosphorylation status was also confirmed using anti-pY39 or pY125 antibodies in a separate protein blot. Actin was used as a loading control. The α-syn protein levels were evaluated by densitometry quantification (right-hand-side panels). The band intensities were normalized in the following manner: (α-syn/actin). The bars represent the mean ± SD of three independent experiments. P > 0.05 (Student's t -test:GFP versus c-Abl infected cells); * P

    Article Snippet: The sections were incubated overnight at 4°C ( ) with the following antibodies: rabbit polyclonal anti-c-Abl (K-12), rabbit polyclonal anti-pY39-α-syn, rabbit anti-pY125-α-syn, FL α-syn rabbit polyclonal (Millipore) followed by tagged secondary antibody, avidin biotin HRP and diaminobenzidine reactivity.

    Techniques: Infection, SDS Page, Expressing, Western Blot

    In vitro DPV-µCFE scans obtained in PBS, 0.1 M; pH 7.4 (LEFT) or in a PBS solution containing a mixture of ascorbic acid (AA) 5 mM; DOPAC, 50 µM; 5HIAA, 25 µM and SRIF 1 mM; in PBS, 0.1 M; pH 7.4. Peak 1: AA at −50 mV; Peak 2: DOPAC at +100 mV; Peak 3: 5HIAA at +300 mV; and Peak 5: SRIF at +800 mV (modified from [ 18 ]).

    Journal: Biosensors

    Article Title: Central [CNS] and Peripheral [Gastric Tissue] Selective Monitoring of Somatostatin (SRIF) with Micro-Sensor and Voltammetry in Rats: Influence of Growth Factors (GH, EGF)

    doi: 10.3390/bios7040053

    Figure Lengend Snippet: In vitro DPV-µCFE scans obtained in PBS, 0.1 M; pH 7.4 (LEFT) or in a PBS solution containing a mixture of ascorbic acid (AA) 5 mM; DOPAC, 50 µM; 5HIAA, 25 µM and SRIF 1 mM; in PBS, 0.1 M; pH 7.4. Peak 1: AA at −50 mV; Peak 2: DOPAC at +100 mV; Peak 3: 5HIAA at +300 mV; and Peak 5: SRIF at +800 mV (modified from [ 18 ]).

    Article Snippet: Each antrum was then divided, and each part was incubated during 120 min with: (1) Vehicle (PBS), or (2) Antibodies for SRIF (rabbit polyclonal, IgG antiSRIF AB5494); Millipore (MERCK S.p.A., Vimodrone, Milan, Italy) or with non-specific antibodies as described by Funato, et al. [ ], or (3) Cysteamine 1 mM, or (4) Epidermal growth factor (EGF) 1 mM.

    Techniques: In Vitro, Modification

    Typical DPVoltammograms monitored in the striatum of anesthetized rats following local injection of SRIF (top, n = 1), SRIF antisera (S.A.), i.e., antibodies for SRIF (rabbit polyclonal, IgG antiSRIF AB5494); Millipore (MERCK) (middle, n = 1); or control antisera (C.A.), i.e., non specific antibodies in the striatum of a single animal (bottom, n = 1). See Table 2 for data obtained in groups of rats treated as above (n = 7 each treatment), as well as with NaCl 0.9% (control group, n = 9), Bacitracin (n = 5), GH (n = 5) and Cysteamine (n = 5).

    Journal: Biosensors

    Article Title: Central [CNS] and Peripheral [Gastric Tissue] Selective Monitoring of Somatostatin (SRIF) with Micro-Sensor and Voltammetry in Rats: Influence of Growth Factors (GH, EGF)

    doi: 10.3390/bios7040053

    Figure Lengend Snippet: Typical DPVoltammograms monitored in the striatum of anesthetized rats following local injection of SRIF (top, n = 1), SRIF antisera (S.A.), i.e., antibodies for SRIF (rabbit polyclonal, IgG antiSRIF AB5494); Millipore (MERCK) (middle, n = 1); or control antisera (C.A.), i.e., non specific antibodies in the striatum of a single animal (bottom, n = 1). See Table 2 for data obtained in groups of rats treated as above (n = 7 each treatment), as well as with NaCl 0.9% (control group, n = 9), Bacitracin (n = 5), GH (n = 5) and Cysteamine (n = 5).

    Article Snippet: Each antrum was then divided, and each part was incubated during 120 min with: (1) Vehicle (PBS), or (2) Antibodies for SRIF (rabbit polyclonal, IgG antiSRIF AB5494); Millipore (MERCK S.p.A., Vimodrone, Milan, Italy) or with non-specific antibodies as described by Funato, et al. [ ], or (3) Cysteamine 1 mM, or (4) Epidermal growth factor (EGF) 1 mM.

    Techniques: Injection

    ( A ) Left: DPV-µCFE scans obtained in ( a ) vehicle (PBS); ( b ) in the gastric tissue of a single animal incubated with vehicle; ( c ) in the gastric tissue of a single animal incubated with aspecific IgG antisera (asp IgG). Note that the oxidation signal monitored at approximately .8 V (i.e., 800 mV) and measuring approximately 60 nanoAmperes (nA) superimposes the signal detected in the gastric tissue incubated with vehicle. Right, data obtained in antral preparation from 5 rats, incubated with vehicle or aspecific IgG antisera [asp IgG]; ( B ) Left: DPV-µCFE scans obtained in ( a ) vehicle (PBS); ( b ) in the gastric tissue of a single animal incubated with vehicle; ( c ) in the gastric tissue of a single animal incubated with specific IgG antisera (SRIF IgG); note that the peak monitored at +800 mV (denoted as peak 3 in this figure) and having a size of approximately 60 nA is no longer detected following incubation with specific IgG antiSRIF [peaks 1 and 2 are detected at lower oxidation potential, i.e., +200 or +400 mV, respectively, and are not affected by SRIF IgG]. Right, data obtained in antral preparation from 5 rats, incubated with vehicle or specific IgG antisera; ( C ) Left: DPV-µCFE scans obtained in ( a ) vehicle (PBS); ( b ) in the gastric tissue of a single animal incubated with vehicle; ( c ) in the gastric tissue of a single animal incubated with cysteamine; note that the peak monitored at +800 mV is greatly decreased. Right, data obtained in antral preparation from 5 rats, incubated with vehicle or cysteamine; ( D ) Left: DPV-µCFE scans obtained in ( a ) vehicle (PBS); ( b ) in the gastric tissue of a single animal incubated with vehicle; ( c ) in the gastric tissue of a single animal incubated with EGF: note that the peak monitored at +800 mV is significantly increased, i.e., from approximately 60 nA to approximately 85 nA.

    Journal: Biosensors

    Article Title: Central [CNS] and Peripheral [Gastric Tissue] Selective Monitoring of Somatostatin (SRIF) with Micro-Sensor and Voltammetry in Rats: Influence of Growth Factors (GH, EGF)

    doi: 10.3390/bios7040053

    Figure Lengend Snippet: ( A ) Left: DPV-µCFE scans obtained in ( a ) vehicle (PBS); ( b ) in the gastric tissue of a single animal incubated with vehicle; ( c ) in the gastric tissue of a single animal incubated with aspecific IgG antisera (asp IgG). Note that the oxidation signal monitored at approximately .8 V (i.e., 800 mV) and measuring approximately 60 nanoAmperes (nA) superimposes the signal detected in the gastric tissue incubated with vehicle. Right, data obtained in antral preparation from 5 rats, incubated with vehicle or aspecific IgG antisera [asp IgG]; ( B ) Left: DPV-µCFE scans obtained in ( a ) vehicle (PBS); ( b ) in the gastric tissue of a single animal incubated with vehicle; ( c ) in the gastric tissue of a single animal incubated with specific IgG antisera (SRIF IgG); note that the peak monitored at +800 mV (denoted as peak 3 in this figure) and having a size of approximately 60 nA is no longer detected following incubation with specific IgG antiSRIF [peaks 1 and 2 are detected at lower oxidation potential, i.e., +200 or +400 mV, respectively, and are not affected by SRIF IgG]. Right, data obtained in antral preparation from 5 rats, incubated with vehicle or specific IgG antisera; ( C ) Left: DPV-µCFE scans obtained in ( a ) vehicle (PBS); ( b ) in the gastric tissue of a single animal incubated with vehicle; ( c ) in the gastric tissue of a single animal incubated with cysteamine; note that the peak monitored at +800 mV is greatly decreased. Right, data obtained in antral preparation from 5 rats, incubated with vehicle or cysteamine; ( D ) Left: DPV-µCFE scans obtained in ( a ) vehicle (PBS); ( b ) in the gastric tissue of a single animal incubated with vehicle; ( c ) in the gastric tissue of a single animal incubated with EGF: note that the peak monitored at +800 mV is significantly increased, i.e., from approximately 60 nA to approximately 85 nA.

    Article Snippet: Each antrum was then divided, and each part was incubated during 120 min with: (1) Vehicle (PBS), or (2) Antibodies for SRIF (rabbit polyclonal, IgG antiSRIF AB5494); Millipore (MERCK S.p.A., Vimodrone, Milan, Italy) or with non-specific antibodies as described by Funato, et al. [ ], or (3) Cysteamine 1 mM, or (4) Epidermal growth factor (EGF) 1 mM.

    Techniques: Incubation

    B6 SLy1− NK cells demonstrate multiple functional defects. (A) Clearance of RMA lymphoma expressing the NKG2D ligand Rae-1 from lungs of B6 wt or B6 SLy1− mice 18 h after i.v. injection. Comparison performed by unpaired t-test. (B) Growth of LLC injected into the flank of B6 SLy1− (red line) and B6 wt mice (black line) in untreated mice (left), those depleted of CD4 + and CD8 + lymphocytes (middle) or NK1.1 + cells (right). Comparison performed by unpaired t-test at each time point. Isotype control-treated animals demonstrated tumor growth identical to unmanipulated mice (data not shown). (C) In vitro lysis of LLC at a 50:1 effector:target ratio. Data representative of four separate experiments with comparison performed by unpaired t-test. (D) Average number of durable contacts between B6 SLy1− (top) and B6 wt (bottom) NK (crossed to express tomato cherry red on NK1.1 promoter 56 ) cells with GFP-expressing LLC injected into the lung. Yellow arrow points to 4 B6 wt NK cells establishing contact with one tumor cell. Data representative of three separate mice per group with comparison performed by unpaired t-test. (E) In vivo clearance of CFSE labeled B6 CD45.1+ wild-type or B6 CD45.2+ m157-expressing splenocytes injected into B6 SLy1− (top) and B6 wt mice (bottom) recipient mice. Data derived from flow cytometric analysis of the spleen, demonstrating one representative experiment on the left and summary of five animals on the right compared by unpaired t-test. (F) Degranulation of B6 SLy1− (red) and B6 wt (black) NK cells after plate bound stimulation with NKp46 as measured by surface CD107a expression. Left panel representative of one experiment with heavy black line representative of Cd107a in B6 wt NK cells after co-culture with plate bound NKp46 and heavy red line representative of B6 SLy1− NK cells after co-culture with plate bound NKp46. Shaded black line representative of plate-bound isotype control. Comparison performed by unpaired t-test. Summary of five separate experiments. * p

    Journal: Oncoimmunology

    Article Title: Deficiency of the adaptor protein SLy1 results in a natural killer cell ribosomopathy affecting tumor clearance

    doi: 10.1080/2162402X.2016.1238543

    Figure Lengend Snippet: B6 SLy1− NK cells demonstrate multiple functional defects. (A) Clearance of RMA lymphoma expressing the NKG2D ligand Rae-1 from lungs of B6 wt or B6 SLy1− mice 18 h after i.v. injection. Comparison performed by unpaired t-test. (B) Growth of LLC injected into the flank of B6 SLy1− (red line) and B6 wt mice (black line) in untreated mice (left), those depleted of CD4 + and CD8 + lymphocytes (middle) or NK1.1 + cells (right). Comparison performed by unpaired t-test at each time point. Isotype control-treated animals demonstrated tumor growth identical to unmanipulated mice (data not shown). (C) In vitro lysis of LLC at a 50:1 effector:target ratio. Data representative of four separate experiments with comparison performed by unpaired t-test. (D) Average number of durable contacts between B6 SLy1− (top) and B6 wt (bottom) NK (crossed to express tomato cherry red on NK1.1 promoter 56 ) cells with GFP-expressing LLC injected into the lung. Yellow arrow points to 4 B6 wt NK cells establishing contact with one tumor cell. Data representative of three separate mice per group with comparison performed by unpaired t-test. (E) In vivo clearance of CFSE labeled B6 CD45.1+ wild-type or B6 CD45.2+ m157-expressing splenocytes injected into B6 SLy1− (top) and B6 wt mice (bottom) recipient mice. Data derived from flow cytometric analysis of the spleen, demonstrating one representative experiment on the left and summary of five animals on the right compared by unpaired t-test. (F) Degranulation of B6 SLy1− (red) and B6 wt (black) NK cells after plate bound stimulation with NKp46 as measured by surface CD107a expression. Left panel representative of one experiment with heavy black line representative of Cd107a in B6 wt NK cells after co-culture with plate bound NKp46 and heavy red line representative of B6 SLy1− NK cells after co-culture with plate bound NKp46. Shaded black line representative of plate-bound isotype control. Comparison performed by unpaired t-test. Summary of five separate experiments. * p

    Article Snippet: Immunoprecipitation was performed on wild-type littermate NK cells using SLy1 Ab (rabbit polyclonal custom-generated by Squarix) or anti-Mdm2 (clone 2A10)(Millipore) cross-linked to magnetic bead immunoprecipitation with Dynabead Protein G Immunoprecipitation kit according to manufacturer instructions (Life Technologies).

    Techniques: Functional Assay, Expressing, Mouse Assay, Injection, In Vitro, Lysis, In Vivo, Labeling, Derivative Assay, Flow Cytometry, Co-Culture Assay

    In the absence of SLy1 NK cells upregulate p53. (A) Western blot analysis of freshly isolated NK cells for Mdm2, PRL5 and p53. Left panel representative of one Western blot and right panel demonstrated relative ratios of blot intensities from 5–7 separate experiments. (B) Western blot analysis of Mdm2 co-immunoprecipitates of freshly isolated NK cells for Mdm2, PRL5 and p53. Left panel representative of one Western blot and right panel demonstrated relative ratios of blot intensities from 5–6 separate experiments. Statistical comparison performed by Mann–Whitney U test to the null hypothesis assuming relative ratio to equal 1. 30% of NK cell lysate loaded for Western blot analysis of input lysate as described in panel (A) and 70% used for Mdm2 Co-IP as demonstrated in panel (B). * p

    Journal: Oncoimmunology

    Article Title: Deficiency of the adaptor protein SLy1 results in a natural killer cell ribosomopathy affecting tumor clearance

    doi: 10.1080/2162402X.2016.1238543

    Figure Lengend Snippet: In the absence of SLy1 NK cells upregulate p53. (A) Western blot analysis of freshly isolated NK cells for Mdm2, PRL5 and p53. Left panel representative of one Western blot and right panel demonstrated relative ratios of blot intensities from 5–7 separate experiments. (B) Western blot analysis of Mdm2 co-immunoprecipitates of freshly isolated NK cells for Mdm2, PRL5 and p53. Left panel representative of one Western blot and right panel demonstrated relative ratios of blot intensities from 5–6 separate experiments. Statistical comparison performed by Mann–Whitney U test to the null hypothesis assuming relative ratio to equal 1. 30% of NK cell lysate loaded for Western blot analysis of input lysate as described in panel (A) and 70% used for Mdm2 Co-IP as demonstrated in panel (B). * p

    Article Snippet: Immunoprecipitation was performed on wild-type littermate NK cells using SLy1 Ab (rabbit polyclonal custom-generated by Squarix) or anti-Mdm2 (clone 2A10)(Millipore) cross-linked to magnetic bead immunoprecipitation with Dynabead Protein G Immunoprecipitation kit according to manufacturer instructions (Life Technologies).

    Techniques: Western Blot, Isolation, MANN-WHITNEY, Co-Immunoprecipitation Assay

    Gene expression and viability of splenic NK Cells. (A) Relative mRNA expression of NK activating receptors in freshly isolated B6 SLy1− and B6 wt NK cells. (B) Viability, defined as 7AAD − Annexin − , NK cells in the spleen of B6 SLy1− (red) and B6 wt mice (black). Representative gated flow cytometry plots on the left and summary of data (middle). Comparison performed by unpaired t-test. Electron microscopy of cytospin preparations of splenic B6 wt and B6 Sly1− NK cells. Arrows point to apoptotic cells (right).

    Journal: Oncoimmunology

    Article Title: Deficiency of the adaptor protein SLy1 results in a natural killer cell ribosomopathy affecting tumor clearance

    doi: 10.1080/2162402X.2016.1238543

    Figure Lengend Snippet: Gene expression and viability of splenic NK Cells. (A) Relative mRNA expression of NK activating receptors in freshly isolated B6 SLy1− and B6 wt NK cells. (B) Viability, defined as 7AAD − Annexin − , NK cells in the spleen of B6 SLy1− (red) and B6 wt mice (black). Representative gated flow cytometry plots on the left and summary of data (middle). Comparison performed by unpaired t-test. Electron microscopy of cytospin preparations of splenic B6 wt and B6 Sly1− NK cells. Arrows point to apoptotic cells (right).

    Article Snippet: Immunoprecipitation was performed on wild-type littermate NK cells using SLy1 Ab (rabbit polyclonal custom-generated by Squarix) or anti-Mdm2 (clone 2A10)(Millipore) cross-linked to magnetic bead immunoprecipitation with Dynabead Protein G Immunoprecipitation kit according to manufacturer instructions (Life Technologies).

    Techniques: Expressing, Isolation, Mouse Assay, Flow Cytometry, Cytometry, Electron Microscopy

    Phenotype of activated B6 SLy1− and B6 wt NK cells. (A) MCMV and m157 − MCMV viral titers in the spleens of B6 SLy1− (red) and B6 wt (black) mice seventy-two hours after infection. (B) In vivo clearance of B6 CD45.1+ wild-type or B6 CD45.2+ m157-expressing splenocytes injected into IL-2 treated B6 SLy1− (top) and B6 wt mice (bottom) recipient mice. Data derived from flow cytometric analysis of the spleen, demonstrating one representative experiment on the left and summary of five animals on the right. Comparison performed by unpaired t-test between ratios. (C) In vitro lysis of LLC at a 3.125:1 effector:target ratio with NK cells isolated from the spleens of B6 SLy1− (red) or B6 wt (black) mice and expanded in high dose IL-2 for one week in vitro . (D) Growth of LLC injected into the flank of B6 SLy1− (red line) and B6 wt mice (black line) treated with 10 doses of 75,000 IU of wild-type IL-2. Representative of two separate experiments with four mice per group per experiment. (E) Comparison of p53 levels in resting and IL-2 activated B6 SLy1− and B6 wt NK cells (representative of two separate experiments). (F) Relative mRNA expression of various activating receptors, adhesion molecules and signaling intermediates in IL-2 activated NK cells from B6 SLy1− and B6 wt mice.

    Journal: Oncoimmunology

    Article Title: Deficiency of the adaptor protein SLy1 results in a natural killer cell ribosomopathy affecting tumor clearance

    doi: 10.1080/2162402X.2016.1238543

    Figure Lengend Snippet: Phenotype of activated B6 SLy1− and B6 wt NK cells. (A) MCMV and m157 − MCMV viral titers in the spleens of B6 SLy1− (red) and B6 wt (black) mice seventy-two hours after infection. (B) In vivo clearance of B6 CD45.1+ wild-type or B6 CD45.2+ m157-expressing splenocytes injected into IL-2 treated B6 SLy1− (top) and B6 wt mice (bottom) recipient mice. Data derived from flow cytometric analysis of the spleen, demonstrating one representative experiment on the left and summary of five animals on the right. Comparison performed by unpaired t-test between ratios. (C) In vitro lysis of LLC at a 3.125:1 effector:target ratio with NK cells isolated from the spleens of B6 SLy1− (red) or B6 wt (black) mice and expanded in high dose IL-2 for one week in vitro . (D) Growth of LLC injected into the flank of B6 SLy1− (red line) and B6 wt mice (black line) treated with 10 doses of 75,000 IU of wild-type IL-2. Representative of two separate experiments with four mice per group per experiment. (E) Comparison of p53 levels in resting and IL-2 activated B6 SLy1− and B6 wt NK cells (representative of two separate experiments). (F) Relative mRNA expression of various activating receptors, adhesion molecules and signaling intermediates in IL-2 activated NK cells from B6 SLy1− and B6 wt mice.

    Article Snippet: Immunoprecipitation was performed on wild-type littermate NK cells using SLy1 Ab (rabbit polyclonal custom-generated by Squarix) or anti-Mdm2 (clone 2A10)(Millipore) cross-linked to magnetic bead immunoprecipitation with Dynabead Protein G Immunoprecipitation kit according to manufacturer instructions (Life Technologies).

    Techniques: Mouse Assay, Infection, In Vivo, Expressing, Injection, Derivative Assay, Flow Cytometry, In Vitro, Lysis, Isolation

    Strain-specific differences in cancer susceptibility and SLy1 levels. (A) Strain-specific differences in the number of splenic NK cells (defined as NKp46 + CD3 − ). Graphic data showing one representative FACS plot from three to six animals. (B) Strain-specific differences in in vitro specific lysis of Lewis Lung Carcinoma (LLC) by splenic NK cells at a 25:1 effector:target ratio by 51 Cr-release assay (representative of four separate experiments with comparison performed by unpaired t-test). (C) Structure of SLy1 (top) and relative levels of SLy1 in splenic NK cells of various strains of mice as measured by mRNA gene expression array (bottom). (D) SLy1 levels in freshly isolated splenic NK cells as measured by Western blot analysis (four animals per group). Comparison performed by unpaired t-test. * p

    Journal: Oncoimmunology

    Article Title: Deficiency of the adaptor protein SLy1 results in a natural killer cell ribosomopathy affecting tumor clearance

    doi: 10.1080/2162402X.2016.1238543

    Figure Lengend Snippet: Strain-specific differences in cancer susceptibility and SLy1 levels. (A) Strain-specific differences in the number of splenic NK cells (defined as NKp46 + CD3 − ). Graphic data showing one representative FACS plot from three to six animals. (B) Strain-specific differences in in vitro specific lysis of Lewis Lung Carcinoma (LLC) by splenic NK cells at a 25:1 effector:target ratio by 51 Cr-release assay (representative of four separate experiments with comparison performed by unpaired t-test). (C) Structure of SLy1 (top) and relative levels of SLy1 in splenic NK cells of various strains of mice as measured by mRNA gene expression array (bottom). (D) SLy1 levels in freshly isolated splenic NK cells as measured by Western blot analysis (four animals per group). Comparison performed by unpaired t-test. * p

    Article Snippet: Immunoprecipitation was performed on wild-type littermate NK cells using SLy1 Ab (rabbit polyclonal custom-generated by Squarix) or anti-Mdm2 (clone 2A10)(Millipore) cross-linked to magnetic bead immunoprecipitation with Dynabead Protein G Immunoprecipitation kit according to manufacturer instructions (Life Technologies).

    Techniques: FACS, In Vitro, Lysis, Release Assay, Mouse Assay, Expressing, Isolation, Western Blot

    B6 SLy1− NK cells demonstrate multiple phenotypic defects. (A) Quantification of splenic NK cells defined as either NK1.1 + CD3 − or NKp46 + CD3 − . Comparison performed by unpaired t-test. (B) Comparison of four-stage maturation program of splenic NK1.1 + CD3 − NK cells in B6 SLy1− and B6 wt mice as defined by surface expression of CD27 and CD11b. Representative flow cytometry plots at the top and summary data of five separate experiments at the bottom. (C) Expression of NK1.1, CD122, Tbet and EOMES in B6 SLy1− and B6 wt splenic CD3 − DX5 + NK cells. (D) Representative histograms and relative ratios (right upper corner) of NKG2D, NKp46, NK1.1 and Ly49H in DX5 + CD3 − splenic NK cells in in B6 SLy− and B6 wt mice. Representative of five separate experiments performed on different animals from different litters. Statistical analysis performed by unpaired t-test to the null hypothesis considering the relative ratio to be 1. (E) Representative histograms and relative ratios (right upper corner) of NKG2D, NKp46, NK1.1 and Ly49H in DX5 + CD3 − splenic NK cells in in B6 SLy1− and B6 wt mice after activation in vitro with 1,000 IU/mL of IL-2. Statistical analysis of (D) and (E) performed by unpaired t-test to the null hypothesis which assumes the relative ratio to be 1.

    Journal: Oncoimmunology

    Article Title: Deficiency of the adaptor protein SLy1 results in a natural killer cell ribosomopathy affecting tumor clearance

    doi: 10.1080/2162402X.2016.1238543

    Figure Lengend Snippet: B6 SLy1− NK cells demonstrate multiple phenotypic defects. (A) Quantification of splenic NK cells defined as either NK1.1 + CD3 − or NKp46 + CD3 − . Comparison performed by unpaired t-test. (B) Comparison of four-stage maturation program of splenic NK1.1 + CD3 − NK cells in B6 SLy1− and B6 wt mice as defined by surface expression of CD27 and CD11b. Representative flow cytometry plots at the top and summary data of five separate experiments at the bottom. (C) Expression of NK1.1, CD122, Tbet and EOMES in B6 SLy1− and B6 wt splenic CD3 − DX5 + NK cells. (D) Representative histograms and relative ratios (right upper corner) of NKG2D, NKp46, NK1.1 and Ly49H in DX5 + CD3 − splenic NK cells in in B6 SLy− and B6 wt mice. Representative of five separate experiments performed on different animals from different litters. Statistical analysis performed by unpaired t-test to the null hypothesis considering the relative ratio to be 1. (E) Representative histograms and relative ratios (right upper corner) of NKG2D, NKp46, NK1.1 and Ly49H in DX5 + CD3 − splenic NK cells in in B6 SLy1− and B6 wt mice after activation in vitro with 1,000 IU/mL of IL-2. Statistical analysis of (D) and (E) performed by unpaired t-test to the null hypothesis which assumes the relative ratio to be 1.

    Article Snippet: Immunoprecipitation was performed on wild-type littermate NK cells using SLy1 Ab (rabbit polyclonal custom-generated by Squarix) or anti-Mdm2 (clone 2A10)(Millipore) cross-linked to magnetic bead immunoprecipitation with Dynabead Protein G Immunoprecipitation kit according to manufacturer instructions (Life Technologies).

    Techniques: Mouse Assay, Expressing, Flow Cytometry, Cytometry, Activation Assay, In Vitro

    SLy1 contributes to ribosome stability in NK cells. (A) Mass spectroscopy of anti-SLy1 co-immunoprecipitation of B6 wt and B6 SLy1− NK cells. Proteins quantitated by spectral counts of peptides. Ribosomal proteins associated with the small 40S subunit are defined as RPS (ribosomal protein small), with the appropriate protein number after the RPS designation. Ribosomal proteins associated with the large 60S subunit are defined as RPL (ribosomal protein large), with the appropriate protein number after the RPL designation. (B) Sucrose density gradient fractionation and Western blot analysis of SLy1 localization of freshly isolated NK cells (left panel) and T lymphocytes (right panel). Representative of four separate experiments. (C) Western blot analysis of cytoplasm and nucleolar SLy1 in the KY1.1 NK cell line utilizing nucleophosmin (NPM) and superoxide dismutase (SOD) for nucleolar and cytoplasmic localization, respectively. (D) Spectral counts of free ribosomal components, not associated with the ribosome, in B6 SLy1− in B6 wt NK cells by mass spectroscopy. Comparison performed by t-test. (E) Sucrose density gradient fractionation with A254 absorbance evaluation of ribosome formation in freshly-isolated B6 wt (black) and B6 SLy1− (red) NK cells. Representative of three separate experiments. (F) RT-PCR of 18S rRNA expression in freshly isolated NK cells from B6 wt (black) and B6 SLy1− (red) mice. (G) RT-PCR analysis of 47S rRNA copy number in freshly isolated NK cells from B6 wt (black) and B6 SLy1− (red) mice. Comparison performed by unpaired t-test. (H) RT-PCR of 18S rRNA levels in freshly isolated T cells, B cells and NK cells from B6 mice. Expression normalized to β-actin. Comparison performed by One-way ANOVA followed by Bonferroni post-test. * p

    Journal: Oncoimmunology

    Article Title: Deficiency of the adaptor protein SLy1 results in a natural killer cell ribosomopathy affecting tumor clearance

    doi: 10.1080/2162402X.2016.1238543

    Figure Lengend Snippet: SLy1 contributes to ribosome stability in NK cells. (A) Mass spectroscopy of anti-SLy1 co-immunoprecipitation of B6 wt and B6 SLy1− NK cells. Proteins quantitated by spectral counts of peptides. Ribosomal proteins associated with the small 40S subunit are defined as RPS (ribosomal protein small), with the appropriate protein number after the RPS designation. Ribosomal proteins associated with the large 60S subunit are defined as RPL (ribosomal protein large), with the appropriate protein number after the RPL designation. (B) Sucrose density gradient fractionation and Western blot analysis of SLy1 localization of freshly isolated NK cells (left panel) and T lymphocytes (right panel). Representative of four separate experiments. (C) Western blot analysis of cytoplasm and nucleolar SLy1 in the KY1.1 NK cell line utilizing nucleophosmin (NPM) and superoxide dismutase (SOD) for nucleolar and cytoplasmic localization, respectively. (D) Spectral counts of free ribosomal components, not associated with the ribosome, in B6 SLy1− in B6 wt NK cells by mass spectroscopy. Comparison performed by t-test. (E) Sucrose density gradient fractionation with A254 absorbance evaluation of ribosome formation in freshly-isolated B6 wt (black) and B6 SLy1− (red) NK cells. Representative of three separate experiments. (F) RT-PCR of 18S rRNA expression in freshly isolated NK cells from B6 wt (black) and B6 SLy1− (red) mice. (G) RT-PCR analysis of 47S rRNA copy number in freshly isolated NK cells from B6 wt (black) and B6 SLy1− (red) mice. Comparison performed by unpaired t-test. (H) RT-PCR of 18S rRNA levels in freshly isolated T cells, B cells and NK cells from B6 mice. Expression normalized to β-actin. Comparison performed by One-way ANOVA followed by Bonferroni post-test. * p

    Article Snippet: Immunoprecipitation was performed on wild-type littermate NK cells using SLy1 Ab (rabbit polyclonal custom-generated by Squarix) or anti-Mdm2 (clone 2A10)(Millipore) cross-linked to magnetic bead immunoprecipitation with Dynabead Protein G Immunoprecipitation kit according to manufacturer instructions (Life Technologies).

    Techniques: Mass Spectrometry, Immunoprecipitation, Fractionation, Western Blot, Isolation, Reverse Transcription Polymerase Chain Reaction, Expressing, Mouse Assay