peroxidase conjugated streptavidin  (Thermo Fisher)


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    Structured Review

    Thermo Fisher peroxidase conjugated streptavidin
    In vitro selection and binding of aptamers. ( a ) Schematic representation of the SELEX process used within this study. ( b ) Schematic illustration of the enzyme linked apta-sorbent assay (ELASA) setup. ( c ) Folded biotinylated aptamers (RA1-RA9, c = 500 nM) were tested for rituximab binding. Bound aptamers were detected by <t>streptavidin-HRP,</t> chemiluminescence ELISA substrate was used for detection and luminescence was measured. Measurements were performed in six replicates; means and standard errors of the mean are given.
    Peroxidase Conjugated Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 1041 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Aptamers as quality control tool for production, storage and biosimilarity of the anti-CD20 biopharmaceutical rituximab"

    Article Title: Aptamers as quality control tool for production, storage and biosimilarity of the anti-CD20 biopharmaceutical rituximab

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-37624-1

    In vitro selection and binding of aptamers. ( a ) Schematic representation of the SELEX process used within this study. ( b ) Schematic illustration of the enzyme linked apta-sorbent assay (ELASA) setup. ( c ) Folded biotinylated aptamers (RA1-RA9, c = 500 nM) were tested for rituximab binding. Bound aptamers were detected by streptavidin-HRP, chemiluminescence ELISA substrate was used for detection and luminescence was measured. Measurements were performed in six replicates; means and standard errors of the mean are given.
    Figure Legend Snippet: In vitro selection and binding of aptamers. ( a ) Schematic representation of the SELEX process used within this study. ( b ) Schematic illustration of the enzyme linked apta-sorbent assay (ELASA) setup. ( c ) Folded biotinylated aptamers (RA1-RA9, c = 500 nM) were tested for rituximab binding. Bound aptamers were detected by streptavidin-HRP, chemiluminescence ELISA substrate was used for detection and luminescence was measured. Measurements were performed in six replicates; means and standard errors of the mean are given.

    Techniques Used: In Vitro, Selection, Binding Assay, Enzyme-linked Immunosorbent Assay

    2) Product Images from "The Heparan Sulfate Proteoglycan Form of Epithelial CD44v3 Serves as a CD11b/CD18 Counter-receptor during Polymorphonuclear Leukocyte Transepithelial Migration"

    Article Title: The Heparan Sulfate Proteoglycan Form of Epithelial CD44v3 Serves as a CD11b/CD18 Counter-receptor during Polymorphonuclear Leukocyte Transepithelial Migration

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M807805200

    Localization of C3H7 antigen to the basolateral and lateral membranes of epithelium. A , immunofluorescence labeling and confocal microscopy was used to define the pattern of C3H7 antigen expression. Shown here are an X-Z orientation series and an en face image demonstrating nearly complete localization to the basolateral and subapical lateral membranes. B , immunofluorescence labeling of mAb C3H7 in normal human colon tissue sections. Scale bar , 20 μm. C , Western blot analysis of equal amounts of lysates from indicated cell types with mAb C3H7. D , confluent T84 monolayers were biotinylated on the apical ( Ap ) or basolateral ( BL ) surface. Lysates were immunoprecipitated with either C3H7 or isotype-matched control mAb C1F3. Proteins were visualized with streptavidin-HRP to identify only surface biotinylated C3H7 antigen ( upper panel ) or with mAb C3H7 to demonstrate total C3H7 antigen ( lower panel ). The control lane ( lane C ) represents mAb C1F3 immunoprecipitation.
    Figure Legend Snippet: Localization of C3H7 antigen to the basolateral and lateral membranes of epithelium. A , immunofluorescence labeling and confocal microscopy was used to define the pattern of C3H7 antigen expression. Shown here are an X-Z orientation series and an en face image demonstrating nearly complete localization to the basolateral and subapical lateral membranes. B , immunofluorescence labeling of mAb C3H7 in normal human colon tissue sections. Scale bar , 20 μm. C , Western blot analysis of equal amounts of lysates from indicated cell types with mAb C3H7. D , confluent T84 monolayers were biotinylated on the apical ( Ap ) or basolateral ( BL ) surface. Lysates were immunoprecipitated with either C3H7 or isotype-matched control mAb C1F3. Proteins were visualized with streptavidin-HRP to identify only surface biotinylated C3H7 antigen ( upper panel ) or with mAb C3H7 to demonstrate total C3H7 antigen ( lower panel ). The control lane ( lane C ) represents mAb C1F3 immunoprecipitation.

    Techniques Used: Immunofluorescence, Labeling, Confocal Microscopy, Expressing, Western Blot, Immunoprecipitation

    3) Product Images from "Transglutaminase 2 inhibits Rb binding of human papillomavirus E7 by incorporating polyamine"

    Article Title: Transglutaminase 2 inhibits Rb binding of human papillomavirus E7 by incorporating polyamine

    Journal: The EMBO Journal

    doi: 10.1093/emboj/cdg495

    Fig. 4. TGase 2 catalyzes polyamination of HPV18 E7, but not HPV16 E7. ( A ) One hundred micrograms of His-tagged HPV18 E7 and HPV16 E7 were incubated with TGase 2 in the presence of 14 C putrescine. Radioactivity bound to each protein was measured by a liquid scintillation counter. BSA and casein were employed as negative and a positive controls respectively. The figure shows the mean values and SD of three independent experiments. ( B ) HPV18 E7 was incubated with TGase 2 in the presence of 0.1 mM of biotinylated spermine and 0, 0.1, 1 and 10 mM of spermine (lane 1–4). The incorporation of biotinylated spermine was analyzed by dot-blotting using HRP-conjugated streptavidin (SA-HRP). HPV18 E7 was probed with anti-HPV18 E7 antibody. ( C ) The reaction mixtures were subjected to 15% SDS–PAGE and polyaminated HPV E7 was probed with SA-HRP. ( D ) HeLa and Caski cells were treated with 1 mM of biotinylated pentylamine for 1 h in the presence of A23187. Biotinylated pentylamine-incorporated E7s were precipitated by streptavidin-conjugated magnetic beads. Proteins bound to beads were analyzed by western blot using antibodies to HPV18 E7 or HPV16 E7. C33A cells were used as a negative control.
    Figure Legend Snippet: Fig. 4. TGase 2 catalyzes polyamination of HPV18 E7, but not HPV16 E7. ( A ) One hundred micrograms of His-tagged HPV18 E7 and HPV16 E7 were incubated with TGase 2 in the presence of 14 C putrescine. Radioactivity bound to each protein was measured by a liquid scintillation counter. BSA and casein were employed as negative and a positive controls respectively. The figure shows the mean values and SD of three independent experiments. ( B ) HPV18 E7 was incubated with TGase 2 in the presence of 0.1 mM of biotinylated spermine and 0, 0.1, 1 and 10 mM of spermine (lane 1–4). The incorporation of biotinylated spermine was analyzed by dot-blotting using HRP-conjugated streptavidin (SA-HRP). HPV18 E7 was probed with anti-HPV18 E7 antibody. ( C ) The reaction mixtures were subjected to 15% SDS–PAGE and polyaminated HPV E7 was probed with SA-HRP. ( D ) HeLa and Caski cells were treated with 1 mM of biotinylated pentylamine for 1 h in the presence of A23187. Biotinylated pentylamine-incorporated E7s were precipitated by streptavidin-conjugated magnetic beads. Proteins bound to beads were analyzed by western blot using antibodies to HPV18 E7 or HPV16 E7. C33A cells were used as a negative control.

    Techniques Used: Incubation, Radioactivity, SDS Page, Magnetic Beads, Western Blot, Negative Control

    4) Product Images from "Development of viral nanoparticles for efficient intracellular delivery †"

    Article Title: Development of viral nanoparticles for efficient intracellular delivery †

    Journal: Nanoscale

    doi: 10.1039/c2nr30366c

    Characterization of CPMV labeling with the biotinylated R5 peptide. (A) Size exclusion chromatography of wild-type CPMV, CPMV–R5L and CPMV–R5Hat 280 nm. (B) ECL dot blot of purified CPMV particles. The number of biotin labels per particle was determined using standardized biotin concentrations and Chemidoc XRS software. (C) Native gel electrophoresis of intact CPMV particles (10 µg) using a 0.8% (w/v) agarose gel. Particles were visualized under UV light. Lane 1 = CPMV, 2 = CPMV–4FB, 3 = CPMV–R5H, 4 = CPMV–PFB, 5 = CPMV–R5L. (D) SDS–PAGE of CPMV particles (10 µg) using a 4–12% Bis-Tris gel and western blotting using streptavidin–alkaline phosphatase to detect the N-terminal biotin tag of the R5 peptide. (E) Zeta potential of CPMV wild type, CPMV–R5L and CPMV–R5H formulations.
    Figure Legend Snippet: Characterization of CPMV labeling with the biotinylated R5 peptide. (A) Size exclusion chromatography of wild-type CPMV, CPMV–R5L and CPMV–R5Hat 280 nm. (B) ECL dot blot of purified CPMV particles. The number of biotin labels per particle was determined using standardized biotin concentrations and Chemidoc XRS software. (C) Native gel electrophoresis of intact CPMV particles (10 µg) using a 0.8% (w/v) agarose gel. Particles were visualized under UV light. Lane 1 = CPMV, 2 = CPMV–4FB, 3 = CPMV–R5H, 4 = CPMV–PFB, 5 = CPMV–R5L. (D) SDS–PAGE of CPMV particles (10 µg) using a 4–12% Bis-Tris gel and western blotting using streptavidin–alkaline phosphatase to detect the N-terminal biotin tag of the R5 peptide. (E) Zeta potential of CPMV wild type, CPMV–R5L and CPMV–R5H formulations.

    Techniques Used: Labeling, Size-exclusion Chromatography, Dot Blot, Purification, Software, Nucleic Acid Electrophoresis, Agarose Gel Electrophoresis, SDS Page, Western Blot

    5) Product Images from "Maladaptive Sexual Behavior Following Concurrent Methamphetamine and Sexual Experience in Male Rats is Associated with Altered Neural Activity in Frontal Cortex"

    Article Title: Maladaptive Sexual Behavior Following Concurrent Methamphetamine and Sexual Experience in Male Rats is Associated with Altered Neural Activity in Frontal Cortex

    Journal: Neuropsychopharmacology

    doi: 10.1038/npp.2017.1

    Phosphorylated MAP kinase (pERK) is localized in CaMKII neurons in medial prefrontal cortex (mPFC) and orbitofrontal cortex (OFC). (a) Numbers of pERK-immunofluorescent neurons per mm 2 (mean±SEM) in mPFC subregions (ACA, PL, IL) and OFC in males (from experiment 2) pretreated with Saline/sex (saline), or Meth/sex (Meth) and removed from home cage (control; white bars; n =4 saline and n =4 Meth) or exposed to the female (female; black bars; n =4 saline and n =3 Meth). (b) Numbers of neurons co-labeled for pERK and CaMKII per mm 2 (mean±SEM). (c–k) Representative images illustrating the co-localization (c, f; arrows indicate dual labeled neurons) of pERK (green; d,g,j) and CaMKII (red; e,h) and the lack of co-localization (i; arrow heads indicate pERK single-labeled neurons) of pERK (green; j) and GAD (red; k) in PL of representative Meth+Sex Home Cage control (c–e), or Saline/sex exposed to female (f–k) males. Scale bar indicates 50 μm.
    Figure Legend Snippet: Phosphorylated MAP kinase (pERK) is localized in CaMKII neurons in medial prefrontal cortex (mPFC) and orbitofrontal cortex (OFC). (a) Numbers of pERK-immunofluorescent neurons per mm 2 (mean±SEM) in mPFC subregions (ACA, PL, IL) and OFC in males (from experiment 2) pretreated with Saline/sex (saline), or Meth/sex (Meth) and removed from home cage (control; white bars; n =4 saline and n =4 Meth) or exposed to the female (female; black bars; n =4 saline and n =3 Meth). (b) Numbers of neurons co-labeled for pERK and CaMKII per mm 2 (mean±SEM). (c–k) Representative images illustrating the co-localization (c, f; arrows indicate dual labeled neurons) of pERK (green; d,g,j) and CaMKII (red; e,h) and the lack of co-localization (i; arrow heads indicate pERK single-labeled neurons) of pERK (green; j) and GAD (red; k) in PL of representative Meth+Sex Home Cage control (c–e), or Saline/sex exposed to female (f–k) males. Scale bar indicates 50 μm.

    Techniques Used: Labeling

    Representative images of pERK expression in home cage control males (a,c,e,g) and following exposure to the conditioned aversive cue, ie, the female (b,d,f,h) in experiment 1. CeA, central amygdala; cc, corpus callosum; CPu, caudate putamen; LV, lateral ventricle; mPFC, medial prefrontal cortex; OFC, orbitofrontal cortex (l: lateral; m: medial); st, stria terminalis. Circle in h indicates location of CeA. Scale bar indicates 200 μm.
    Figure Legend Snippet: Representative images of pERK expression in home cage control males (a,c,e,g) and following exposure to the conditioned aversive cue, ie, the female (b,d,f,h) in experiment 1. CeA, central amygdala; cc, corpus callosum; CPu, caudate putamen; LV, lateral ventricle; mPFC, medial prefrontal cortex; OFC, orbitofrontal cortex (l: lateral; m: medial); st, stria terminalis. Circle in h indicates location of CeA. Scale bar indicates 200 μm.

    Techniques Used: Expressing

    6) Product Images from "Overexpression of platelet-derived growth factor receptor ? in breast cancer is associated with tumour progression"

    Article Title: Overexpression of platelet-derived growth factor receptor ? in breast cancer is associated with tumour progression

    Journal: Breast Cancer Research

    doi: 10.1186/bcr1304

    PDGFR-α expression in invasive breast carcinomas by immunohistochemistry (streptavidin-biotin-peroxidase). (a) Platelet-derived growth factor receptor α (PDGFR-α) expression in pericytes and smooth muscle cells of a blood vessel: internal control (original magnification × 200); (b) Absence of PDGFR-α expression in neoplastic cells (original magnification × 200); (c) PDGFR-α diffuse cytoplasmic expression in neoplastic cells (original magnification × 200; inset × 400); (d) Neoplastic cells showing strong and diffuse cytoplasmic PDGFR-α expression (original magnification × 400).
    Figure Legend Snippet: PDGFR-α expression in invasive breast carcinomas by immunohistochemistry (streptavidin-biotin-peroxidase). (a) Platelet-derived growth factor receptor α (PDGFR-α) expression in pericytes and smooth muscle cells of a blood vessel: internal control (original magnification × 200); (b) Absence of PDGFR-α expression in neoplastic cells (original magnification × 200); (c) PDGFR-α diffuse cytoplasmic expression in neoplastic cells (original magnification × 200; inset × 400); (d) Neoplastic cells showing strong and diffuse cytoplasmic PDGFR-α expression (original magnification × 400).

    Techniques Used: Expressing, Immunohistochemistry, Derivative Assay

    7) Product Images from "Follicular helper T cells are required for systemic autoimmunity"

    Article Title: Follicular helper T cells are required for systemic autoimmunity

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20081886

    Lack of IL-21 does not affect the phenotype, T FH cell accumulation, or GC formation of Roquin san/san mice. (a) IgG ANAs in the serum of mice of the genotypes indicated, detected by immunofluorescence using Hep-2 substrate. Data are representative of three independent experiments ( n ≥ 5 mice per group). (b) Score of ANA staining intensity by confocal microscopy from sera taken from mice of the indicated genotypes. Data are representative of three independent experiments ( n ≥ 5 mice per group). (c) Basal serum total IgG, IgG1, and IgE measured by ELISA. Data are representative of two independent experiments ( n = 5 mice per group). (d) Lymph node and spleen weight in grams for mice of the indicated genotypes. Data are representative of two independent experiments ( n ≥ 5 per group). (e) Flow cytometric contour plots and dot plots of PD-1 high CXCR5 + CD4 + T FH cells and (f) GL-7 + CD95 + B220 + GC cells from mice of the indicated genotypes. Data are representative of two independent experiments ( n ≥ 5 mice per group). In e and f, the numbers in the plots represent percentages.
    Figure Legend Snippet: Lack of IL-21 does not affect the phenotype, T FH cell accumulation, or GC formation of Roquin san/san mice. (a) IgG ANAs in the serum of mice of the genotypes indicated, detected by immunofluorescence using Hep-2 substrate. Data are representative of three independent experiments ( n ≥ 5 mice per group). (b) Score of ANA staining intensity by confocal microscopy from sera taken from mice of the indicated genotypes. Data are representative of three independent experiments ( n ≥ 5 mice per group). (c) Basal serum total IgG, IgG1, and IgE measured by ELISA. Data are representative of two independent experiments ( n = 5 mice per group). (d) Lymph node and spleen weight in grams for mice of the indicated genotypes. Data are representative of two independent experiments ( n ≥ 5 per group). (e) Flow cytometric contour plots and dot plots of PD-1 high CXCR5 + CD4 + T FH cells and (f) GL-7 + CD95 + B220 + GC cells from mice of the indicated genotypes. Data are representative of two independent experiments ( n ≥ 5 mice per group). In e and f, the numbers in the plots represent percentages.

    Techniques Used: Mouse Assay, Immunofluorescence, Staining, Confocal Microscopy, Enzyme-linked Immunosorbent Assay, Flow Cytometry

    Spontaneous GC and T FH formation are corrected by loss of SAP in Roquin san/san mice. (a) CD4 + CXCR5 + PD-1 high T FH cells in unimmunized 10-wk-old Roquin san/san and Roquin san/san Sap −/− mice (P = 0.0056). Representative flow cytometric contour plots are shown (right). Data are representative of four independent experiments ( n = 4 per group). (b) B220 + GL-7 + CD95 + GC B cells in unimmunized 10-wk-old Roquin san/san Sap +/+ and Roquin san/san Sap −/− mice (P = 0.0007). Representative flow cytometric contour plots are shown (right). Data are representative of four independent experiments ( n = 4 per group). (c) Photomicrographs of frozen spleen sections from unimmunized 6-mo-old mice of the indicated genotypes stained with IgD (brown; all panels), PNA (blue; left), TCRβ (blue; middle), and PD-1 (blue; right). Bars, 200 µm.
    Figure Legend Snippet: Spontaneous GC and T FH formation are corrected by loss of SAP in Roquin san/san mice. (a) CD4 + CXCR5 + PD-1 high T FH cells in unimmunized 10-wk-old Roquin san/san and Roquin san/san Sap −/− mice (P = 0.0056). Representative flow cytometric contour plots are shown (right). Data are representative of four independent experiments ( n = 4 per group). (b) B220 + GL-7 + CD95 + GC B cells in unimmunized 10-wk-old Roquin san/san Sap +/+ and Roquin san/san Sap −/− mice (P = 0.0007). Representative flow cytometric contour plots are shown (right). Data are representative of four independent experiments ( n = 4 per group). (c) Photomicrographs of frozen spleen sections from unimmunized 6-mo-old mice of the indicated genotypes stained with IgD (brown; all panels), PNA (blue; left), TCRβ (blue; middle), and PD-1 (blue; right). Bars, 200 µm.

    Techniques Used: Mouse Assay, Flow Cytometry, Staining

    Th1 and Th2 cells are present in Roquin san/san mice in the absence of SAP and T FH cells, but not non-T FH effector cells, induce a GC response in wild-type mice. (a) Representative flow cytometric contour plots and (b) graphical analysis of GATA3 + CD44 high CD4 + and Tbet + CD44 high CD4 + cells in mice of the indicated genotypes. Data are representative of two independent experiments ( n ≥ 4 per group). (c) Representative dot plots of lymph node CD4 + PD-1 high CXCR5 + (left), Tbet + CD44 high CD4 + (middle), and GATA3 + CD44 high CD4 + (right) cells. (d) Experimental outline for adoptive transfer of Roquin san/san CD4 + CD45.2 CD44 high PD-1 high CXCR5 + or CD4 + CD45.2 CD44 high PD-1 − CXCR5 − T cells into CD45.1 C57BL/6 mice. (e) Flow cytometric contour plots and (f) dot plots of B220 + GL-7 + CD95 + GC B cells from CD45.1 C57BL/6 recipients 3 wk after adoptive transfer of the indicated cell type. Data were generated from three mice per group (**, P > 0.001). In a, d, and e, the numbers in the plots represent percentages.
    Figure Legend Snippet: Th1 and Th2 cells are present in Roquin san/san mice in the absence of SAP and T FH cells, but not non-T FH effector cells, induce a GC response in wild-type mice. (a) Representative flow cytometric contour plots and (b) graphical analysis of GATA3 + CD44 high CD4 + and Tbet + CD44 high CD4 + cells in mice of the indicated genotypes. Data are representative of two independent experiments ( n ≥ 4 per group). (c) Representative dot plots of lymph node CD4 + PD-1 high CXCR5 + (left), Tbet + CD44 high CD4 + (middle), and GATA3 + CD44 high CD4 + (right) cells. (d) Experimental outline for adoptive transfer of Roquin san/san CD4 + CD45.2 CD44 high PD-1 high CXCR5 + or CD4 + CD45.2 CD44 high PD-1 − CXCR5 − T cells into CD45.1 C57BL/6 mice. (e) Flow cytometric contour plots and (f) dot plots of B220 + GL-7 + CD95 + GC B cells from CD45.1 C57BL/6 recipients 3 wk after adoptive transfer of the indicated cell type. Data were generated from three mice per group (**, P > 0.001). In a, d, and e, the numbers in the plots represent percentages.

    Techniques Used: Mouse Assay, Flow Cytometry, Adoptive Transfer Assay, Generated

    Heterozygosity for Bcl6 reduces the magnitude of the GC response in Roquin +/+ and Roquin san/san mice and ameliorates the lupus-like phenotype of Roquin san/san mice. (a) Flow cytometric contour plots (left) and graphical analysis (right) of B220 + GL-7 + CD95 + GC B cells in 10-wk-old wild-type ( Bcl6 +/+ ) and Bcl6 +/− mice 8 d after SRBC immunization (P = 0.0011). Data are representative of four independent experiments ( n = 4 per group). (b) Flow cytometric contour plots (left) and dot plots (right) showing B220 + GL-7 + CD95 + GC B cells from 10-wk-old naive Roquin san/san Bcl6 +/+ and Roquin san/san Bcl6 +/− mice. Data are representative of five independent experiments ( n ≥ 4 per group). (c) Representative determination of serum IgG anti-dsDNA from 6-mo-old female Roquin +/+ Bcl6 +/+ , Roquin san/san Bcl6 +/+ , and Roquin san/san Bcl6 +/− mice, determined by immunofluorescence staining of C. luciliae substrate. Data shown reflect the occurrence ( n ≥ 6 mice per group); three out of six Roquin san/san Bcl6 +/− mice had low intensity staining (illustrated in the fourth panel from left), and three out of six were negative (illustrated in the third panel from left). (d) Representative images of kidney sections stained with H E (left) or viewed under an electron microscope (right) from 6-mo-old mice of the indicated genotypes. Roquin san/san animals show widespread mesangial proliferative lesions with moderate interstitial infiltrate. There are multiple electron-dense deposits (arrows). Histological changes in Roquin san/san Bcl6 +/− mice were mild, with occasional electron-dense deposits visible on electron microscopy in two individuals (far right). Images are representative ( n ≥ 4 per group). Bars: (H E) 100 µm in all panels; (electron microscopy) 5 µm in the Roquin +/+ Bcl6 +/+ and Roquin san/san Bcl6 +/+ panels, 2 µm in the left Roquin san/san Bcl6 +/− panels, and 10 µm in the right Roquin san/san Bcl6 +/− panel. (e) Nephritis severity score of 6-mo-old female Roquin +/+ , Roquin san/san , and Roquin san/san Bcl6 +/− mice as determined by histological analysis according to the criteria given in Table S1 (available at http://www.jem.org/cgi/content/full/jem.20081886/DC1 ). Horizontal bars indicate medians. In a, b, and e, each symbol represents one mouse; p-values are indicated on the graphs, and the numbers in the plots represent percentages.
    Figure Legend Snippet: Heterozygosity for Bcl6 reduces the magnitude of the GC response in Roquin +/+ and Roquin san/san mice and ameliorates the lupus-like phenotype of Roquin san/san mice. (a) Flow cytometric contour plots (left) and graphical analysis (right) of B220 + GL-7 + CD95 + GC B cells in 10-wk-old wild-type ( Bcl6 +/+ ) and Bcl6 +/− mice 8 d after SRBC immunization (P = 0.0011). Data are representative of four independent experiments ( n = 4 per group). (b) Flow cytometric contour plots (left) and dot plots (right) showing B220 + GL-7 + CD95 + GC B cells from 10-wk-old naive Roquin san/san Bcl6 +/+ and Roquin san/san Bcl6 +/− mice. Data are representative of five independent experiments ( n ≥ 4 per group). (c) Representative determination of serum IgG anti-dsDNA from 6-mo-old female Roquin +/+ Bcl6 +/+ , Roquin san/san Bcl6 +/+ , and Roquin san/san Bcl6 +/− mice, determined by immunofluorescence staining of C. luciliae substrate. Data shown reflect the occurrence ( n ≥ 6 mice per group); three out of six Roquin san/san Bcl6 +/− mice had low intensity staining (illustrated in the fourth panel from left), and three out of six were negative (illustrated in the third panel from left). (d) Representative images of kidney sections stained with H E (left) or viewed under an electron microscope (right) from 6-mo-old mice of the indicated genotypes. Roquin san/san animals show widespread mesangial proliferative lesions with moderate interstitial infiltrate. There are multiple electron-dense deposits (arrows). Histological changes in Roquin san/san Bcl6 +/− mice were mild, with occasional electron-dense deposits visible on electron microscopy in two individuals (far right). Images are representative ( n ≥ 4 per group). Bars: (H E) 100 µm in all panels; (electron microscopy) 5 µm in the Roquin +/+ Bcl6 +/+ and Roquin san/san Bcl6 +/+ panels, 2 µm in the left Roquin san/san Bcl6 +/− panels, and 10 µm in the right Roquin san/san Bcl6 +/− panel. (e) Nephritis severity score of 6-mo-old female Roquin +/+ , Roquin san/san , and Roquin san/san Bcl6 +/− mice as determined by histological analysis according to the criteria given in Table S1 (available at http://www.jem.org/cgi/content/full/jem.20081886/DC1 ). Horizontal bars indicate medians. In a, b, and e, each symbol represents one mouse; p-values are indicated on the graphs, and the numbers in the plots represent percentages.

    Techniques Used: Mouse Assay, Flow Cytometry, Immunofluorescence, Staining, Microscopy, Electron Microscopy

    8) Product Images from "Isolation of Single-Stranded DNA Aptamers That Distinguish Influenza Virus Hemagglutinin Subtype H1 from H5"

    Article Title: Isolation of Single-Stranded DNA Aptamers That Distinguish Influenza Virus Hemagglutinin Subtype H1 from H5

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0125060

    Western blot analysis using aptamers. Various amounts of GST-H1-HA1, GST-H5-HA1, and GST proteins were separated by SDS-PAGE, incubated with 5′-biotinylated aptamers, detected by streptavidin-HRP and ECL visualization (−, 0 μg; +, 1 μg; ++, 5 μg; +++, 10 μg).
    Figure Legend Snippet: Western blot analysis using aptamers. Various amounts of GST-H1-HA1, GST-H5-HA1, and GST proteins were separated by SDS-PAGE, incubated with 5′-biotinylated aptamers, detected by streptavidin-HRP and ECL visualization (−, 0 μg; +, 1 μg; ++, 5 μg; +++, 10 μg).

    Techniques Used: Western Blot, SDS Page, Incubation

    Affinity measurements of biotinylated ssDNA aptamers by SPR. (A) Biotinylated aptamers immobilized on an NLC sensor chip and interacting with various amounts of GST-H1-HA1. Equilibrium dissociation constants K D were calculated from association and dissociation rate constants (K D = k d /k a ) as 96.6 nM for ApI, 1.09 μM for ApII, and 293 nM for ApIII. (B) Interaction of 10 μM H1-HA1, H5-HA1, or GST with aptamers immobilized on an NLC sensor chip, as measured by SPR.
    Figure Legend Snippet: Affinity measurements of biotinylated ssDNA aptamers by SPR. (A) Biotinylated aptamers immobilized on an NLC sensor chip and interacting with various amounts of GST-H1-HA1. Equilibrium dissociation constants K D were calculated from association and dissociation rate constants (K D = k d /k a ) as 96.6 nM for ApI, 1.09 μM for ApII, and 293 nM for ApIII. (B) Interaction of 10 μM H1-HA1, H5-HA1, or GST with aptamers immobilized on an NLC sensor chip, as measured by SPR.

    Techniques Used: SPR Assay, Chromatin Immunoprecipitation

    Binding analysis of selected ssDNA aptamers by ELISA. (A) Affinity measurements of selected ssDNA aptamers to H1-HA1 by ELISA. Immobilized biotinylated ssDNA aptamers were incubated with increasing concentrations of GST-H1-HA1, and binding was detected with anti-GST antibody-HRP. The biotinylated ssDNA library was used as negative control (◊). Graphs were fitted to the Michaelis-Menten equation, and K d values were calculated as 64.76 ± 18.24 nM for ApI (●), 69.06 ± 12.34 nM for ApII (△), and 50.32 ± 14.07 nM for ApIII (■). (B) 100 nM GST-H1-HA1, GST-H5-HA1, or GST proteins incubated with biotinylated aptamers immobilized on streptavidin-coated plates to compare binding affinities.
    Figure Legend Snippet: Binding analysis of selected ssDNA aptamers by ELISA. (A) Affinity measurements of selected ssDNA aptamers to H1-HA1 by ELISA. Immobilized biotinylated ssDNA aptamers were incubated with increasing concentrations of GST-H1-HA1, and binding was detected with anti-GST antibody-HRP. The biotinylated ssDNA library was used as negative control (◊). Graphs were fitted to the Michaelis-Menten equation, and K d values were calculated as 64.76 ± 18.24 nM for ApI (●), 69.06 ± 12.34 nM for ApII (△), and 50.32 ± 14.07 nM for ApIII (■). (B) 100 nM GST-H1-HA1, GST-H5-HA1, or GST proteins incubated with biotinylated aptamers immobilized on streptavidin-coated plates to compare binding affinities.

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation, Negative Control

    9) Product Images from "Preclinical efficacy and safety of an anti-IL-1β vaccine for the treatment of type 2 diabetes"

    Article Title: Preclinical efficacy and safety of an anti-IL-1β vaccine for the treatment of type 2 diabetes

    Journal: Molecular Therapy. Methods & Clinical Development

    doi: 10.1038/mtm.2014.48

    Characterization of the detoxified vaccine antigen hIL-1b(D145K). ( a ) hIL-1RI binding. Serial dilutions of wild-type hIL-1β or hIL-1b(D145K) were mixed with a constant amount of 1 nmol/l biotinylated hIL-1β and applied to ELISA plates that had been coated with hIL-1RI. Obtained OD values were reciprocally transformed to express % receptor binding of wild-type hIL-1β or hIL-1b(D145K), respectively. ( b ) Formation of the ternary hIL-1RI-hIL-1β-hIL-1RAcP signaling complex. Wild-type human IL-1β (0.4 µg/ml) or hIL-1b(D145K) (100 µg/ml) were incubated with hIL-1RAcP (1 µg/ml) and applied to ELISA plates that had been coated with 1 µg/ml of human IL-1RI. Formation of the ternary complex was detected with a hIL-1RAcP-specific antibody. ( c ) hIL-1RII binding. Experimental conditions were as in a but with hIL-1RII coated on the ELISA plate. ( d ) IL-6 secretion. HeLa cells were incubated with serial dilutions of wild-type hIL-1β or hIL-1b (D145K). After an incubation of 4 hours, IL-6 was quantified in supernatants by Sandwich ELISA. ( e ) Cytopathic effect. A375 cells were incubated with serial dilutions of either wild-type hIL-1β or hIL-1b(D145K). After 7 days, viable adherent cells were stained with crystal violet and quantified by measuring optical densities at 600 nm. Shown are mean values from triplicate measurements ± SEM. ( f ) In vivo inflammatory activity. Groups of female C57BL/6 mice ( n = 4) were injected i.p. with 1 µg of either wild-type hIL-1β or hIL-1b(D145K) or s.c. with 25 µg of the conjugate vaccine Qβ-hIL-1b(D145K). Three hours after injection, sera were collected, and IL-6 levels were quantified with a Quantikine ELISA kit (* P
    Figure Legend Snippet: Characterization of the detoxified vaccine antigen hIL-1b(D145K). ( a ) hIL-1RI binding. Serial dilutions of wild-type hIL-1β or hIL-1b(D145K) were mixed with a constant amount of 1 nmol/l biotinylated hIL-1β and applied to ELISA plates that had been coated with hIL-1RI. Obtained OD values were reciprocally transformed to express % receptor binding of wild-type hIL-1β or hIL-1b(D145K), respectively. ( b ) Formation of the ternary hIL-1RI-hIL-1β-hIL-1RAcP signaling complex. Wild-type human IL-1β (0.4 µg/ml) or hIL-1b(D145K) (100 µg/ml) were incubated with hIL-1RAcP (1 µg/ml) and applied to ELISA plates that had been coated with 1 µg/ml of human IL-1RI. Formation of the ternary complex was detected with a hIL-1RAcP-specific antibody. ( c ) hIL-1RII binding. Experimental conditions were as in a but with hIL-1RII coated on the ELISA plate. ( d ) IL-6 secretion. HeLa cells were incubated with serial dilutions of wild-type hIL-1β or hIL-1b (D145K). After an incubation of 4 hours, IL-6 was quantified in supernatants by Sandwich ELISA. ( e ) Cytopathic effect. A375 cells were incubated with serial dilutions of either wild-type hIL-1β or hIL-1b(D145K). After 7 days, viable adherent cells were stained with crystal violet and quantified by measuring optical densities at 600 nm. Shown are mean values from triplicate measurements ± SEM. ( f ) In vivo inflammatory activity. Groups of female C57BL/6 mice ( n = 4) were injected i.p. with 1 µg of either wild-type hIL-1β or hIL-1b(D145K) or s.c. with 25 µg of the conjugate vaccine Qβ-hIL-1b(D145K). Three hours after injection, sera were collected, and IL-6 levels were quantified with a Quantikine ELISA kit (* P

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay, Transformation Assay, Incubation, Sandwich ELISA, Staining, In Vivo, Activity Assay, Mouse Assay, Injection

    10) Product Images from "On the Role of Protein Disulfide Isomerase in the Retrograde Cell Transport of Secreted Phospholipases A2"

    Article Title: On the Role of Protein Disulfide Isomerase in the Retrograde Cell Transport of Secreted Phospholipases A2

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0120692

    Atx interacts with several domains of yPDI. (A) yPDI is a multi-domain protein. It consists of domains a, a’, b, b’ and c. Domain b’ is linked to domain a’ by a linker sequence x. Separate domains and their fusions, as schematically presented, were employed in affinity-labelling experiments using photo-reactive sulfo-SBED-Atx. Atx photo-probe was incubated with the wt yPDI (wt-PDI) (B) or its various parts (C) for 30 min, followed by the UV light-initiated reaction of the photo-probe with proteins in close contact, which were thus biotinylated. Samples were analysed on SDS-PAGE under reducing conditions, electro-transferred to a PVDF membrane. Biotin-containing bands on the PVDF membrane were detected using streptavidin-HRP. Labelling specificity in (B) was verified with the addition of 100-fold unlabelled Atx over sulfo-SBED-Atx (wt-PDI+). Black arrowheads in (A) point at those elements of yPDI which bind and react with sulfo-SBED-Atx. In (B) and (C) black arrowheads are pointing at biotinylated yPDI-structures. Positions on the membrane with biotinylated monomer and dimer of Atx are pointed at by white arrowheads. Experimental details are described in section Materials and Methods. Experiment was performed in triplicate.
    Figure Legend Snippet: Atx interacts with several domains of yPDI. (A) yPDI is a multi-domain protein. It consists of domains a, a’, b, b’ and c. Domain b’ is linked to domain a’ by a linker sequence x. Separate domains and their fusions, as schematically presented, were employed in affinity-labelling experiments using photo-reactive sulfo-SBED-Atx. Atx photo-probe was incubated with the wt yPDI (wt-PDI) (B) or its various parts (C) for 30 min, followed by the UV light-initiated reaction of the photo-probe with proteins in close contact, which were thus biotinylated. Samples were analysed on SDS-PAGE under reducing conditions, electro-transferred to a PVDF membrane. Biotin-containing bands on the PVDF membrane were detected using streptavidin-HRP. Labelling specificity in (B) was verified with the addition of 100-fold unlabelled Atx over sulfo-SBED-Atx (wt-PDI+). Black arrowheads in (A) point at those elements of yPDI which bind and react with sulfo-SBED-Atx. In (B) and (C) black arrowheads are pointing at biotinylated yPDI-structures. Positions on the membrane with biotinylated monomer and dimer of Atx are pointed at by white arrowheads. Experimental details are described in section Materials and Methods. Experiment was performed in triplicate.

    Techniques Used: Sequencing, Incubation, SDS Page

    11) Product Images from "Uptake of the antifungal cationic peptide Histatin 5 by Candida albicans Ssa2p requires binding to non-conventional sites within the ATPase domain"

    Article Title: Uptake of the antifungal cationic peptide Histatin 5 by Candida albicans Ssa2p requires binding to non-conventional sites within the ATPase domain

    Journal: Molecular Microbiology

    doi: 10.1111/j.1365-2958.2008.06480.x

    BHst 5 co-elutes with rSsa2p. Purified rSsa2p (5 μM) was mixed with excess BHst 5 (100 μM) for 30 min before loading onto Superose column. Fractions were collected (top) and subjected to 7.5% SDS-PAGE and silver-stained to detect rSsa2p (bottom left). rSsa2p polymer peak and monomer peak fractions were pooled to generate pool A (∼640 μl) and pool B (∼1300 μl) respectively. Both co-eluted BHst 5 and rSsa2 proteins from each pool were quantified by slot blot with corresponding linear loading standards (bottom right). Two slots of each pool were loaded (lane 4) corresponding to 25% and 5% of total volume in order to be within the linear range for quantification. Lane 1: rSsa2p loading standards (300, 150, 75, 37.5, 18.8, 9.4, 4.7 ng); lane 2: BHst 5 loading standards: (125, 100, 50, 25, 12.5, 6.3 ng); lane 3: BHst 5 co-eluted in pool A or pool B was quantified following detection by Streptavidin-HRP antibody; lane 4: quantification of rSsa2p was performed on the same membrane after being stripped and re-probed with Anti-Xpress antibody to detect rSsa2p. Co-eluted BHst 5 and rSsa2p under nucleotide treatment conditions were similarly quantified.
    Figure Legend Snippet: BHst 5 co-elutes with rSsa2p. Purified rSsa2p (5 μM) was mixed with excess BHst 5 (100 μM) for 30 min before loading onto Superose column. Fractions were collected (top) and subjected to 7.5% SDS-PAGE and silver-stained to detect rSsa2p (bottom left). rSsa2p polymer peak and monomer peak fractions were pooled to generate pool A (∼640 μl) and pool B (∼1300 μl) respectively. Both co-eluted BHst 5 and rSsa2 proteins from each pool were quantified by slot blot with corresponding linear loading standards (bottom right). Two slots of each pool were loaded (lane 4) corresponding to 25% and 5% of total volume in order to be within the linear range for quantification. Lane 1: rSsa2p loading standards (300, 150, 75, 37.5, 18.8, 9.4, 4.7 ng); lane 2: BHst 5 loading standards: (125, 100, 50, 25, 12.5, 6.3 ng); lane 3: BHst 5 co-eluted in pool A or pool B was quantified following detection by Streptavidin-HRP antibody; lane 4: quantification of rSsa2p was performed on the same membrane after being stripped and re-probed with Anti-Xpress antibody to detect rSsa2p. Co-eluted BHst 5 and rSsa2p under nucleotide treatment conditions were similarly quantified.

    Techniques Used: Purification, SDS Page, Staining, Dot Blot

    Design and expression of mutated Ssa2 proteins in C. albicans strains. Regions selected for site-directed mutagenesis within Hst 5 binding regions are based upon results of thermolysin limited digestion (green bars, also indicated as T1 and T2 regions) and peptide array (blue bar, also indicated as P1 region). Amino acids selected for Ala substitution are shown in red (top). Cell wall and cytoplasmic proteins were isolated from each strain (bottom): 25 μg of each protein was loaded on 7.5% SDS-PAGE gel and immunoblotted with Hsp70/Hsc70 monoclonal antibody. C. albicans wt cells display two distinct Hsp70 proteins – Ssa1p (lane 1, lower band) and Ssa2p (lane 1, upper band). Only Ssa1p is expressed in the ssa2 Δ knockout (lower band, lane 2), while the gene restoration strain ( ssa2 Δ /SSA2 ) expressed both Ssa2p and Ssa1p comparable with wt cells (lane 3). Gene restoration constructs of SSA2 with three to four amino acid substitutions (lane 4 to lane 7) expressed similar amounts of Ssa2 proteins as wt. However, significantly less mutant Ssa2 (334−338A3) protein was observed in cell wall fraction (lane 7, bottom).
    Figure Legend Snippet: Design and expression of mutated Ssa2 proteins in C. albicans strains. Regions selected for site-directed mutagenesis within Hst 5 binding regions are based upon results of thermolysin limited digestion (green bars, also indicated as T1 and T2 regions) and peptide array (blue bar, also indicated as P1 region). Amino acids selected for Ala substitution are shown in red (top). Cell wall and cytoplasmic proteins were isolated from each strain (bottom): 25 μg of each protein was loaded on 7.5% SDS-PAGE gel and immunoblotted with Hsp70/Hsc70 monoclonal antibody. C. albicans wt cells display two distinct Hsp70 proteins – Ssa1p (lane 1, lower band) and Ssa2p (lane 1, upper band). Only Ssa1p is expressed in the ssa2 Δ knockout (lower band, lane 2), while the gene restoration strain ( ssa2 Δ /SSA2 ) expressed both Ssa2p and Ssa1p comparable with wt cells (lane 3). Gene restoration constructs of SSA2 with three to four amino acid substitutions (lane 4 to lane 7) expressed similar amounts of Ssa2 proteins as wt. However, significantly less mutant Ssa2 (334−338A3) protein was observed in cell wall fraction (lane 7, bottom).

    Techniques Used: Expressing, Mutagenesis, Binding Assay, Peptide Microarray, Isolation, SDS Page, Knock-Out, Construct

    Mutations in Ssa2p Hst 5-binding epitopes reduce Hst 5 uptake and cytotoxicity. C. albicans constructs of SSA2 mutations were tested for sensitivity to Hst 5 (top) and ability to translocate Hst 5 to the cytosol (bottom). Candidacidal assays were performed by incubating cells with 15 μM Hst 5 for 1 h at 30°C, and percentage cell death was calculated compared with untreated cells. Cytosolic translocation of Biotin-Hst 5 (15 μM) was measured for each C. albicans construct using the same conditions as for candidacidal assays. Cytosolic proteins (10 μg) from each construct were subjected to 16% Tricine SDS-PAGE, immunoblotted with Streptavidin-HRP to detect BHst 5, and quantified. Control proteins from each construct are shown to verify equal protein loading. Mutations in Ssa2 (128−132A3) resulted in complete loss of killing and translocation (lane 4) equivalent to the ssa2 Δ knockout, while mutations in Ssa2 (334−338A3) (lane 7) had significant loss of cytotoxic and transport functions. Mutations in Ssa2 (131−135A3) and Ssa2 (150−156A4) (lane 5 and lane 6) resulted in mild to moderate loss of function.
    Figure Legend Snippet: Mutations in Ssa2p Hst 5-binding epitopes reduce Hst 5 uptake and cytotoxicity. C. albicans constructs of SSA2 mutations were tested for sensitivity to Hst 5 (top) and ability to translocate Hst 5 to the cytosol (bottom). Candidacidal assays were performed by incubating cells with 15 μM Hst 5 for 1 h at 30°C, and percentage cell death was calculated compared with untreated cells. Cytosolic translocation of Biotin-Hst 5 (15 μM) was measured for each C. albicans construct using the same conditions as for candidacidal assays. Cytosolic proteins (10 μg) from each construct were subjected to 16% Tricine SDS-PAGE, immunoblotted with Streptavidin-HRP to detect BHst 5, and quantified. Control proteins from each construct are shown to verify equal protein loading. Mutations in Ssa2 (128−132A3) resulted in complete loss of killing and translocation (lane 4) equivalent to the ssa2 Δ knockout, while mutations in Ssa2 (334−338A3) (lane 7) had significant loss of cytotoxic and transport functions. Mutations in Ssa2 (131−135A3) and Ssa2 (150−156A4) (lane 5 and lane 6) resulted in mild to moderate loss of function.

    Techniques Used: Binding Assay, Construct, Translocation Assay, SDS Page, Knock-Out

    Digestion products of rSsa2p are substantially altered by BHst 5 binding. Purified rSsa2p (10 μg) was incubated with 20 μg of BHst 5 in the presence of 2 mM ATP-Mg 2+ and cross-linker for 30 min at room temperature. Stabilized rSsa2p–BHst 5 complexes were isolated by SA beads pull-down. The complexes was then subjected to 5 μg ml −1 thermolysin digestion at 30°C for 1 h. Digestion fragments not associated with the complex binding site were removed by washing, and the remaining bead-retained rSsa2p–BHst 5 complex was recovered and half was subjected to 15% SDS-PAGE and detected by silver staining (lane 4). Ssa2p digestion products differed quite substantially in the presence of Hst 5. Two peptides within the ATPase domain were identified by MS analysis of a digestion fragment (arrow). Lane 1: rSsa2p (0.5 μg); lane 2: thermolysin (0.5 μg); lane 3: thermolysin digestion of rSsa2p without BHst 5; lane 4: thermolysin digestion products from rSsa2p–BHst 5 complex.
    Figure Legend Snippet: Digestion products of rSsa2p are substantially altered by BHst 5 binding. Purified rSsa2p (10 μg) was incubated with 20 μg of BHst 5 in the presence of 2 mM ATP-Mg 2+ and cross-linker for 30 min at room temperature. Stabilized rSsa2p–BHst 5 complexes were isolated by SA beads pull-down. The complexes was then subjected to 5 μg ml −1 thermolysin digestion at 30°C for 1 h. Digestion fragments not associated with the complex binding site were removed by washing, and the remaining bead-retained rSsa2p–BHst 5 complex was recovered and half was subjected to 15% SDS-PAGE and detected by silver staining (lane 4). Ssa2p digestion products differed quite substantially in the presence of Hst 5. Two peptides within the ATPase domain were identified by MS analysis of a digestion fragment (arrow). Lane 1: rSsa2p (0.5 μg); lane 2: thermolysin (0.5 μg); lane 3: thermolysin digestion of rSsa2p without BHst 5; lane 4: thermolysin digestion products from rSsa2p–BHst 5 complex.

    Techniques Used: Binding Assay, Purification, Incubation, Isolation, SDS Page, Silver Staining, Mass Spectrometry

    Expression and purification of full-length and truncated Ssa2 proteins. A. Schematic representation of the domain structure of Candida albicans Ssa2p and the design for truncated Ssa2 proteins. B. Each purified recombinant protein obtained from a yeast expression system (1 μg) was subjected to 10% SDS-PAGE and Coomassie blue-stained to visualize C. albicans full-length, rSsa2 1−630 , rSsa2 1−385 , rSsa2 386−645 proteins.
    Figure Legend Snippet: Expression and purification of full-length and truncated Ssa2 proteins. A. Schematic representation of the domain structure of Candida albicans Ssa2p and the design for truncated Ssa2 proteins. B. Each purified recombinant protein obtained from a yeast expression system (1 μg) was subjected to 10% SDS-PAGE and Coomassie blue-stained to visualize C. albicans full-length, rSsa2 1−630 , rSsa2 1−385 , rSsa2 386−645 proteins.

    Techniques Used: Expressing, Purification, Recombinant, SDS Page, Staining

    ATPase domain of Ssa2 is necessary for complex formation with BHst 5. A. Purified full length, rSsa2 1−630 , rSsa2 1−385 or rSsa2 386−645 (input) were incubated with Biotin-Hst 5 (BHst 5) for 2 h at 4°C with or without the presence of cross-linker to allow complex formation, and Streptavidin-agarose (SA) beads were added to the mixture. Resulting complexes were isolated by centrifugation of the SA beads and washed to remove non-specifically bound proteins. Recovered protein complexes were subjected to SDS-PAGE and detected by Western blotting with anti-Xpress-HRP monoclonal antibody and enhanced chemiluminescence (ECL). Lane 1: 10% of input Ssa2 proteins; lane 2: negative control, pull-down Ssa2 proteins without BHst 5; lane 3: pull-down Ssa2 proteins with BHst 5 without cross-linker; lane 4: pull-down Ssa2 proteins by BHst 5 with cross-linker . Complex formation was detected only with proteins containing the ATPase domain. B. A six fold molar excess of Ssa2p C-terminal anchor domain peptide 13mer (EPSNDGPTVEEVD) or 4mer (EEVD) was pre-incubated with BHst 5 for 30 min at 4°C prior to addition of full-length rSsa2p for the pull-down assay described in (A). No inhibition of interactions between Ssa2p and Hst 5 was observed with either peptide (+) compared with Hst 5 and Ssa2p alone (−).
    Figure Legend Snippet: ATPase domain of Ssa2 is necessary for complex formation with BHst 5. A. Purified full length, rSsa2 1−630 , rSsa2 1−385 or rSsa2 386−645 (input) were incubated with Biotin-Hst 5 (BHst 5) for 2 h at 4°C with or without the presence of cross-linker to allow complex formation, and Streptavidin-agarose (SA) beads were added to the mixture. Resulting complexes were isolated by centrifugation of the SA beads and washed to remove non-specifically bound proteins. Recovered protein complexes were subjected to SDS-PAGE and detected by Western blotting with anti-Xpress-HRP monoclonal antibody and enhanced chemiluminescence (ECL). Lane 1: 10% of input Ssa2 proteins; lane 2: negative control, pull-down Ssa2 proteins without BHst 5; lane 3: pull-down Ssa2 proteins with BHst 5 without cross-linker; lane 4: pull-down Ssa2 proteins by BHst 5 with cross-linker . Complex formation was detected only with proteins containing the ATPase domain. B. A six fold molar excess of Ssa2p C-terminal anchor domain peptide 13mer (EPSNDGPTVEEVD) or 4mer (EEVD) was pre-incubated with BHst 5 for 30 min at 4°C prior to addition of full-length rSsa2p for the pull-down assay described in (A). No inhibition of interactions between Ssa2p and Hst 5 was observed with either peptide (+) compared with Hst 5 and Ssa2p alone (−).

    Techniques Used: Purification, Incubation, Isolation, Centrifugation, SDS Page, Western Blot, Negative Control, Pull Down Assay, Inhibition

    12) Product Images from "?-Catenin Phosphorylated at Serine 45 Is Spatially Uncoupled from ?-Catenin Phosphorylated in the GSK3 Domain: Implications for Signaling"

    Article Title: ?-Catenin Phosphorylated at Serine 45 Is Spatially Uncoupled from ?-Catenin Phosphorylated in the GSK3 Domain: Implications for Signaling

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0010184

    N-terminally unphoshorylated β-catenin is a minor, nuclear form of β-catenin. A) Immunofluorescence of SW480 cells for total β-catenin or ABC (active β-catenin). Both monoclonal (mAb) and polyclonal (pAb) antibodies against total β-catenin produce a pan-cellular staining, while ABC predominantly stains nuclei. B) SW480 lysate (20 or 60 µg) was compared to known quantities of purified GST-β-catenin to estimate the relative abundance of total β-catenin to ABC. ABC is approximately 100 times less abundant than total β-catenin. Bars, 10 µm.
    Figure Legend Snippet: N-terminally unphoshorylated β-catenin is a minor, nuclear form of β-catenin. A) Immunofluorescence of SW480 cells for total β-catenin or ABC (active β-catenin). Both monoclonal (mAb) and polyclonal (pAb) antibodies against total β-catenin produce a pan-cellular staining, while ABC predominantly stains nuclei. B) SW480 lysate (20 or 60 µg) was compared to known quantities of purified GST-β-catenin to estimate the relative abundance of total β-catenin to ABC. ABC is approximately 100 times less abundant than total β-catenin. Bars, 10 µm.

    Techniques Used: Immunofluorescence, Staining, Purification

    β-catenin phosphorylated at S552 or S675 localizes to cell contacts and associates with E-cadherin. A) Cadherin-free β-catenin was isolated from an SW480 lysate by affinity precipitation with GST-ICAT, as previously described [48] . LC-MS/MS analysis identified S552 and S675 as two phosphorylation sites in β-catenin. Peptide abundance is plotted as a function of mass/charge (m/z). Identified phospho-sites are shown in red. B) Immunofluorescence of SW480 cells with antibodies to total β-catenin and phospho-S552 or -S675 reveals that phospho-S552 appears punctate, while phospho-S675 and total β-catenin localize uniformly to sites of cell-cell contact. C) Detergent-free lysis of membrane and cytosolic fractions from SW480/E-cad cells. D) Sucrose gradient density centrifugation of the detergent-free membrane preparation from SW480/E-cadherin cells. Note that phospho-S552 or -S675 float with cadherins. E) Cell surface biotinylation of SW480/E-cadherin cells followed by immunoprecipitation with the indicated antibodies and detection with streptavidin-HRP reveals that phospho-S552 and -S675 coimmunoprecipitate with a cell surface protein the same size as E-cadherin. Western blot for total β-catenin demonstrates immunoprecipitation efficiency. Mouse IgG (mIgG) and rabbit IgG (rIgG) controls are shown, as well as a positive control for E-cadherin (IP: E-cad). An LRP6 immunoprecipitation was also performed and neither phospho-S552, -S675 nor N-terminal phospho-forms of β-catenin coimmunoprecipitate with a surface protein of this size. Bars, 10 µm.
    Figure Legend Snippet: β-catenin phosphorylated at S552 or S675 localizes to cell contacts and associates with E-cadherin. A) Cadherin-free β-catenin was isolated from an SW480 lysate by affinity precipitation with GST-ICAT, as previously described [48] . LC-MS/MS analysis identified S552 and S675 as two phosphorylation sites in β-catenin. Peptide abundance is plotted as a function of mass/charge (m/z). Identified phospho-sites are shown in red. B) Immunofluorescence of SW480 cells with antibodies to total β-catenin and phospho-S552 or -S675 reveals that phospho-S552 appears punctate, while phospho-S675 and total β-catenin localize uniformly to sites of cell-cell contact. C) Detergent-free lysis of membrane and cytosolic fractions from SW480/E-cad cells. D) Sucrose gradient density centrifugation of the detergent-free membrane preparation from SW480/E-cadherin cells. Note that phospho-S552 or -S675 float with cadherins. E) Cell surface biotinylation of SW480/E-cadherin cells followed by immunoprecipitation with the indicated antibodies and detection with streptavidin-HRP reveals that phospho-S552 and -S675 coimmunoprecipitate with a cell surface protein the same size as E-cadherin. Western blot for total β-catenin demonstrates immunoprecipitation efficiency. Mouse IgG (mIgG) and rabbit IgG (rIgG) controls are shown, as well as a positive control for E-cadherin (IP: E-cad). An LRP6 immunoprecipitation was also performed and neither phospho-S552, -S675 nor N-terminal phospho-forms of β-catenin coimmunoprecipitate with a surface protein of this size. Bars, 10 µm.

    Techniques Used: Isolation, Affinity Precipitation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Immunofluorescence, Lysis, Centrifugation, Immunoprecipitation, Western Blot, Positive Control

    N-terminally unphosphorylated β-catenin appears highly sensitive to cadherin expression. A) Control (i. and iii.) and E-cadherin-restored SW480 cells (ii. and iv.) were stained with antibodies against total β-catenin or ABC. Note that ABC appears selectively recruited to sites of cell-cell contact upon cadherin expression relative to the total pool of β-catenin. B) A detergent-free cytosolic fraction from SW480/E-cadherin cells was subjected to gel filtration chromatography. Peak fractions are marked with arrows. The presence of E-cadherin reduces the abundance of monomeric ABC relative to control cells (compare to Fig. 2A ). C) Detergent-free preparations of membrane and cytosolic fractions isolated from control and E-cadherin-restored SW480 cells. Note that E-cadherin appears to selectively recruit ABC to the membrane, leaving a portion of total β-catenin in the cytosol. A non-specific band (*) recognized by ABC [47] serves as a loading control. Bars, 10 µm.
    Figure Legend Snippet: N-terminally unphosphorylated β-catenin appears highly sensitive to cadherin expression. A) Control (i. and iii.) and E-cadherin-restored SW480 cells (ii. and iv.) were stained with antibodies against total β-catenin or ABC. Note that ABC appears selectively recruited to sites of cell-cell contact upon cadherin expression relative to the total pool of β-catenin. B) A detergent-free cytosolic fraction from SW480/E-cadherin cells was subjected to gel filtration chromatography. Peak fractions are marked with arrows. The presence of E-cadherin reduces the abundance of monomeric ABC relative to control cells (compare to Fig. 2A ). C) Detergent-free preparations of membrane and cytosolic fractions isolated from control and E-cadherin-restored SW480 cells. Note that E-cadherin appears to selectively recruit ABC to the membrane, leaving a portion of total β-catenin in the cytosol. A non-specific band (*) recognized by ABC [47] serves as a loading control. Bars, 10 µm.

    Techniques Used: Expressing, Staining, Filtration, Chromatography, Isolation

    Cytosolic N-terminally unphosphorylated β-catenin is primarily monomeric. A detergent-free cytosolic fraction from SW480 control (A) or α-catenin shRNA knock-down (B) cells was subjected to gel filtration chromatography and immunoblot analysis. A) ABC sizes as a monomer (compared to calibration standards (not shown) and purified β-catenin ( Fig. S1 ); peak fraction #42), while total β-catenin sizes evenly between monomer- and β-catenin/α-catenin dimer fractions (peak fraction #36). Peak fractions are marked with arrows. B–D) β-catenin dimer fraction is due to association with α-catenin. B) Size fractionation of cytosol from SW480 cells depleted of α-catenin by shRNA. C) Immunoblot of SW480 control and α-catenin knock down lysates. D) [ 35 S]-methionine/cysteine-labeling of SW480 cells and immunoprecipitation of α-catenin and β-catenin (1∶100, lanes 3 and 5; 1∶300, lanes 4 and 6). Autoradiogram reveals the major binding partner of β-catenin in this cell type is α-catenin. No antibody (lane 1) or non-immune control (lane 2) are also shown.
    Figure Legend Snippet: Cytosolic N-terminally unphosphorylated β-catenin is primarily monomeric. A detergent-free cytosolic fraction from SW480 control (A) or α-catenin shRNA knock-down (B) cells was subjected to gel filtration chromatography and immunoblot analysis. A) ABC sizes as a monomer (compared to calibration standards (not shown) and purified β-catenin ( Fig. S1 ); peak fraction #42), while total β-catenin sizes evenly between monomer- and β-catenin/α-catenin dimer fractions (peak fraction #36). Peak fractions are marked with arrows. B–D) β-catenin dimer fraction is due to association with α-catenin. B) Size fractionation of cytosol from SW480 cells depleted of α-catenin by shRNA. C) Immunoblot of SW480 control and α-catenin knock down lysates. D) [ 35 S]-methionine/cysteine-labeling of SW480 cells and immunoprecipitation of α-catenin and β-catenin (1∶100, lanes 3 and 5; 1∶300, lanes 4 and 6). Autoradiogram reveals the major binding partner of β-catenin in this cell type is α-catenin. No antibody (lane 1) or non-immune control (lane 2) are also shown.

    Techniques Used: shRNA, Filtration, Chromatography, Purification, Fractionation, Labeling, Immunoprecipitation, Binding Assay

    N-terminally phosphorylated β-catenin is largely not associated with E-cadherin. A) SW480 cell lysates were sequentially incubated with GST-cadherin cytoplasmic domain coupled-glutathione sepharose beads. Non-binding lane reflects 5% of the total unbound fraction. Note that while ABC can be affinity precipitated by GST-cadherin, β-catenin phosphorylated at S45, T41/S45, and S33/37/T41 bind to a lesser extent. B) Sucrose gradient density centrifugation of the detergent-free membrane preparation from SW480/E-cadherin cells reveals that N-terminally phosphorylated β-catenin does not appreciably co-fractionate (i.e., float) with cadherins. C) Immunoprecipitation of Axin or E-cadherin from SW480/E-cadherin lysates reveals that β-catenin phosphorylated at S33/37/T41 does not associate with E-cadherin. D) Cell surface biotinylation of SW480/E-cadherin cells followed by immunoprecipitation with the indicated antibodies and detection by streptavidin-HRP reveals that ABC coimmunoprecipitates with a cell surface protein the same size as E-cadherin, while β-catenin phosphorylated at S33/37/T41 does not. Western blot analysis for total β-catenin confirms that the same amount of β-catenin was immunopreciptated with antibodies against T41/S45 and S33/37/T41. Mouse IgG (mIgG) and rabbit IgG (rIgG) controls are shown, as well as a positive control for E-cadherin (IP: E-cad).
    Figure Legend Snippet: N-terminally phosphorylated β-catenin is largely not associated with E-cadherin. A) SW480 cell lysates were sequentially incubated with GST-cadherin cytoplasmic domain coupled-glutathione sepharose beads. Non-binding lane reflects 5% of the total unbound fraction. Note that while ABC can be affinity precipitated by GST-cadherin, β-catenin phosphorylated at S45, T41/S45, and S33/37/T41 bind to a lesser extent. B) Sucrose gradient density centrifugation of the detergent-free membrane preparation from SW480/E-cadherin cells reveals that N-terminally phosphorylated β-catenin does not appreciably co-fractionate (i.e., float) with cadherins. C) Immunoprecipitation of Axin or E-cadherin from SW480/E-cadherin lysates reveals that β-catenin phosphorylated at S33/37/T41 does not associate with E-cadherin. D) Cell surface biotinylation of SW480/E-cadherin cells followed by immunoprecipitation with the indicated antibodies and detection by streptavidin-HRP reveals that ABC coimmunoprecipitates with a cell surface protein the same size as E-cadherin, while β-catenin phosphorylated at S33/37/T41 does not. Western blot analysis for total β-catenin confirms that the same amount of β-catenin was immunopreciptated with antibodies against T41/S45 and S33/37/T41. Mouse IgG (mIgG) and rabbit IgG (rIgG) controls are shown, as well as a positive control for E-cadherin (IP: E-cad).

    Techniques Used: Incubation, Binding Assay, Centrifugation, Immunoprecipitation, Western Blot, Positive Control

    β-catenin phosphorylated at T41/S45 is spatially uncoupled from β-catenin phosphorylated at S33/37/T41. SW480 cells were co-stained with antibodies against β-catenin phosphorylated at S33/37/T41 or T41/S45 and ABC. Merged images reveal that phospho-S33/37/T41 appears excluded from the nucleus, while phospho-T41/S45 is largely nuclear, as is ABC. Bars, 10 µm.
    Figure Legend Snippet: β-catenin phosphorylated at T41/S45 is spatially uncoupled from β-catenin phosphorylated at S33/37/T41. SW480 cells were co-stained with antibodies against β-catenin phosphorylated at S33/37/T41 or T41/S45 and ABC. Merged images reveal that phospho-S33/37/T41 appears excluded from the nucleus, while phospho-T41/S45 is largely nuclear, as is ABC. Bars, 10 µm.

    Techniques Used: Staining

    13) Product Images from "Sulforaphane Improves Ischemia-Induced Detrusor Overactivity by Downregulating the Enhancement of Associated Endoplasmic Reticulum Stress, Autophagy, and Apoptosis in Rat Bladder"

    Article Title: Sulforaphane Improves Ischemia-Induced Detrusor Overactivity by Downregulating the Enhancement of Associated Endoplasmic Reticulum Stress, Autophagy, and Apoptosis in Rat Bladder

    Journal: Scientific Reports

    doi: 10.1038/srep36110

    Changes in bladder expression of ( A ) c-Nrf2, ( B ) Keap1, ( C ) n-Nrf2, ( D ) GRP78 (ER stress), ( E ) CHOP (ER stress), ( F ) caspase 3, ( G ) Beclin-1, ( H ) p62, ( I ) LC3 II, ( J ) muscarinic M 2 receptor, ( K ) muscarinic M 3 receptor, ( L ) purinergic P 2 X 1 receptor, ( M ) purinergic P 2 X 2 receptor, and ( N ) purinergic P 2 X 3 receptor in response to 2WBI, 4WBI, and treatments. All of the experiments were performed in three rats of each group. * p
    Figure Legend Snippet: Changes in bladder expression of ( A ) c-Nrf2, ( B ) Keap1, ( C ) n-Nrf2, ( D ) GRP78 (ER stress), ( E ) CHOP (ER stress), ( F ) caspase 3, ( G ) Beclin-1, ( H ) p62, ( I ) LC3 II, ( J ) muscarinic M 2 receptor, ( K ) muscarinic M 3 receptor, ( L ) purinergic P 2 X 1 receptor, ( M ) purinergic P 2 X 2 receptor, and ( N ) purinergic P 2 X 3 receptor in response to 2WBI, 4WBI, and treatments. All of the experiments were performed in three rats of each group. * p

    Techniques Used: Expressing

    Representative histological findings in response to bladder ischemia. The figure shows ( A – C ) GRP78 staining, ( D – F ) CHOP staining, ( G – I ) Beclin-1 staining, ( J – L ) LC3 II, and ( M – O ) TUNEL staining in the sham, 2WBI, and 4WBI groups. All images are magnified with x400. The scale bar is 50 μm. Respective statistic data are shown in ( P ) GRP78, ( Q ) CHOP, ( R ) Beclin-1, ( S ) LC3 II and ( T ) TUNEL stain (n = 6 in each test) in each group. * p
    Figure Legend Snippet: Representative histological findings in response to bladder ischemia. The figure shows ( A – C ) GRP78 staining, ( D – F ) CHOP staining, ( G – I ) Beclin-1 staining, ( J – L ) LC3 II, and ( M – O ) TUNEL staining in the sham, 2WBI, and 4WBI groups. All images are magnified with x400. The scale bar is 50 μm. Respective statistic data are shown in ( P ) GRP78, ( Q ) CHOP, ( R ) Beclin-1, ( S ) LC3 II and ( T ) TUNEL stain (n = 6 in each test) in each group. * p

    Techniques Used: Staining, TUNEL Assay

    14) Product Images from "Genome-wide methylation analysis reveals differentially methylated loci that are associated with an age-dependent increase in bovine fibroblast response to LPS"

    Article Title: Genome-wide methylation analysis reveals differentially methylated loci that are associated with an age-dependent increase in bovine fibroblast response to LPS

    Journal: BMC Genomics

    doi: 10.1186/s12864-017-3796-1

    Fibroblast Response to LPS in Young versus Old Cultures. Interleukin-8 ( a c ) and Interleukin-6 ( b d ) protein production and gene expression were measured in young and old dermal fibroblasts (n = 6 per group) at various time points post LPS. Protein production is presented in pg/ml following 36 h of LPS stimulation. Gene expression was measured by RT-qPCR and is presented as the change in cycles to threshold (dCT) of gene expression at 0, 2 8 h post LPS in comparison to β-actin. Fold change gene expression (Old > Young) is presented in parentheses above each significant time point. All values are displayed as mean (+/− SEM). Significance was measured using a paired Student’s t test at each time point and ** = P
    Figure Legend Snippet: Fibroblast Response to LPS in Young versus Old Cultures. Interleukin-8 ( a c ) and Interleukin-6 ( b d ) protein production and gene expression were measured in young and old dermal fibroblasts (n = 6 per group) at various time points post LPS. Protein production is presented in pg/ml following 36 h of LPS stimulation. Gene expression was measured by RT-qPCR and is presented as the change in cycles to threshold (dCT) of gene expression at 0, 2 8 h post LPS in comparison to β-actin. Fold change gene expression (Old > Young) is presented in parentheses above each significant time point. All values are displayed as mean (+/− SEM). Significance was measured using a paired Student’s t test at each time point and ** = P

    Techniques Used: Expressing, Quantitative RT-PCR

    15) Product Images from "Mouse Bestrophin-2 Is a Bona fide Cl− Channel"

    Article Title: Mouse Bestrophin-2 Is a Bona fide Cl− Channel

    Journal: The Journal of General Physiology

    doi: 10.1085/jgp.200409031

    Localization of mBest2 in the plasma membrane of HEK-293 cells. Lanes 1 and 2: streptavidin labeling of immunoprecipitated proteins. Nontransfected (lane 2) or mBest2-transfected (lane 1) HEK-293 cells were labeled with membrane-impermeant NHS-LC-biotin and lysed. mBest2 was immunoprecipitated with A7116 antibody, run on SDS-PAGE, and transferred to nitrocellulose. The nitrocellulose was then probed with HRP-conjugated streptavidin. A 64kD band was observed in transfected, but not nontransfected cells. Lanes 3–5: Western blots. Lane 3: total extract from mBest2-transfected cells was probed with antibody to α-actin. Lanes 4 and 5: the extract from mBest2-transfected cells was incubated with streptavidin-beads to collect biotinylated proteins. Biotinylated proteins were then probed with antibody to α-actin (Lane 4) or B4947 mBest2-specific antibody (Lane 5).
    Figure Legend Snippet: Localization of mBest2 in the plasma membrane of HEK-293 cells. Lanes 1 and 2: streptavidin labeling of immunoprecipitated proteins. Nontransfected (lane 2) or mBest2-transfected (lane 1) HEK-293 cells were labeled with membrane-impermeant NHS-LC-biotin and lysed. mBest2 was immunoprecipitated with A7116 antibody, run on SDS-PAGE, and transferred to nitrocellulose. The nitrocellulose was then probed with HRP-conjugated streptavidin. A 64kD band was observed in transfected, but not nontransfected cells. Lanes 3–5: Western blots. Lane 3: total extract from mBest2-transfected cells was probed with antibody to α-actin. Lanes 4 and 5: the extract from mBest2-transfected cells was incubated with streptavidin-beads to collect biotinylated proteins. Biotinylated proteins were then probed with antibody to α-actin (Lane 4) or B4947 mBest2-specific antibody (Lane 5).

    Techniques Used: Labeling, Immunoprecipitation, Transfection, SDS Page, Western Blot, Incubation

    16) Product Images from "Development of a Sphingosylphosphorylcholine Detection System Using RNA Aptamers"

    Article Title: Development of a Sphingosylphosphorylcholine Detection System Using RNA Aptamers

    Journal: Molecules

    doi: 10.3390/molecules15085742

    ELAA. (a) Sensitivity of the m012 aptamer for biotinylated SPC. The amount of biotinylated SPC captured by immobilized m012 was shown as the absorbance at 405 nm. The experiment was repeated three times with each data point measured in duplicates, and a representative result is shown. (b) Competitive ELAA using m012 and biotinylated and unmodified SPC. The biotinylated and serially diluted unmodified SPC were added to the m012-immobilized plate. The amount of biotinylated SPC captured by the immobilized m012 was shown as the absorbance at 405 nm. The experiment was repeated three times with each data point measured in duplicates, and a representative result is shown. (c) Specificity of the m012 aptamer for SPC and S1P. The amount of the biotinylated m012 reacted with the immobilized biotinylated SPC and S1P was shown as the absorbance at 405 nm. The experiment was repeated three times in triplicates, and a representative result is shown. Error bars show standard deviations.
    Figure Legend Snippet: ELAA. (a) Sensitivity of the m012 aptamer for biotinylated SPC. The amount of biotinylated SPC captured by immobilized m012 was shown as the absorbance at 405 nm. The experiment was repeated three times with each data point measured in duplicates, and a representative result is shown. (b) Competitive ELAA using m012 and biotinylated and unmodified SPC. The biotinylated and serially diluted unmodified SPC were added to the m012-immobilized plate. The amount of biotinylated SPC captured by the immobilized m012 was shown as the absorbance at 405 nm. The experiment was repeated three times with each data point measured in duplicates, and a representative result is shown. (c) Specificity of the m012 aptamer for SPC and S1P. The amount of the biotinylated m012 reacted with the immobilized biotinylated SPC and S1P was shown as the absorbance at 405 nm. The experiment was repeated three times in triplicates, and a representative result is shown. Error bars show standard deviations.

    Techniques Used:

    Bindinganalysis of the m012 aptamer to SPC and S1P using SPR. The biotinylated SPC and S1P were immobilized on each flow cell at similar amounts (~250 RU). 400 nM aptamer was injected over the ligands for 2 min.
    Figure Legend Snippet: Bindinganalysis of the m012 aptamer to SPC and S1P using SPR. The biotinylated SPC and S1P were immobilized on each flow cell at similar amounts (~250 RU). 400 nM aptamer was injected over the ligands for 2 min.

    Techniques Used: SPR Assay, Flow Cytometry, Injection

    17) Product Images from "Glypican-1 Mediates Both Prion Protein Lipid Raft Association and Disease Isoform Formation"

    Article Title: Glypican-1 Mediates Both Prion Protein Lipid Raft Association and Disease Isoform Formation

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1000666

    Depletion of glypican-1 stimulates the endocytosis of PrP C . SH-SY5Y cells expressing wild type PrP C were treated with either control or glypican-1 siRNA and then incubated for 60 h. Cells were surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Where indicated, cells were treated with trypsin to remove remaining cell surface PrP C . Cells were then lysed and total PrP C immunoprecipitated from the sample using antibody 3F4. ( A ) Samples were subjected to western blot analysis and the biotin-labelled PrP C fraction was detected with peroxidase-conjugated streptavidin. ( B ) Densitometric analysis (mean ± s.e.m.) of multiple blots from three separate experiments in (A) is shown. ( C ) Expression of glypican-1 (in lysate samples treated with heparinase I and heparinase III) and PrP C in the cell lysates from (A). β-actin was used as a loading control. ( D ) SH-SY5Y cells expressing PrP C were treated with either control siRNA or glypican-1 siRNA and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( E ) Densitometric analysis of the proportion of total PrP C present in the detergent soluble fractions of the plasma membrane after siRNA treatment from three independent experiments. ( F ) SH-SY5Y cells expressing PrP C were seeded onto glass coverslips and grown to 50% confluency. Cells were fixed, and then incubated with anti-PrP antibody 3F4 and a glypican-1 polyclonal antibody. Finally, cells were incubated with Alexa488-conjugated rabbit anti-mouse and Alexa594-conjugated goat anti-rabbit antibodies and viewed using a DeltaVision Optical Restoration Microscopy System. Images are representative of three individual experiments. Scale bars equal 10 µm. * P
    Figure Legend Snippet: Depletion of glypican-1 stimulates the endocytosis of PrP C . SH-SY5Y cells expressing wild type PrP C were treated with either control or glypican-1 siRNA and then incubated for 60 h. Cells were surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Where indicated, cells were treated with trypsin to remove remaining cell surface PrP C . Cells were then lysed and total PrP C immunoprecipitated from the sample using antibody 3F4. ( A ) Samples were subjected to western blot analysis and the biotin-labelled PrP C fraction was detected with peroxidase-conjugated streptavidin. ( B ) Densitometric analysis (mean ± s.e.m.) of multiple blots from three separate experiments in (A) is shown. ( C ) Expression of glypican-1 (in lysate samples treated with heparinase I and heparinase III) and PrP C in the cell lysates from (A). β-actin was used as a loading control. ( D ) SH-SY5Y cells expressing PrP C were treated with either control siRNA or glypican-1 siRNA and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( E ) Densitometric analysis of the proportion of total PrP C present in the detergent soluble fractions of the plasma membrane after siRNA treatment from three independent experiments. ( F ) SH-SY5Y cells expressing PrP C were seeded onto glass coverslips and grown to 50% confluency. Cells were fixed, and then incubated with anti-PrP antibody 3F4 and a glypican-1 polyclonal antibody. Finally, cells were incubated with Alexa488-conjugated rabbit anti-mouse and Alexa594-conjugated goat anti-rabbit antibodies and viewed using a DeltaVision Optical Restoration Microscopy System. Images are representative of three individual experiments. Scale bars equal 10 µm. * P

    Techniques Used: Expressing, Incubation, Immunoprecipitation, Western Blot, Gradient Centrifugation, Microscopy

    Depletion of glypican-1 does not affect cell division or surface levels of PrP C . ( A ) ScN2a cells were seeded into 96 well plates and treated with transfection reagent only or incubated with either control siRNA or one of the four siRNAs targeted to glypican-1. Those experiments exceeding 48 h were dosed with a second treatment of the indicated siRNAs. Cells were then rinsed with PBS and fixed with 70% (v/v) ethanol. Plates were allowed to dry, stained with Hoescht 33342 and the fluorescence measured. ( B ) ScN2a cells were treated with control or glypican-1 siRNA. After 96 h, cell monolayers were labelled with a membrane impermeable biotin reagent. Biotin-labelled cell surface PrP was detected by immunoprecipitation using 6D11 and subsequent immunoblotting using HRP-conjugated streptavidin. Total PrP and PK-resistant PrP (PrP Sc ) were detected by immunoblotting using antibody 6D11. ( C ) Densitometric analysis of the proportion of the relative amount of biotinylated cell surface PrP in the absence or presence of glypican-1 siRNA from three independent experiments.
    Figure Legend Snippet: Depletion of glypican-1 does not affect cell division or surface levels of PrP C . ( A ) ScN2a cells were seeded into 96 well plates and treated with transfection reagent only or incubated with either control siRNA or one of the four siRNAs targeted to glypican-1. Those experiments exceeding 48 h were dosed with a second treatment of the indicated siRNAs. Cells were then rinsed with PBS and fixed with 70% (v/v) ethanol. Plates were allowed to dry, stained with Hoescht 33342 and the fluorescence measured. ( B ) ScN2a cells were treated with control or glypican-1 siRNA. After 96 h, cell monolayers were labelled with a membrane impermeable biotin reagent. Biotin-labelled cell surface PrP was detected by immunoprecipitation using 6D11 and subsequent immunoblotting using HRP-conjugated streptavidin. Total PrP and PK-resistant PrP (PrP Sc ) were detected by immunoblotting using antibody 6D11. ( C ) Densitometric analysis of the proportion of the relative amount of biotinylated cell surface PrP in the absence or presence of glypican-1 siRNA from three independent experiments.

    Techniques Used: Transfection, Incubation, Staining, Fluorescence, Immunoprecipitation

    Heparin stimulates the endocytosis of PrP C in a dose-dependent manner and displaces it from detergent-resistant lipid rafts. ( A ) SH-SY5Y cells expressing PrP C were surface biotinylated and then incubated for 1 h at 37°C in the absence or presence of various concentrations of heparin diluted in OptiMEM. Prior to lysis cells were, where indicated, incubated with trypsin to digest cell surface PrP C . Cells were then lysed and PrP C immunoprecipitated from the sample using antibody 3F4. Samples were subjected to SDS PAGE and western blot analysis and the biotin-labelled PrP C detected with peroxidase-conjugated streptavidin. ( B ) Densitometric analysis of multiple blots from four separate experiments as described in (A) is shown. ( C ) SH-SY5Y cells expressing PrP C were surface biotinylated and then incubated in the absence or presence of 50 µM heparin prepared in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. PrP C was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and subjected to SDS-PAGE and western blotting. The gradient fractions from both the untreated and heparin treated cells were analysed on the same SDS gel and immunoblotted under identical conditions. The biotin-labelled PrP C was detected with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions, respectively. ( D ) Densitometric analysis of the proportion of total PrP C in the detergent soluble fractions of the plasma membrane. ( E ) Untransfected SH-SY5Y cells and SH-SY5Y cells expressing either PrP C or PrP-TM were grown to confluence and then incubated for 1 h in the presence or absence of 50 µM heparin prepared in OptiMEM. Media samples were collected and concentrated and cells harvested and lysed. Cell lysate samples were immunoblotted for PrP C using antibody 3F4, with β-actin used as a loading control. ( F ) Quantification of PrP C and PrP-TM levels after treatment of cells with heparin as in (E). Experiments were performed in triplicate and repeated on three occasions. * P
    Figure Legend Snippet: Heparin stimulates the endocytosis of PrP C in a dose-dependent manner and displaces it from detergent-resistant lipid rafts. ( A ) SH-SY5Y cells expressing PrP C were surface biotinylated and then incubated for 1 h at 37°C in the absence or presence of various concentrations of heparin diluted in OptiMEM. Prior to lysis cells were, where indicated, incubated with trypsin to digest cell surface PrP C . Cells were then lysed and PrP C immunoprecipitated from the sample using antibody 3F4. Samples were subjected to SDS PAGE and western blot analysis and the biotin-labelled PrP C detected with peroxidase-conjugated streptavidin. ( B ) Densitometric analysis of multiple blots from four separate experiments as described in (A) is shown. ( C ) SH-SY5Y cells expressing PrP C were surface biotinylated and then incubated in the absence or presence of 50 µM heparin prepared in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. PrP C was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and subjected to SDS-PAGE and western blotting. The gradient fractions from both the untreated and heparin treated cells were analysed on the same SDS gel and immunoblotted under identical conditions. The biotin-labelled PrP C was detected with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions, respectively. ( D ) Densitometric analysis of the proportion of total PrP C in the detergent soluble fractions of the plasma membrane. ( E ) Untransfected SH-SY5Y cells and SH-SY5Y cells expressing either PrP C or PrP-TM were grown to confluence and then incubated for 1 h in the presence or absence of 50 µM heparin prepared in OptiMEM. Media samples were collected and concentrated and cells harvested and lysed. Cell lysate samples were immunoblotted for PrP C using antibody 3F4, with β-actin used as a loading control. ( F ) Quantification of PrP C and PrP-TM levels after treatment of cells with heparin as in (E). Experiments were performed in triplicate and repeated on three occasions. * P

    Techniques Used: Expressing, Incubation, Lysis, Immunoprecipitation, SDS Page, Western Blot, Gradient Centrifugation, SDS-Gel

    Depletion of glypican-1 inhibits the association of PrP-TM with DRMs. SH-SY5Y cells expressing PrP-TM were treated with either control siRNA or siRNA targeted to glypican-1 and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C in the presence of Tyrphostin A23 to block endocytosis. The media was removed and the cells washed in phosphate-buffered saline prior to homogenisation in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( A ) Quantification of glypican-1 and PrP-TM expression in cell lysates. To detect glypican-1, cell lysate samples were treated with heparinase I and heparinase III prior to electrophoresis as described in the materials and methods section. ( B ) PrP-TM was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and then subjected to western blotting with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions, respectively. ( C ) Densitometric analysis of the proportion of total PrP-TM present in the detergent soluble fractions of the plasma membrane after siRNA treatment from multiple blots from three independent experiments. * P
    Figure Legend Snippet: Depletion of glypican-1 inhibits the association of PrP-TM with DRMs. SH-SY5Y cells expressing PrP-TM were treated with either control siRNA or siRNA targeted to glypican-1 and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C in the presence of Tyrphostin A23 to block endocytosis. The media was removed and the cells washed in phosphate-buffered saline prior to homogenisation in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( A ) Quantification of glypican-1 and PrP-TM expression in cell lysates. To detect glypican-1, cell lysate samples were treated with heparinase I and heparinase III prior to electrophoresis as described in the materials and methods section. ( B ) PrP-TM was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and then subjected to western blotting with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions, respectively. ( C ) Densitometric analysis of the proportion of total PrP-TM present in the detergent soluble fractions of the plasma membrane after siRNA treatment from multiple blots from three independent experiments. * P

    Techniques Used: Expressing, Incubation, Blocking Assay, Homogenization, Gradient Centrifugation, Electrophoresis, Immunoprecipitation, Western Blot

    The association of PrP-TM with DRMs is disrupted by treatment of cells with either heparin or bacterial PI-PLC. SH-SY5Y cells expressing PrP-TM were surface biotinylated and then ( A ) incubated in the absence or presence of 50 µM heparin prepared in OptiMEM for 1 h at 37°C or ( B ) incubated in the absence or presence of 1 U/ml bacterial PI-PLC for 1 h at 4°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. PrP-TM was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and subjected to western blotting. The biotin-labelled PrP-TM fraction was detected with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions respectively. ( C ) Densitometric analysis of the proportion of total PrP-TM present in the detergent soluble fractions of the plasma membrane after heparin and PI-PLC treatment. Experiments were performed in triplicate and repeated on three occasions. * P
    Figure Legend Snippet: The association of PrP-TM with DRMs is disrupted by treatment of cells with either heparin or bacterial PI-PLC. SH-SY5Y cells expressing PrP-TM were surface biotinylated and then ( A ) incubated in the absence or presence of 50 µM heparin prepared in OptiMEM for 1 h at 37°C or ( B ) incubated in the absence or presence of 1 U/ml bacterial PI-PLC for 1 h at 4°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. PrP-TM was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and subjected to western blotting. The biotin-labelled PrP-TM fraction was detected with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions respectively. ( C ) Densitometric analysis of the proportion of total PrP-TM present in the detergent soluble fractions of the plasma membrane after heparin and PI-PLC treatment. Experiments were performed in triplicate and repeated on three occasions. * P

    Techniques Used: Planar Chromatography, Expressing, Incubation, Gradient Centrifugation, Immunoprecipitation, Western Blot

    18) Product Images from "Human and murine inhibitory natural killer cell receptors transfer from natural killer cells to target cells"

    Article Title: Human and murine inhibitory natural killer cell receptors transfer from natural killer cells to target cells

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

    doi: 10.1073/pnas.0406240101

    Factors influencing the amount of KIR2DL1-GFP transfer to target cells. ( A ) Conjugates of YTS-TG- and PKH-26-labeled 221/Cw6 cells were isolated by FACS, disrupted with EDTA, and analyzed for expression of MHC class I, MHC class II, CD53, or CD54. Dot
    Figure Legend Snippet: Factors influencing the amount of KIR2DL1-GFP transfer to target cells. ( A ) Conjugates of YTS-TG- and PKH-26-labeled 221/Cw6 cells were isolated by FACS, disrupted with EDTA, and analyzed for expression of MHC class I, MHC class II, CD53, or CD54. Dot

    Techniques Used: Labeling, Isolation, FACS, Expressing

    19) Product Images from "Quail egg homogenate alleviates food allergy induced eosinophilic esophagitis like disease through modulating PAR-2 transduction pathway in peanut sensitized mice"

    Article Title: Quail egg homogenate alleviates food allergy induced eosinophilic esophagitis like disease through modulating PAR-2 transduction pathway in peanut sensitized mice

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-19309-x

    Oral quail egg treatment reduced PPE specific IgE, IgG1, and the level release of allergic mediators (n = 10). ( A ) PPE and quail egg specific IgE levels ( B ) PPE and quail egg specific IgG1 levels; Allergic mediators: ( C ) histamine, ( D ) tryptase; ( E ) ECP. Results are expressed as mean ± SEM. * P
    Figure Legend Snippet: Oral quail egg treatment reduced PPE specific IgE, IgG1, and the level release of allergic mediators (n = 10). ( A ) PPE and quail egg specific IgE levels ( B ) PPE and quail egg specific IgG1 levels; Allergic mediators: ( C ) histamine, ( D ) tryptase; ( E ) ECP. Results are expressed as mean ± SEM. * P

    Techniques Used:

    20) Product Images from "Chemical labelling for visualizing native AMPA receptors in live neurons"

    Article Title: Chemical labelling for visualizing native AMPA receptors in live neurons

    Journal: Nature Communications

    doi: 10.1038/ncomms14850

    Chemical labelling of native AMPARs in cultured neurons. ( a ) Western blot analyses of cultured neurons after labelling using CAM reagents. Cultured cortical neurons were treated with 1 μM of CAM2(OG) , CAM2(Fl) , CAM2(Ax488) , or CAM2(Bt) in the absence or presence of 10 μM NBQX in serum free Neurobasal medium. The cell lysates were analyzed by western blot using anti-Fl/OG, anti-Ax488, or anti-GluA2 antibody, or by biotin blotting using streptavidin-HRP. * indicates biotinylated proteins endogenously expressed in the neurons. ( b ) Effect of competitive antagonists for glutamate receptors on chemical labelling of native AMPARs in cultured neurons. Western blot analyses of cultured neurons after labelling using CAM reagents are shown. Cultured cortical neurons were treated with 1 μM of CAM2(OG) in the absence or presence of 10 μM NBQX, 10 μM AP5, or 10 μM (2S,4R)-4-methyl glutamate (4MG) to examine selective labelling of AMPARs among the ionotropic glutamate receptor family. ( c ) Analyses of labelled proteins in cultured neurons by immunoprecipitation using anti-Fl/OG antibody. Chemical labelling was conducted with the same procedure described in a . After lysis of labelled cultured neurons by CAM2(Fl) , the cell lysate was immunoprecipitated with anti-Fl/OG antibodies. The immunoprecipitates were analyzed by western blot using glutamate receptor-specific antibodies. ( d – h ) Confocal imaging of cultured neurons after labelling using CAM reagents. Cultured hippocampal neurons labelled with 1 μM CAM2(Fl) were fixed, permeabilized and immunostained using anti-MAP2 (in d , f ), anti-GluA2 (in e , g ) or anti-PSD95 antibody (in h ). White square ROIs indicated in d , e are expanded in f , g , respectively. Scale bars, 10 μm ( d , e ) and 5 μm ( f – h ). Full blots for b and c are shown in Supplementary Fig. 22 .
    Figure Legend Snippet: Chemical labelling of native AMPARs in cultured neurons. ( a ) Western blot analyses of cultured neurons after labelling using CAM reagents. Cultured cortical neurons were treated with 1 μM of CAM2(OG) , CAM2(Fl) , CAM2(Ax488) , or CAM2(Bt) in the absence or presence of 10 μM NBQX in serum free Neurobasal medium. The cell lysates were analyzed by western blot using anti-Fl/OG, anti-Ax488, or anti-GluA2 antibody, or by biotin blotting using streptavidin-HRP. * indicates biotinylated proteins endogenously expressed in the neurons. ( b ) Effect of competitive antagonists for glutamate receptors on chemical labelling of native AMPARs in cultured neurons. Western blot analyses of cultured neurons after labelling using CAM reagents are shown. Cultured cortical neurons were treated with 1 μM of CAM2(OG) in the absence or presence of 10 μM NBQX, 10 μM AP5, or 10 μM (2S,4R)-4-methyl glutamate (4MG) to examine selective labelling of AMPARs among the ionotropic glutamate receptor family. ( c ) Analyses of labelled proteins in cultured neurons by immunoprecipitation using anti-Fl/OG antibody. Chemical labelling was conducted with the same procedure described in a . After lysis of labelled cultured neurons by CAM2(Fl) , the cell lysate was immunoprecipitated with anti-Fl/OG antibodies. The immunoprecipitates were analyzed by western blot using glutamate receptor-specific antibodies. ( d – h ) Confocal imaging of cultured neurons after labelling using CAM reagents. Cultured hippocampal neurons labelled with 1 μM CAM2(Fl) were fixed, permeabilized and immunostained using anti-MAP2 (in d , f ), anti-GluA2 (in e , g ) or anti-PSD95 antibody (in h ). White square ROIs indicated in d , e are expanded in f , g , respectively. Scale bars, 10 μm ( d , e ) and 5 μm ( f – h ). Full blots for b and c are shown in Supplementary Fig. 22 .

    Techniques Used: Cell Culture, Western Blot, Chick Chorioallantoic Membrane Assay, Immunoprecipitation, Lysis, Imaging

    21) Product Images from "Sequence Variation in Promoter of Ica1 Gene, Which Encodes Protein Implicated in Type 1 Diabetes, Causes Transcription Factor Autoimmune Regulator (AIRE) to Increase Its Binding and Down-regulate Expression *"

    Article Title: Sequence Variation in Promoter of Ica1 Gene, Which Encodes Protein Implicated in Type 1 Diabetes, Causes Transcription Factor Autoimmune Regulator (AIRE) to Increase Its Binding and Down-regulate Expression *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.319020

    ICA69 autoantigen expression in the human thymus ( A and B ), spleen ( C ), and lymph node ( D ) denoted with arrows . A , a microphotograph of ICA69 using the ICA69 mAb and alkaline phosphatase immunoenzymatic detection. Staining of a paraformaldehyde-fixed section of human thymus with the ICA69 mAb shows selective staining for a cell subtype that is present predominantly in the medulla (×200). B , ICA69-specific staining disappears after absorbing out the ICA69 mAb with the recombinant human ICA69 molecule against which this mAb was produced (×200). C and D , immunostaining of spleen (×400) and lymph node (×200) frozen sections after incubation with the ICA69 mAb detected with the streptavidin-biotin-peroxidase method and the aminoethylcarbazole substrate ( red ) and counterstaining with hematoxylin.
    Figure Legend Snippet: ICA69 autoantigen expression in the human thymus ( A and B ), spleen ( C ), and lymph node ( D ) denoted with arrows . A , a microphotograph of ICA69 using the ICA69 mAb and alkaline phosphatase immunoenzymatic detection. Staining of a paraformaldehyde-fixed section of human thymus with the ICA69 mAb shows selective staining for a cell subtype that is present predominantly in the medulla (×200). B , ICA69-specific staining disappears after absorbing out the ICA69 mAb with the recombinant human ICA69 molecule against which this mAb was produced (×200). C and D , immunostaining of spleen (×400) and lymph node (×200) frozen sections after incubation with the ICA69 mAb detected with the streptavidin-biotin-peroxidase method and the aminoethylcarbazole substrate ( red ) and counterstaining with hematoxylin.

    Techniques Used: Expressing, Staining, Recombinant, Produced, Immunostaining, Incubation

    22) Product Images from "Colonic epithelial cells are a major site of macrophage inflammatory protein 3? (MIP-3?) production in normal colon and inflammatory bowel disease"

    Article Title: Colonic epithelial cells are a major site of macrophage inflammatory protein 3? (MIP-3?) production in normal colon and inflammatory bowel disease

    Journal: Gut

    doi:

    Kinetics of increased steady state macrophage inflammatory protein (MIP)-3α mRNA levels in cytokine stimulated Caco-2 and HT-29 cells. (A) Confluent Caco-2 cell monolayers were exposed to recombinant interleukin (IL)-1β (1 ng/ml) for 0–24 hours. Steady state levels of MIP-3α mRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA were then determined by reverse transcription-polymerase chain reaction (RT-PCR). (B) Confluent HT-29 cell monolayers were exposed to recombinant tumour necrosis factor α (TNF-α 10 ng/ml) for 0–24 hours. Steady state levels of MIP-3α mRNA and GAPDH mRNA were determined by RT-PCR.
    Figure Legend Snippet: Kinetics of increased steady state macrophage inflammatory protein (MIP)-3α mRNA levels in cytokine stimulated Caco-2 and HT-29 cells. (A) Confluent Caco-2 cell monolayers were exposed to recombinant interleukin (IL)-1β (1 ng/ml) for 0–24 hours. Steady state levels of MIP-3α mRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA were then determined by reverse transcription-polymerase chain reaction (RT-PCR). (B) Confluent HT-29 cell monolayers were exposed to recombinant tumour necrosis factor α (TNF-α 10 ng/ml) for 0–24 hours. Steady state levels of MIP-3α mRNA and GAPDH mRNA were determined by RT-PCR.

    Techniques Used: Recombinant, Reverse Transcription Polymerase Chain Reaction

    Time course of macrophage inflammatory protein (MIP)-3α protein production by cytokine stimulated Caco-2 and HT-29 cells. (A) Confluent Caco-2 cell monolayers were incubated with or without recombinant interleukin (IL)-1β (1 ng/ml) for 0–24 hours. Accumulated MIP-3α protein levels in conditioned media from non-stimulated cells or cells treated with IL-1β were then measured by ELISA. Data are expressed as mean (SD) (n=4). (B) Confluent HT-29 cell monolayers were incubated with or without recombinant tumour necrosis factor α (TNF-α 10 ng/ml) for 0–24 h. Accumulated MIP-3α protein levels in conditioned media from non-stimulated cells or cells treated with TNF-α were then measured by ELISA. Data are expressed as mean (SD) (n=4).
    Figure Legend Snippet: Time course of macrophage inflammatory protein (MIP)-3α protein production by cytokine stimulated Caco-2 and HT-29 cells. (A) Confluent Caco-2 cell monolayers were incubated with or without recombinant interleukin (IL)-1β (1 ng/ml) for 0–24 hours. Accumulated MIP-3α protein levels in conditioned media from non-stimulated cells or cells treated with IL-1β were then measured by ELISA. Data are expressed as mean (SD) (n=4). (B) Confluent HT-29 cell monolayers were incubated with or without recombinant tumour necrosis factor α (TNF-α 10 ng/ml) for 0–24 h. Accumulated MIP-3α protein levels in conditioned media from non-stimulated cells or cells treated with TNF-α were then measured by ELISA. Data are expressed as mean (SD) (n=4).

    Techniques Used: Incubation, Recombinant, Enzyme-linked Immunosorbent Assay

    Enterocytes are the primary site of macrophage inflammatory protein (MIP)-3α production in normal colon and inflammatory bowel disease. (A, B) Normal colon. MIP-3α immunoreactivity is principally associated with colonic epithelial cells (B). Minimal staining of enterocytes was observed in normal colon using an isotype control monoclonal IgG 1 antibody (A). (C, D) Ulcerative colitis. Similar to normal colon, MIP-3α immunoreactivity in ulcerative colitis was mainly associated with epithelial cells (D). Little staining of epithelial cells was seen in ulcerative colitis tissue samples stained with the isotype control monoclonal antibody (C). (E, F) Crohn’s disease. Again, in colonic tissues from patients with Crohn’s disease MIP-3α immunoreactivity was primarily observed in epithelial cells (F). In contrast, Crohn’s disease sections treated with the isotype control monoclonal antibody showed little immunostaining (E). (G, H) Antibody controls. Minimal staining of epithelial cells was seen in Crohn’s disease tissue samples when the primary antibody was omitted (G). As expected, no staining of Crohn’s disease tissues was observed on omission of both the primary and secondary antibodies (H). Magnification for each panel, ×200.
    Figure Legend Snippet: Enterocytes are the primary site of macrophage inflammatory protein (MIP)-3α production in normal colon and inflammatory bowel disease. (A, B) Normal colon. MIP-3α immunoreactivity is principally associated with colonic epithelial cells (B). Minimal staining of enterocytes was observed in normal colon using an isotype control monoclonal IgG 1 antibody (A). (C, D) Ulcerative colitis. Similar to normal colon, MIP-3α immunoreactivity in ulcerative colitis was mainly associated with epithelial cells (D). Little staining of epithelial cells was seen in ulcerative colitis tissue samples stained with the isotype control monoclonal antibody (C). (E, F) Crohn’s disease. Again, in colonic tissues from patients with Crohn’s disease MIP-3α immunoreactivity was primarily observed in epithelial cells (F). In contrast, Crohn’s disease sections treated with the isotype control monoclonal antibody showed little immunostaining (E). (G, H) Antibody controls. Minimal staining of epithelial cells was seen in Crohn’s disease tissue samples when the primary antibody was omitted (G). As expected, no staining of Crohn’s disease tissues was observed on omission of both the primary and secondary antibodies (H). Magnification for each panel, ×200.

    Techniques Used: Staining, Immunostaining

    Macrophage inflammatory protein (MIP)-3α mRNA and protein levels are elevated in Crohn’s disease. Colonic tissue samples were obtained from patients undergoing colectomy for ulcerative colitis or Crohn’s colitis and from patients undergoing colectomy for colon cancer to serve as non-inflamed controls. (A) Total RNA derived from each tissue was reverse transcribed and subjected to real time quantitative reverse transcription-polymerase chain reaction (RT-PCR) to evaluate MIP-3α and GAPDH mRNA levels. Results are expressed as the number of MIP-3α amplicons per 10 4 glyceraldehyde-3-phosphate dehydrogenase (GAPDH) amplicons. The horizontal bar represents the mean value for each group. (B) Tissue homogenates from each specimen were subjected to ELISA to quantify MIP-3α protein levels. Results are expressed as ng of MIP-3α per mg of total homogenate protein. The horizontal bar represents the mean value for each group.
    Figure Legend Snippet: Macrophage inflammatory protein (MIP)-3α mRNA and protein levels are elevated in Crohn’s disease. Colonic tissue samples were obtained from patients undergoing colectomy for ulcerative colitis or Crohn’s colitis and from patients undergoing colectomy for colon cancer to serve as non-inflamed controls. (A) Total RNA derived from each tissue was reverse transcribed and subjected to real time quantitative reverse transcription-polymerase chain reaction (RT-PCR) to evaluate MIP-3α and GAPDH mRNA levels. Results are expressed as the number of MIP-3α amplicons per 10 4 glyceraldehyde-3-phosphate dehydrogenase (GAPDH) amplicons. The horizontal bar represents the mean value for each group. (B) Tissue homogenates from each specimen were subjected to ELISA to quantify MIP-3α protein levels. Results are expressed as ng of MIP-3α per mg of total homogenate protein. The horizontal bar represents the mean value for each group.

    Techniques Used: Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    Macrophage inflammatory protein (MIP)-3α protein production by primary epithelial cells is increased during inflammatory bowel disease. Epithelial cells were isolated from tissue samples obtained from patients undergoing colectomy for ulcerative colitis (UC) or Crohn’s disease (CD), and from tissue samples obtained from patients undergoing colectomy for colon cancer to serve as non-inflamed controls. Homogenates of each tissue were prepared and subjected to ELISA to quantify MIP-3α protein levels. Results are expressed as ng of MIP-3α per mg of total homogenate protein. The horizontal bar represents the mean value for each group.
    Figure Legend Snippet: Macrophage inflammatory protein (MIP)-3α protein production by primary epithelial cells is increased during inflammatory bowel disease. Epithelial cells were isolated from tissue samples obtained from patients undergoing colectomy for ulcerative colitis (UC) or Crohn’s disease (CD), and from tissue samples obtained from patients undergoing colectomy for colon cancer to serve as non-inflamed controls. Homogenates of each tissue were prepared and subjected to ELISA to quantify MIP-3α protein levels. Results are expressed as ng of MIP-3α per mg of total homogenate protein. The horizontal bar represents the mean value for each group.

    Techniques Used: Isolation, Enzyme-linked Immunosorbent Assay

    Human gastrointestinal epithelial cell lines produce macrophage inflammatory protein (MIP)-3α in response to cytokine stimulation. (A) Confluent Caco-2 cell monolayers were exposed to recombinant interleukin (IL)-1β (0.01–100 ng/ml) for 24 hours. MIP-3α protein levels in conditioned media were then measured by ELISA. (B) Confluent HT-29 cell monolayers were exposed to recombinant tumour necrosis factor α (TNF-α 0.01–100 ng/ml) for 24 hours and MIP-3α protein levels in conditioned media were then measured by ELISA. Data are expressed as mean (SEM) (n=4). *p
    Figure Legend Snippet: Human gastrointestinal epithelial cell lines produce macrophage inflammatory protein (MIP)-3α in response to cytokine stimulation. (A) Confluent Caco-2 cell monolayers were exposed to recombinant interleukin (IL)-1β (0.01–100 ng/ml) for 24 hours. MIP-3α protein levels in conditioned media were then measured by ELISA. (B) Confluent HT-29 cell monolayers were exposed to recombinant tumour necrosis factor α (TNF-α 0.01–100 ng/ml) for 24 hours and MIP-3α protein levels in conditioned media were then measured by ELISA. Data are expressed as mean (SEM) (n=4). *p

    Techniques Used: Recombinant, Enzyme-linked Immunosorbent Assay

    23) Product Images from "Real-time in vivo detection of biomaterial-induced reactive oxygen species"

    Article Title: Real-time in vivo detection of biomaterial-induced reactive oxygen species

    Journal: Biomaterials

    doi: 10.1016/j.biomaterials.2010.11.029

    Characterization of myeloperoxidase expression
    Figure Legend Snippet: Characterization of myeloperoxidase expression

    Techniques Used: Expressing

    24) Product Images from "Prion Nucleation Site Unmasked by Transient Interaction with Phospholipid Cofactor"

    Article Title: Prion Nucleation Site Unmasked by Transient Interaction with Phospholipid Cofactor

    Journal: Biochemistry

    doi: 10.1021/bi4014825

    Photoaffinity labeling of various PrP species. Streptavidin-HRP-probed blots of samples photoaffinity labeled with PA-PBD peptide. (A) Samples containing PrP Int1 or PrP C were incubated with or without PA-PBD and exposed to UV light for varying time periods, as indicated. (B) Samples containing α -helical PrP or PrP Int1 were incubated with PA-PBD and exposed to UV light for 5 min. (C) Samples of PrP Int1 were incubated with varying concentrations of PA-PBD, as indicated, and exposed to UV light for 0 or 5 min, as indicated. (D) Sample containing 7 μ g of PrP Int1 photoaffinity labeled with PA-PBD (PA-PrP Int1 ) is compared to a standard curve of biotinylated AviTag PrP for reference.
    Figure Legend Snippet: Photoaffinity labeling of various PrP species. Streptavidin-HRP-probed blots of samples photoaffinity labeled with PA-PBD peptide. (A) Samples containing PrP Int1 or PrP C were incubated with or without PA-PBD and exposed to UV light for varying time periods, as indicated. (B) Samples containing α -helical PrP or PrP Int1 were incubated with PA-PBD and exposed to UV light for 5 min. (C) Samples of PrP Int1 were incubated with varying concentrations of PA-PBD, as indicated, and exposed to UV light for 0 or 5 min, as indicated. (D) Sample containing 7 μ g of PrP Int1 photoaffinity labeled with PA-PBD (PA-PrP Int1 ) is compared to a standard curve of biotinylated AviTag PrP for reference.

    Techniques Used: Labeling, Incubation

    25) Product Images from "Glycogen synthase kinase 3β ubiquitination by TRAF6 regulates TLR3-mediated pro-inflammatory cytokine production"

    Article Title: Glycogen synthase kinase 3β ubiquitination by TRAF6 regulates TLR3-mediated pro-inflammatory cytokine production

    Journal: Nature Communications

    doi: 10.1038/ncomms7765

    TRAF6-mediated GSK3β ubiquitination at lysine 183 is critical for TLR3-dependent cytokine production. ( a ) BMDMs were stimulated with 10 μg ml −1 poly I:C for 10 min and subjected to immunoprecipitation with an anti-Ub antibody followed by western blotting with an anti-GSK3β antibody. ( b ) HEK293T cells transfected with HA-GSK3β and HA-Ub along with Flag-TRAF6 plasmids were subjected to immunoprecipitation with an anti-GSK3β antibody followed by western blotting with an anti-HA antibody. ( c ) HEK293T cells were transfected with HA-GSK3β and HA-Ub along with TRAF6 (WT) or TRAF6 (C70A) plasmids. These experiments were performed as described in b . ( d ) Traf6 +/+ and Traf6 −/− 3T3 cells stimulated with 10 μg ml −1 poly I:C for 10 min were subjected to immunoprecipitation with an anti-GSK3β antibody followed by western blotting with an anti-Ub antibody. ( e ) GSK3β proteins were incubated with E1, E2 and biotinylated-Ub (Bt-Ub) in the presence or absence of Flag-TRAF6 proteins for in vitro ubiquitination of GSK3β. Ubiquitination of GSK3β was analysed by western blotting with streptavidin-HRP. ( f ) HEK293T cells transfected with Ub and Flag-TRAF6 along with HA-GSK3β WT or various HA-GSK3β mutants were subjected to immunoprecipitation with an anti-HA antibody followed by western blotting with an anti-Ub antibody. ( g ) HEK293-TLR3 cells were transiently transfected with GSK3β (WT) or GSK3β (K183R) plasmids. The levels of IL-6, TNF-α and c-Fos mRNA were determined by real-time PCR analysis (top). GSK3β expression levels were confirmed by western blotting with an anti-HA antibody (bottom). A longer exposure of the HA blot shows the presence of ubiquitin ladder. Data are presented as the mean±s.d. from at least three independent experiments. Statistical analyses were calculated using the Student’s t -test (** P
    Figure Legend Snippet: TRAF6-mediated GSK3β ubiquitination at lysine 183 is critical for TLR3-dependent cytokine production. ( a ) BMDMs were stimulated with 10 μg ml −1 poly I:C for 10 min and subjected to immunoprecipitation with an anti-Ub antibody followed by western blotting with an anti-GSK3β antibody. ( b ) HEK293T cells transfected with HA-GSK3β and HA-Ub along with Flag-TRAF6 plasmids were subjected to immunoprecipitation with an anti-GSK3β antibody followed by western blotting with an anti-HA antibody. ( c ) HEK293T cells were transfected with HA-GSK3β and HA-Ub along with TRAF6 (WT) or TRAF6 (C70A) plasmids. These experiments were performed as described in b . ( d ) Traf6 +/+ and Traf6 −/− 3T3 cells stimulated with 10 μg ml −1 poly I:C for 10 min were subjected to immunoprecipitation with an anti-GSK3β antibody followed by western blotting with an anti-Ub antibody. ( e ) GSK3β proteins were incubated with E1, E2 and biotinylated-Ub (Bt-Ub) in the presence or absence of Flag-TRAF6 proteins for in vitro ubiquitination of GSK3β. Ubiquitination of GSK3β was analysed by western blotting with streptavidin-HRP. ( f ) HEK293T cells transfected with Ub and Flag-TRAF6 along with HA-GSK3β WT or various HA-GSK3β mutants were subjected to immunoprecipitation with an anti-HA antibody followed by western blotting with an anti-Ub antibody. ( g ) HEK293-TLR3 cells were transiently transfected with GSK3β (WT) or GSK3β (K183R) plasmids. The levels of IL-6, TNF-α and c-Fos mRNA were determined by real-time PCR analysis (top). GSK3β expression levels were confirmed by western blotting with an anti-HA antibody (bottom). A longer exposure of the HA blot shows the presence of ubiquitin ladder. Data are presented as the mean±s.d. from at least three independent experiments. Statistical analyses were calculated using the Student’s t -test (** P

    Techniques Used: Immunoprecipitation, Western Blot, Transfection, Incubation, In Vitro, Real-time Polymerase Chain Reaction, Expressing

    26) Product Images from "Longitudinal analysis of antigen specific response in individuals with Schistosoma mansoni infection in an endemic area of Minas Gerais, Brazil"

    Article Title: Longitudinal analysis of antigen specific response in individuals with Schistosoma mansoni infection in an endemic area of Minas Gerais, Brazil

    Journal: Transactions of the Royal Society of Tropical Medicine and Hygiene

    doi: 10.1093/trstmh/trt091

    A receiver operating characteristic (ROC) plot, illustrating the ability of IgG4/IgE anti-SEA antibodies to discriminate infected from uninfected individuals. Higher areas under the curve indicate better discrimination.
    Figure Legend Snippet: A receiver operating characteristic (ROC) plot, illustrating the ability of IgG4/IgE anti-SEA antibodies to discriminate infected from uninfected individuals. Higher areas under the curve indicate better discrimination.

    Techniques Used: Infection

    A receiver operating characteristic (ROC) plot, illustrating the ability of IgG anti-SEA antibodies to discriminate infected from uninfected individuals. Higher areas under the curve indicate better discrimination.
    Figure Legend Snippet: A receiver operating characteristic (ROC) plot, illustrating the ability of IgG anti-SEA antibodies to discriminate infected from uninfected individuals. Higher areas under the curve indicate better discrimination.

    Techniques Used: Infection

    27) Product Images from "Proteomic Analysis of Early-Responsive Redox-Sensitive Proteins in Arabidopsis"

    Article Title: Proteomic Analysis of Early-Responsive Redox-Sensitive Proteins in Arabidopsis

    Journal: Journal of Proteome Research

    doi: 10.1021/pr200918f

    Oxidative modification of identified proteins in planta upon H 2 O 2 treatment. Transgenic plants expressing the protein of interest fused with the FLAG tag were vacuum infiltrated with either water (mock) or 5 mM H 2 O 2 . For analysis of AtCIAPIN1, eEF1α, and AtPTP1, free thiols in the total protein were labeled with BIAM during protein extraction. For analysis of AtNAP1;1 and AtPDIL1-1, free thiols in the samples were first alkylated by IAM. Samples were then treated with DTT and newly generated free thiols were labeled by BIAM. After that, FLAG-tagged protein from each sample was affinity purified, separated by SDS-PAGE, and detected by HRP-Conjugated Streptavidin (to determine the amount of BIAM attached to the FLAG-tagged protein) or by the anti-FLAG M2 antibody (to determine the amount of the total recombinant protein).
    Figure Legend Snippet: Oxidative modification of identified proteins in planta upon H 2 O 2 treatment. Transgenic plants expressing the protein of interest fused with the FLAG tag were vacuum infiltrated with either water (mock) or 5 mM H 2 O 2 . For analysis of AtCIAPIN1, eEF1α, and AtPTP1, free thiols in the total protein were labeled with BIAM during protein extraction. For analysis of AtNAP1;1 and AtPDIL1-1, free thiols in the samples were first alkylated by IAM. Samples were then treated with DTT and newly generated free thiols were labeled by BIAM. After that, FLAG-tagged protein from each sample was affinity purified, separated by SDS-PAGE, and detected by HRP-Conjugated Streptavidin (to determine the amount of BIAM attached to the FLAG-tagged protein) or by the anti-FLAG M2 antibody (to determine the amount of the total recombinant protein).

    Techniques Used: Modification, Transgenic Assay, Expressing, FLAG-tag, Labeling, Protein Extraction, Generated, Affinity Purification, SDS Page, Recombinant

    28) Product Images from "Identification of Michael Acceptor-Centric Pharmacophores with Substituents That Yield Strong Thioredoxin Reductase Inhibitory Character Correlated to Antiproliferative Activity"

    Article Title: Identification of Michael Acceptor-Centric Pharmacophores with Substituents That Yield Strong Thioredoxin Reductase Inhibitory Character Correlated to Antiproliferative Activity

    Journal: Antioxidants & Redox Signaling

    doi: 10.1089/ars.2012.4909

    Irreversibility of TrxR inhibition and selenocysteine (Sec) residue targeting by the DPPen and DPPro lead analogs. (A) Oxidized recombinant rat TrxR (110 n M ) in the presence or absence of 200 μ M NADPH was incubated with different concentrations of 2,2′-diOH-5,5′-diF-DPPen or 2-OH-5-F-DPPro for 20 min, and the TrxR activity was determined by the DTNB assay. All data points are means of two independent experiments. (B) Recombinant rat TrxR (0.9 μ M ) was incubated with 20 μ M of 2,2′-diOH-5,5′-diF-DPPen or 2-OH-5-F-DPPro for 20 min in a 50 mM Tris-HCl, 1 mN EDTA, pH 7.5 buffer containing 200 μ M NADPH. The excess drug was removed by passing the protein through a NAP-5-desalting column, followed by determination of the activity of the eluted protein at indicated times using the DTNB assay. (C) Recombinant rat TrxR (0.9 μ M ) was incubated with 200 μ M NADPH and 20 μ M of 2,2′-diOH-5,5′-diF-DPPen or 2-OH-5-F-DPPro. At different timepoints, an aliquot of enzyme mixture was withdrawn for TrxR activity measurement by the DTNB assay and N-(Biotinoyl)-N′-(Iodoacetyl) Ethylenediamine (BIAM) labeling of Sec at pH 6.5 and 8.5. Top panel: time course of TrxR enzyme activity; middle panel: horseradish peroxidase (HRP)-conjugated streptavidin detection of BIAM labeling of free selenol at pH 6.5 at various incubation times; bottom panel: HRP-conjugated streptavidin detection of BIAM labeling of free selenol and sulfhydryls at pH 8.5 at various incubation times. Results shown are representative of three independent experiments.
    Figure Legend Snippet: Irreversibility of TrxR inhibition and selenocysteine (Sec) residue targeting by the DPPen and DPPro lead analogs. (A) Oxidized recombinant rat TrxR (110 n M ) in the presence or absence of 200 μ M NADPH was incubated with different concentrations of 2,2′-diOH-5,5′-diF-DPPen or 2-OH-5-F-DPPro for 20 min, and the TrxR activity was determined by the DTNB assay. All data points are means of two independent experiments. (B) Recombinant rat TrxR (0.9 μ M ) was incubated with 20 μ M of 2,2′-diOH-5,5′-diF-DPPen or 2-OH-5-F-DPPro for 20 min in a 50 mM Tris-HCl, 1 mN EDTA, pH 7.5 buffer containing 200 μ M NADPH. The excess drug was removed by passing the protein through a NAP-5-desalting column, followed by determination of the activity of the eluted protein at indicated times using the DTNB assay. (C) Recombinant rat TrxR (0.9 μ M ) was incubated with 200 μ M NADPH and 20 μ M of 2,2′-diOH-5,5′-diF-DPPen or 2-OH-5-F-DPPro. At different timepoints, an aliquot of enzyme mixture was withdrawn for TrxR activity measurement by the DTNB assay and N-(Biotinoyl)-N′-(Iodoacetyl) Ethylenediamine (BIAM) labeling of Sec at pH 6.5 and 8.5. Top panel: time course of TrxR enzyme activity; middle panel: horseradish peroxidase (HRP)-conjugated streptavidin detection of BIAM labeling of free selenol at pH 6.5 at various incubation times; bottom panel: HRP-conjugated streptavidin detection of BIAM labeling of free selenol and sulfhydryls at pH 8.5 at various incubation times. Results shown are representative of three independent experiments.

    Techniques Used: Inhibition, Size-exclusion Chromatography, Recombinant, Incubation, Activity Assay, DTNB Assay, Labeling

    29) Product Images from "Distinct Roles of Adenovirus Vector-Transduced Dendritic Cells, Myoblasts, and Endothelial Cells in Mediating an Immune Response against a Transgene Product"

    Article Title: Distinct Roles of Adenovirus Vector-Transduced Dendritic Cells, Myoblasts, and Endothelial Cells in Mediating an Immune Response against a Transgene Product

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.6.2899-2911.2002

    β-Gal-specific antibody titers in mice immunized with AdβGal or with AdβGal-transduced muscle cells. Mice received one i.m. injection with either 10 9 PFU of AdβGal (Ad) or 5 × 10 4 AdβGal-expressing DC (DC/Ad), myoblasts (Myo/Ad), or EC (EC/Ad). Mice injected with untransduced DC, myoblasts, or EC (DC, Myo, or EC) were used as negative controls. Mice were bled once a week, and β-Gal-specific antibody titers were measured by ELISA. (A) Generation of β-Gal-specific IgG antibodies as a function of time. (B) Levels of IgG1- and IgG2a-specific antibodies at day 42. (C) Kinetics of IgG1 and IgG2a β-Gal-specific antibodies after immunization with AdβGal-transduced DC.
    Figure Legend Snippet: β-Gal-specific antibody titers in mice immunized with AdβGal or with AdβGal-transduced muscle cells. Mice received one i.m. injection with either 10 9 PFU of AdβGal (Ad) or 5 × 10 4 AdβGal-expressing DC (DC/Ad), myoblasts (Myo/Ad), or EC (EC/Ad). Mice injected with untransduced DC, myoblasts, or EC (DC, Myo, or EC) were used as negative controls. Mice were bled once a week, and β-Gal-specific antibody titers were measured by ELISA. (A) Generation of β-Gal-specific IgG antibodies as a function of time. (B) Levels of IgG1- and IgG2a-specific antibodies at day 42. (C) Kinetics of IgG1 and IgG2a β-Gal-specific antibodies after immunization with AdβGal-transduced DC.

    Techniques Used: Mouse Assay, Injection, Expressing, Enzyme-linked Immunosorbent Assay

    30) Product Images from "Functional Mapping of Community Acquired Respiratory Distress Syndrome (CARDS) Toxin of Mycoplasma pneumoniae Defines Regions with ADP-ribosyltransferase, Vacuolating, and Receptor-Binding Activities"

    Article Title: Functional Mapping of Community Acquired Respiratory Distress Syndrome (CARDS) Toxin of Mycoplasma pneumoniae Defines Regions with ADP-ribosyltransferase, Vacuolating, and Receptor-Binding Activities

    Journal: Molecular microbiology

    doi: 10.1111/mmi.12680

    Induction of vacuolization in HeLa cells by carboxy region of CARDS toxin A) Equimolar (~140, 350 and 700 pmol) concentrations of FL, trypsin cleaved (TC), carboxy and amino terminal region truncated (CARDS 249 , 178 CARDS, 264 CARDS and 308 CARDS) proteins were added to 60% confluent monolayer cultures of HeLa cells as indicated in Experimental Procedures and observed at 12 h to 60h. Representative images of vacuoles generated by 140 pmol concentration of proteins at 24 h are shown. Vacuoles were generated in HeLa cells by FL CARDS toxin, TC-CARDS toxin and carboxy region retaining CARDS toxin derivatives and not by CARDS 249 . B) Quantification of numbers of CARDS toxin-induced vacuoles in HeLa cells incubated with FL and carboxy region variants of CARDS toxin ( 178 CARDS, 264 CARDS and 308 CARDS) at 24 h. As indicated in section A, cells were incubated with CARDS toxin and its derivatives and vacuoles per cell were counted and compared as described in Experimental Procedures. C) Quantification of percentage of HeLa cells with CARDS-toxin induced vacuoles. As indicated in panel A, cells were incubated 12–60 h with CARDS toxin and its derivatives (140 pmol) and the number of vacuolated cells in random fields was calculated as described in Experimental Procedures.
    Figure Legend Snippet: Induction of vacuolization in HeLa cells by carboxy region of CARDS toxin A) Equimolar (~140, 350 and 700 pmol) concentrations of FL, trypsin cleaved (TC), carboxy and amino terminal region truncated (CARDS 249 , 178 CARDS, 264 CARDS and 308 CARDS) proteins were added to 60% confluent monolayer cultures of HeLa cells as indicated in Experimental Procedures and observed at 12 h to 60h. Representative images of vacuoles generated by 140 pmol concentration of proteins at 24 h are shown. Vacuoles were generated in HeLa cells by FL CARDS toxin, TC-CARDS toxin and carboxy region retaining CARDS toxin derivatives and not by CARDS 249 . B) Quantification of numbers of CARDS toxin-induced vacuoles in HeLa cells incubated with FL and carboxy region variants of CARDS toxin ( 178 CARDS, 264 CARDS and 308 CARDS) at 24 h. As indicated in section A, cells were incubated with CARDS toxin and its derivatives and vacuoles per cell were counted and compared as described in Experimental Procedures. C) Quantification of percentage of HeLa cells with CARDS-toxin induced vacuoles. As indicated in panel A, cells were incubated 12–60 h with CARDS toxin and its derivatives (140 pmol) and the number of vacuolated cells in random fields was calculated as described in Experimental Procedures.

    Techniques Used: Generated, Concentration Assay, Incubation

    N-terminal region of CARDS toxin houses ADPRT activity A) Schematic representation of the ART activity of CARDS toxin and its derivatives. As indicated in Experimental Procedures, FL CARDS toxin, its N-terminal derivatives (truncations) and APDRT-conserved amino acid site-directed mutagenized (SDM) proteins (Arg 10→Ala , His 36→Ala and Glu 132→Ala ) were analyzed for ADPRT activity, and the results are depicted. PT-S1 indicates pertussis toxin S1 catalytic subunit; FL indicates full length CARDS toxin; B) Comparison of ADP-ribosylating activity of FL and different carboxy region-deleted derivatives of CARDS toxin. Purified FL CARDS toxin and its carboxy region-truncated derivatives were individually incubated with HeLa cell lysate in the presence of 32 P NAD as indicated in Experimental Procedures, and the radiolabeled ADP-ribosylated proteins were detected. Each number indicates the truncated proteins depicted in Fig. 2A; CB represents carrier buffer. The two solid black arrows (~50 kDa) indicate the target ADP-ribosylated proteins of CARDS toxin, and the dotted arrow indicates the inherent ADP-ribosylated protein of mammalian cells.
    Figure Legend Snippet: N-terminal region of CARDS toxin houses ADPRT activity A) Schematic representation of the ART activity of CARDS toxin and its derivatives. As indicated in Experimental Procedures, FL CARDS toxin, its N-terminal derivatives (truncations) and APDRT-conserved amino acid site-directed mutagenized (SDM) proteins (Arg 10→Ala , His 36→Ala and Glu 132→Ala ) were analyzed for ADPRT activity, and the results are depicted. PT-S1 indicates pertussis toxin S1 catalytic subunit; FL indicates full length CARDS toxin; B) Comparison of ADP-ribosylating activity of FL and different carboxy region-deleted derivatives of CARDS toxin. Purified FL CARDS toxin and its carboxy region-truncated derivatives were individually incubated with HeLa cell lysate in the presence of 32 P NAD as indicated in Experimental Procedures, and the radiolabeled ADP-ribosylated proteins were detected. Each number indicates the truncated proteins depicted in Fig. 2A; CB represents carrier buffer. The two solid black arrows (~50 kDa) indicate the target ADP-ribosylated proteins of CARDS toxin, and the dotted arrow indicates the inherent ADP-ribosylated protein of mammalian cells.

    Techniques Used: Activity Assay, Purification, Incubation

    Binding and internalization of CARDS toxin ADPRT mutants HeLa cell monolayers were treated with 10 µg (≈140 pmol) of FL CARDS toxin or ADPRT mutants (Arg 10→Ala , His 36→Ala and Glu 132→Ala ) at 37°C for 1h. Cells were fixed, permeabilized with 0.1% Triton X-100 and incubated with anti-CARDS toxin rabbit primary antibodies. Subsequently, cells were treated with AlexaFluor-633 conjugated goat (polyclonal) anti-rabbit secondary antibodies to detect the cellular binding and trafficking of CARDS toxin by confocal laser scanning microscopy. Cell nuclei were stained with DAPI (4',6-diamidino-2-phenylindole dihydrochloride).
    Figure Legend Snippet: Binding and internalization of CARDS toxin ADPRT mutants HeLa cell monolayers were treated with 10 µg (≈140 pmol) of FL CARDS toxin or ADPRT mutants (Arg 10→Ala , His 36→Ala and Glu 132→Ala ) at 37°C for 1h. Cells were fixed, permeabilized with 0.1% Triton X-100 and incubated with anti-CARDS toxin rabbit primary antibodies. Subsequently, cells were treated with AlexaFluor-633 conjugated goat (polyclonal) anti-rabbit secondary antibodies to detect the cellular binding and trafficking of CARDS toxin by confocal laser scanning microscopy. Cell nuclei were stained with DAPI (4',6-diamidino-2-phenylindole dihydrochloride).

    Techniques Used: Binding Assay, Incubation, Confocal Laser Scanning Microscopy, Staining

    Carboxy region of CARDS toxin mediates binding and internalization A) Comparison of binding and internalization of FL, selected C-terminal truncated CARDS toxin derivatives and 178 CARDS toxin. FL toxin and its derivatives were purified as indicated in Experimental Procedures and biotin labeled. Binding : Proteins were incubated with HeLa cells for 1 h at 4°C. Subsequently, cells were washed and bound proteins were quantified as indicated in Experimental Procedures. Internalization : After 1 h incubation at 4°C, bound proteins (FL, selected C-terminal truncated CARDS toxin derivatives and 178 CARDS toxin) were removed by washing, and cells were shifted to 37°C for 1 h with fresh medium. Then, cells were treated with MESNA to remove surface-bound, biotin-labeled CARDS toxin and its derivatives, and internalized proteins were quantified after permeabilizing cells as indicated in Experimental Procedures. Binding and internalization results of FL and variants of CARDS toxin are shown by closed and open symbols respectively. FL - square, 178 CARDS - circle, CARDS 249 - triangle and CARDS 550 -diamond. B) Endocytosis of 178 CARDS toxin and CARDS 550 toxin. HeLa cells were treated with 140 pmol of 178 CARDS toxin or CARDS 550 toxin for 30 min at 4°C, washed to remove unbound toxins and shifted to 37°C for 1 h. Cell preparations were fixed and permeabilized with 0.1% Triton X-100, followed by incubation with anti-CARDS toxin rabbit primary antibodies. Cells were treated with AlexaFluor-633 conjugated anti-rabbit goat (polyclonal) secondary antibodies to detect cellular binding of CARDS toxin derivatives by confocal laser scanning microscopy. Cell nuclei were stained with DAPI.
    Figure Legend Snippet: Carboxy region of CARDS toxin mediates binding and internalization A) Comparison of binding and internalization of FL, selected C-terminal truncated CARDS toxin derivatives and 178 CARDS toxin. FL toxin and its derivatives were purified as indicated in Experimental Procedures and biotin labeled. Binding : Proteins were incubated with HeLa cells for 1 h at 4°C. Subsequently, cells were washed and bound proteins were quantified as indicated in Experimental Procedures. Internalization : After 1 h incubation at 4°C, bound proteins (FL, selected C-terminal truncated CARDS toxin derivatives and 178 CARDS toxin) were removed by washing, and cells were shifted to 37°C for 1 h with fresh medium. Then, cells were treated with MESNA to remove surface-bound, biotin-labeled CARDS toxin and its derivatives, and internalized proteins were quantified after permeabilizing cells as indicated in Experimental Procedures. Binding and internalization results of FL and variants of CARDS toxin are shown by closed and open symbols respectively. FL - square, 178 CARDS - circle, CARDS 249 - triangle and CARDS 550 -diamond. B) Endocytosis of 178 CARDS toxin and CARDS 550 toxin. HeLa cells were treated with 140 pmol of 178 CARDS toxin or CARDS 550 toxin for 30 min at 4°C, washed to remove unbound toxins and shifted to 37°C for 1 h. Cell preparations were fixed and permeabilized with 0.1% Triton X-100, followed by incubation with anti-CARDS toxin rabbit primary antibodies. Cells were treated with AlexaFluor-633 conjugated anti-rabbit goat (polyclonal) secondary antibodies to detect cellular binding of CARDS toxin derivatives by confocal laser scanning microscopy. Cell nuclei were stained with DAPI.

    Techniques Used: Binding Assay, Purification, Labeling, Incubation, Confocal Laser Scanning Microscopy, Staining

    31) Product Images from "Functional roles of cadherin, aminopeptidase-N and alkaline phosphatase from Helicoverpa armigera (Hübner) in the action mechanism of Bacillus thuringiensis Cry2Aa"

    Article Title: Functional roles of cadherin, aminopeptidase-N and alkaline phosphatase from Helicoverpa armigera (Hübner) in the action mechanism of Bacillus thuringiensis Cry2Aa

    Journal: Scientific Reports

    doi: 10.1038/srep46555

    ELISAs to assess the binding the Cry2Aa toxin with the recombinant CAD, APN4 and ALP2 fragments. Binding ELISAs were performed by fixing 1 μg of the CAD ( A ), APN4 ( B ) and ALP2 ( C ) fragments per well in ELISA plates, followed by incubation with different concentrations of the biotinylated Cry2Aa toxin. K d values obtained through SigmaPlot 12.5 analysis are indicated within the graphs.
    Figure Legend Snippet: ELISAs to assess the binding the Cry2Aa toxin with the recombinant CAD, APN4 and ALP2 fragments. Binding ELISAs were performed by fixing 1 μg of the CAD ( A ), APN4 ( B ) and ALP2 ( C ) fragments per well in ELISA plates, followed by incubation with different concentrations of the biotinylated Cry2Aa toxin. K d values obtained through SigmaPlot 12.5 analysis are indicated within the graphs.

    Techniques Used: Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Incubation

    32) Product Images from "Poly(ADP-ribose) Polymerase-1 Down-regulates BRCA2 Expression through theBRCA2 Promoter *"

    Article Title: Poly(ADP-ribose) Polymerase-1 Down-regulates BRCA2 Expression through theBRCA2 Promoter *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M803693200

    Parp-1 binds to the BRCA2 promoter. A , MCF-7 cell nuclear extracts were mixed with a biotinylated WT probe and a mutant probe ( M ). The DNA-protein complex was isolated with streptavidin-labeled magnetic beads. The magnetic bead column was washed and eluates were collected. Eluted fractions were separated on 10% acrylamide gels and visualized by silver staining. The 120-kDa protein band indicates a prominent DNA-protein complex. BSA was used as a control, and its position is indicated by a black arrow . The experiment was carried out three times. E1, E2, E3 (WT probe eluates); M , mutant probe eluate. B , mass spectrometry of the ∼120-kDa protein. C , EMSAs and antibody super-shifts were performed in MCF-7 nuclear extracts using WT, mutant probes, and Parp-1 antibody. D , EMSAs were carried out in MCF-7 nuclear extracts using the end-streptavidin blocked-biotin-labeled WT and mutant probes. E , luciferase activity of Del-9 in MCF-7 cells treated with 3-AB/NU1025, two Parp-1 inhibitors, or DMSO for 12 h.
    Figure Legend Snippet: Parp-1 binds to the BRCA2 promoter. A , MCF-7 cell nuclear extracts were mixed with a biotinylated WT probe and a mutant probe ( M ). The DNA-protein complex was isolated with streptavidin-labeled magnetic beads. The magnetic bead column was washed and eluates were collected. Eluted fractions were separated on 10% acrylamide gels and visualized by silver staining. The 120-kDa protein band indicates a prominent DNA-protein complex. BSA was used as a control, and its position is indicated by a black arrow . The experiment was carried out three times. E1, E2, E3 (WT probe eluates); M , mutant probe eluate. B , mass spectrometry of the ∼120-kDa protein. C , EMSAs and antibody super-shifts were performed in MCF-7 nuclear extracts using WT, mutant probes, and Parp-1 antibody. D , EMSAs were carried out in MCF-7 nuclear extracts using the end-streptavidin blocked-biotin-labeled WT and mutant probes. E , luciferase activity of Del-9 in MCF-7 cells treated with 3-AB/NU1025, two Parp-1 inhibitors, or DMSO for 12 h.

    Techniques Used: Mutagenesis, Isolation, Labeling, Magnetic Beads, Silver Staining, Mass Spectrometry, Luciferase, Activity Assay

    33) Product Images from "Enhanced Reduction of Helicobacter pylori Load in Precolonized Mice Treated with Combined Famotidine and Urease-Binding Polysaccharides"

    Article Title: Enhanced Reduction of Helicobacter pylori Load in Precolonized Mice Treated with Combined Famotidine and Urease-Binding Polysaccharides

    Journal: Antimicrobial Agents and Chemotherapy

    doi:

    Enhanced inhibition of biotinylated swine gastric mucin adherence to H. pylori native urease protein caused by dextran sulfate in the presence of famotidine. Dextran sulfate exhibited enhanced competitive activity in the presence of a weight-equivalent amount of famotidine using a pH 4.0 adhesion medium containing 20 mM PBS and 0.05% Tween 20. A similar percent inhibition was not observed when the dextran sulfate-omeprazole combination or single-component test inhibitors were used. Symbols: ●, dextran sulfate plus famotidine; ○, dextran sulfate plus omeprazole; ■, dextran sulfate; □, famotidine; ▵, omeprazole; ▴, dextran.
    Figure Legend Snippet: Enhanced inhibition of biotinylated swine gastric mucin adherence to H. pylori native urease protein caused by dextran sulfate in the presence of famotidine. Dextran sulfate exhibited enhanced competitive activity in the presence of a weight-equivalent amount of famotidine using a pH 4.0 adhesion medium containing 20 mM PBS and 0.05% Tween 20. A similar percent inhibition was not observed when the dextran sulfate-omeprazole combination or single-component test inhibitors were used. Symbols: ●, dextran sulfate plus famotidine; ○, dextran sulfate plus omeprazole; ■, dextran sulfate; □, famotidine; ▵, omeprazole; ▴, dextran.

    Techniques Used: Inhibition, Activity Assay

    34) Product Images from "Susceptibility quantitative trait loci for pathogenic leucocytosis in SCG/Kj mice, a spontaneously occurring crescentic glomerulonephritis and vasculitis model"

    Article Title: Susceptibility quantitative trait loci for pathogenic leucocytosis in SCG/Kj mice, a spontaneously occurring crescentic glomerulonephritis and vasculitis model

    Journal: Clinical and Experimental Immunology

    doi: 10.1111/cei.12333

    Increased numbers of Gr-1 + granulocytes (a), F4/80 + macrophages/monocytes (b), dendritic cells (DCs) (c) and CD4 − CD8 − T cells (d) in peripheral blood (PB) of [C57BL/6 × spontaneous crescentic glomerulonephritis-forming/Kinjoh (SCG/Kj)]
    Figure Legend Snippet: Increased numbers of Gr-1 + granulocytes (a), F4/80 + macrophages/monocytes (b), dendritic cells (DCs) (c) and CD4 − CD8 − T cells (d) in peripheral blood (PB) of [C57BL/6 × spontaneous crescentic glomerulonephritis-forming/Kinjoh (SCG/Kj)]

    Techniques Used:

    Distribution of Gr-1 + cells, myeloperoxidase (MPO + ) cells and F4/80 + cells in healthy and glomerulonephritic glomeruli with crescents [Cr (+)] and without crescents [Cr (−)]. Upper photographs: cortical glomeruli, lower photographs: interstitium.
    Figure Legend Snippet: Distribution of Gr-1 + cells, myeloperoxidase (MPO + ) cells and F4/80 + cells in healthy and glomerulonephritic glomeruli with crescents [Cr (+)] and without crescents [Cr (−)]. Upper photographs: cortical glomeruli, lower photographs: interstitium.

    Techniques Used:

    35) Product Images from "Dynamics of clathrin-mediated endocytosis and its requirement for organelle biogenesis in Dictyostelium"

    Article Title: Dynamics of clathrin-mediated endocytosis and its requirement for organelle biogenesis in Dictyostelium

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.108837

    Internalization of dajumin-GFP. ( A ) Biotin internalization assay for AX2, Ap2A1–, apm2–, clc– and chcA– cells. Dajumin-GFP was immunoprecipitated from cell lysates. Membranes were probed for biotin with streptavidin-HRP
    Figure Legend Snippet: Internalization of dajumin-GFP. ( A ) Biotin internalization assay for AX2, Ap2A1–, apm2–, clc– and chcA– cells. Dajumin-GFP was immunoprecipitated from cell lysates. Membranes were probed for biotin with streptavidin-HRP

    Techniques Used: Immunoprecipitation

    36) Product Images from "CD9 Regulates Major Histocompatibility Complex Class II Trafficking in Monocyte-Derived Dendritic Cells"

    Article Title: CD9 Regulates Major Histocompatibility Complex Class II Trafficking in Monocyte-Derived Dendritic Cells

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00202-17

    CD9 regulates MHC-II internalization and recycling in mature MoDCs. (A and B) Immature (A) and LPS-matured (B) WT and CD9 KO MoDCs were incubated with biotinylated MHC-II antibodies for 1 h at 4°C, washed, and incubated for different times at 37°C, and then MHC-II surface expression was detected by flow cytometry after streptavidin labeling in CD11c + cells. Data represent mean fold changes ± SEM of results from three independent experiments performed in triplicate and were analyzed by two-way ANOVA with Bonferroni's post hoc multiple-comparison test. (C and D) Measurement of MHC-II internalization (C) and recycling (D) in mature WT and CD9 KO MoDCs by cell biotinylation assay. After incubation with biotin (blue in the schemes) at 4°C, cells were kept at 37°C (internalization), and the remaining surface biotins were removed by MESNA washing. (D) For recycling experiments, cells were further incubated for different times at 37°C (recycling), and surface biotins removed by MESNA washing. Cell lysates were immunoprecipitated with an anti-MHC-II antibody (M5/114), and biotinylated proteins were detected after membrane incubation with streptavidin-HRP (StrepHRP). Membranes were reprobed with MHC-II antibody for loading measurement. The blots shown are from representative experiments. (C) Only a fractional amount from cells incubated at 4°C in the absence of MESNA (0 min) was loaded. The graph shows the biotinylated MHC-II/total MHC-II signal ratio. The graph shows data determined as follows: [1 − (biotinylated MHC-II/total MHC-II signal ratio)]. Data represent mean fold changes ± SEM of results from four (C) and three (D) independent experiments analyzed by two-way ANOVA with Bonferroni's post hoc multiple-comparison test. **, P
    Figure Legend Snippet: CD9 regulates MHC-II internalization and recycling in mature MoDCs. (A and B) Immature (A) and LPS-matured (B) WT and CD9 KO MoDCs were incubated with biotinylated MHC-II antibodies for 1 h at 4°C, washed, and incubated for different times at 37°C, and then MHC-II surface expression was detected by flow cytometry after streptavidin labeling in CD11c + cells. Data represent mean fold changes ± SEM of results from three independent experiments performed in triplicate and were analyzed by two-way ANOVA with Bonferroni's post hoc multiple-comparison test. (C and D) Measurement of MHC-II internalization (C) and recycling (D) in mature WT and CD9 KO MoDCs by cell biotinylation assay. After incubation with biotin (blue in the schemes) at 4°C, cells were kept at 37°C (internalization), and the remaining surface biotins were removed by MESNA washing. (D) For recycling experiments, cells were further incubated for different times at 37°C (recycling), and surface biotins removed by MESNA washing. Cell lysates were immunoprecipitated with an anti-MHC-II antibody (M5/114), and biotinylated proteins were detected after membrane incubation with streptavidin-HRP (StrepHRP). Membranes were reprobed with MHC-II antibody for loading measurement. The blots shown are from representative experiments. (C) Only a fractional amount from cells incubated at 4°C in the absence of MESNA (0 min) was loaded. The graph shows the biotinylated MHC-II/total MHC-II signal ratio. The graph shows data determined as follows: [1 − (biotinylated MHC-II/total MHC-II signal ratio)]. Data represent mean fold changes ± SEM of results from four (C) and three (D) independent experiments analyzed by two-way ANOVA with Bonferroni's post hoc multiple-comparison test. **, P

    Techniques Used: Incubation, Expressing, Flow Cytometry, Cytometry, Labeling, Cell Surface Biotinylation Assay, Immunoprecipitation

    37) Product Images from "Cross-Reactive HIV-1 Neutralizing Monoclonal Antibodies Selected by Screening of an Immune Human Phage Library against an Envelope Glycoprotein (gp140) Isolated from a Patient (R2) with Broadly HIV-1 Neutralizing Antibodies"

    Article Title: Cross-Reactive HIV-1 Neutralizing Monoclonal Antibodies Selected by Screening of an Immune Human Phage Library against an Envelope Glycoprotein (gp140) Isolated from a Patient (R2) with Broadly HIV-1 Neutralizing Antibodies

    Journal: Virology

    doi: 10.1016/j.virol.2007.01.015

    Competition of m46 with anti-gp41 antibodies. 1 μg/ml gp140 89.6 was coated on 96-well plates. Two-fold serially diluted IgG m46, IgG 2F5 and Fab Z13 were added to the wells and biotinylated mouse monoclonal antibody (mAb) T3 and D3 at a constant concentration corresponding to 70% maximum binding was simultaneously added to the wells. Bound biotinylated T3 (A) and D3 (B) were detected using streptavidin-HRP at 450 nm.
    Figure Legend Snippet: Competition of m46 with anti-gp41 antibodies. 1 μg/ml gp140 89.6 was coated on 96-well plates. Two-fold serially diluted IgG m46, IgG 2F5 and Fab Z13 were added to the wells and biotinylated mouse monoclonal antibody (mAb) T3 and D3 at a constant concentration corresponding to 70% maximum binding was simultaneously added to the wells. Bound biotinylated T3 (A) and D3 (B) were detected using streptavidin-HRP at 450 nm.

    Techniques Used: Concentration Assay, Binding Assay

    Competition of m22 and m24 with other CD4bs and CD4i antibodies. Gp140 R2 was captured by the polyclonal sheep anti-gp120 antibody D7324 (5 μg/ml) coated in 96-well plates. Three-fold serially diluted sCD4, Fabs (m14, m16, m18, X5, m22, m24), and IgGs (b12, 17b) followed by addition of biotinylated m22 (A) or m24 (B), at a constant concentration (which leads to 70% of maximum binding) simultaneously to the wells. Bound antibodies were detected by streptavidin-HRP and measured as optical densities at 450 nm.
    Figure Legend Snippet: Competition of m22 and m24 with other CD4bs and CD4i antibodies. Gp140 R2 was captured by the polyclonal sheep anti-gp120 antibody D7324 (5 μg/ml) coated in 96-well plates. Three-fold serially diluted sCD4, Fabs (m14, m16, m18, X5, m22, m24), and IgGs (b12, 17b) followed by addition of biotinylated m22 (A) or m24 (B), at a constant concentration (which leads to 70% of maximum binding) simultaneously to the wells. Bound antibodies were detected by streptavidin-HRP and measured as optical densities at 450 nm.

    Techniques Used: Concentration Assay, Binding Assay

    Competition of m46 with anti-gp41, CD4bs and CD4i antibodies. Gp140 R2 was captured by the polyclonal sheep anti-gp120 antibody D7324 (5 μg/ml) coated on 96-well plates. Serially diluted sCD4, different Fabs (X5, m43, m45) and IgGs (b12, 2F5, 4E10, 2G12) were added, along with biotinylated m46 at a constant concentration that led to 70% of maximum binding, simultaneously to the wells. Bound antibodies were detected by streptavidin-HRP and measured as optical densities at 405 nm.
    Figure Legend Snippet: Competition of m46 with anti-gp41, CD4bs and CD4i antibodies. Gp140 R2 was captured by the polyclonal sheep anti-gp120 antibody D7324 (5 μg/ml) coated on 96-well plates. Serially diluted sCD4, different Fabs (X5, m43, m45) and IgGs (b12, 2F5, 4E10, 2G12) were added, along with biotinylated m46 at a constant concentration that led to 70% of maximum binding, simultaneously to the wells. Bound antibodies were detected by streptavidin-HRP and measured as optical densities at 405 nm.

    Techniques Used: Concentration Assay, Binding Assay

    38) Product Images from "Porcine monocyte subsets differ in the expression of CCR2 and in their responsiveness to CCL2"

    Article Title: Porcine monocyte subsets differ in the expression of CCR2 and in their responsiveness to CCL2

    Journal: Veterinary Research

    doi: 10.1051/vetres/2010048

    Expression of recombinant porcine CCL2. (A) CHO cell line stably expressing the porcine CCL2 fused to GFP. The expression of GFP fusion protein was directly analysed by flow cytometry. Non transfected CHO cells were used as negative control (grey histogram). 5 000 cells were acquired. (B) Western blot of CCL2-GFP produced by transfected CHO cells. Different dilutions of supernatant were resolved by 15% SDS-PAGE under reducing conditions and revealed with biotinylated anti-GFP and streptavidin-HRP. Numbers on the left indicate the position of MW markers. (C) Chemotactic activity of CCL2-GFP on porcine blood monocytes. Chemotaxis was assessed with the Transwell cell migration system and subsequent flow cytometry counting of migrated cells by a 45 s acquisition. (1) FSC versus SSC dot plot of migrated cells in response to supernatants from CHO cells expressing CCL2-GFP or the inverted sequence of pCCL2 fused to GFP (InvCCL2-GFP, negative control). (2) Results expressed as migration index, calculated as the ratio of the number of cells migrating to the chemokine and the number of cells in the negative control. Results from one representative experiment out of three performed are shown. (A color version of this figure is available at www.vetres.org. )
    Figure Legend Snippet: Expression of recombinant porcine CCL2. (A) CHO cell line stably expressing the porcine CCL2 fused to GFP. The expression of GFP fusion protein was directly analysed by flow cytometry. Non transfected CHO cells were used as negative control (grey histogram). 5 000 cells were acquired. (B) Western blot of CCL2-GFP produced by transfected CHO cells. Different dilutions of supernatant were resolved by 15% SDS-PAGE under reducing conditions and revealed with biotinylated anti-GFP and streptavidin-HRP. Numbers on the left indicate the position of MW markers. (C) Chemotactic activity of CCL2-GFP on porcine blood monocytes. Chemotaxis was assessed with the Transwell cell migration system and subsequent flow cytometry counting of migrated cells by a 45 s acquisition. (1) FSC versus SSC dot plot of migrated cells in response to supernatants from CHO cells expressing CCL2-GFP or the inverted sequence of pCCL2 fused to GFP (InvCCL2-GFP, negative control). (2) Results expressed as migration index, calculated as the ratio of the number of cells migrating to the chemokine and the number of cells in the negative control. Results from one representative experiment out of three performed are shown. (A color version of this figure is available at www.vetres.org. )

    Techniques Used: Expressing, Recombinant, Stable Transfection, Flow Cytometry, Cytometry, Transfection, Negative Control, Western Blot, Produced, SDS Page, Activity Assay, Chemotaxis Assay, Migration, Sequencing

    39) Product Images from "PA1b Inhibitor Binding to Subunits c and e of the Vacuolar ATPase Reveals Its Insecticidal Mechanism *"

    Article Title: PA1b Inhibitor Binding to Subunits c and e of the Vacuolar ATPase Reveals Its Insecticidal Mechanism *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.541250

    Electron microscopy of PA1b-bound V-ATPase. A , representative classes of PA1b-streptavidin-HRP-bound V-ATPase in the absence of ATP. B , as A but in the presence of 2 m m Mg·ATP. The PA1b-streptavidin-HRP density is indicated by an arrow in the far left panel 1 of A. Scale bars in both A and B represent 15 nm. C–E , three-dimensional reconstructions of the V-ATPase viewed perpendicular to the long axis of the complex ( upper image ) and from the extracellular end ( lower image ) bound to PA1b ( C ), bound to PA1b after the addition of Mg·ATP ( D ) and a control with no PA1b ( E ). All models were generated using EMAN, and the picture was produced using Chimera rendered at the same sigma level. In C ( lower ), the decameric c ring (Protein Data Bank ID code 2DB4 ( 53 ) r ainbow colors ) and a subunit model ( red ) have been fitted to the PA1b-streptavidin-HRP V-ATPase reconstruction in the absence of ATP using Chimera. If catalytically active, the c ring would rotate counterclockwise with respect to subunit a when observed from this perspective.
    Figure Legend Snippet: Electron microscopy of PA1b-bound V-ATPase. A , representative classes of PA1b-streptavidin-HRP-bound V-ATPase in the absence of ATP. B , as A but in the presence of 2 m m Mg·ATP. The PA1b-streptavidin-HRP density is indicated by an arrow in the far left panel 1 of A. Scale bars in both A and B represent 15 nm. C–E , three-dimensional reconstructions of the V-ATPase viewed perpendicular to the long axis of the complex ( upper image ) and from the extracellular end ( lower image ) bound to PA1b ( C ), bound to PA1b after the addition of Mg·ATP ( D ) and a control with no PA1b ( E ). All models were generated using EMAN, and the picture was produced using Chimera rendered at the same sigma level. In C ( lower ), the decameric c ring (Protein Data Bank ID code 2DB4 ( 53 ) r ainbow colors ) and a subunit model ( red ) have been fitted to the PA1b-streptavidin-HRP V-ATPase reconstruction in the absence of ATP using Chimera. If catalytically active, the c ring would rotate counterclockwise with respect to subunit a when observed from this perspective.

    Techniques Used: Electron Microscopy, Generated, Produced

    40) Product Images from "Live-cell mapping of organelle-associated RNAs via proximity biotinylation combined with protein-RNA crosslinking"

    Article Title: Live-cell mapping of organelle-associated RNAs via proximity biotinylation combined with protein-RNA crosslinking

    Journal: eLife

    doi: 10.7554/eLife.29224

    Characterization of APEX2 fusion constructs. HEK 293 T cells stably expressing the indicated constructs ( right ) were labeled and crosslinked via Protocol II ( Figure 1—figure supplement 1A ). Cell lysates were analyzed by SDS-PAGE, blotting with streptavidin-HRP, anti-V5 and anti-FLAG. L: ladder; U: untransfected HEK 293T cells. Anti-V5 and anti-FLAG blots ( bottom left ) measure fusion construct expression.
    Figure Legend Snippet: Characterization of APEX2 fusion constructs. HEK 293 T cells stably expressing the indicated constructs ( right ) were labeled and crosslinked via Protocol II ( Figure 1—figure supplement 1A ). Cell lysates were analyzed by SDS-PAGE, blotting with streptavidin-HRP, anti-V5 and anti-FLAG. L: ladder; U: untransfected HEK 293T cells. Anti-V5 and anti-FLAG blots ( bottom left ) measure fusion construct expression.

    Techniques Used: Construct, Stable Transfection, Expressing, Labeling, SDS Page

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    Transfection:

    Article Title: Plectin protects podocytes from adriamycin‐induced apoptosis and F‐actin cytoskeletal disruption through the integrin α6β4/ FAK/p38 MAPK pathway, et al. Plectin protects podocytes from adriamycin‐induced apoptosis and F‐actin cytoskeletal disruption through the integrin α6β4/FAK/p38 MAPK pathway
    Article Snippet: .. To silence plectin expression, we transiently transfected the podocytes with siPlectin or scrambled siRNA at a final concentration of 20 nmol/L for 72 hours using Lipofectamine 2000 (Invitrogen: Waltham, MA). .. We monitored gene silencing efficiency by Western blot and selected the cells with the most efficient gene silencing for subsequent experiments.

    Avidin-Biotin Assay:

    Article Title: The Heparan Sulfate Proteoglycan Form of Epithelial CD44v3 Serves as a CD11b/CD18 Counter-receptor during Polymorphonuclear Leukocyte Transepithelial Migration
    Article Snippet: .. Biotinylated cell surface antigen was detected with avidin-horseradish peroxidase (Pierce). .. Tryptic Digestion and Identification of C3H7 Antigen —Bulk antigen was purified from ∼500 cm2 of confluent T84 cell plasma membranes using mAb C3H7-coupled affinity column (CnBr-activated Sepharose 4B; Pierce).

    Cell Culture:

    Article Title: DRAM1 regulates autophagy and cell proliferation via inhibition of the phosphoinositide 3-kinase-Akt-mTOR-ribosomal protein S6 pathway
    Article Snippet: .. Cell culture and reagents HEK293T, Hela, HCT116 and SW480 cells were cultured at 37 °C in a humidified atmosphere of 95% air, 5% CO2 (v/v) in DMEM medium (GIBCO, Gaithesburger, MD, USA) supplemented with 10% (v/v) fetal bovine serum (GIBCO). .. Bafilomycin A1 was purchased from Enzo Life Sciences (Farmingdale, NY, USA), IGF-1 and EGF were obtained from Invitrogen (Burlingame, CA, USA), while LY294002, MK-2206 and LY2584702 were purchased from Selleck Chemicals (Houston, TX, USA).

    Concentration Assay:

    Article Title: Plectin protects podocytes from adriamycin‐induced apoptosis and F‐actin cytoskeletal disruption through the integrin α6β4/ FAK/p38 MAPK pathway, et al. Plectin protects podocytes from adriamycin‐induced apoptosis and F‐actin cytoskeletal disruption through the integrin α6β4/FAK/p38 MAPK pathway
    Article Snippet: .. To silence plectin expression, we transiently transfected the podocytes with siPlectin or scrambled siRNA at a final concentration of 20 nmol/L for 72 hours using Lipofectamine 2000 (Invitrogen: Waltham, MA). .. We monitored gene silencing efficiency by Western blot and selected the cells with the most efficient gene silencing for subsequent experiments.

    Incubation:

    Article Title: ER stress-induced mediator C/EBP homologous protein thwarts effector T cell activity in tumors through T-bet repression
    Article Snippet: .. After de-paraffinization and antigen retrieval, sections were blocked in 5% goat serum and incubated overnight with mouse monoclonal anti-CD8 (1:100, IgG1, C8/144B, #108M-98, Cell Marque) and mouse monoclonal anti-Chop (1:100, IgG2b, 9C8, #ab11419, Abcam) or mouse monoclonal anti-CD8 and rabbit polyclonal anti-CHOP/GADD153 (1:500, R-20, #sc-793, Santa Cruz Biotechnologies), followed by washes in PBS and incubation in secondary goat anti-mouse IgG1 and IgG2b or goat anti-mouse IgG1 and anti-rabbit IgG labeled with Alexa Fluor® 488 and 647 (all 1:200, ThermoFisher Scientific), respectively. .. For isotype control staining, tissues were similarly incubated with mouse IgG1 isotype (1:100, MG1-45, #401402, Biolegend) and mouse IgG2b isotype (1:100, MPC-11, #400376, Biolegend) or mouse IgG1 (1:100) and rabbit IgG (1:500, DA1E, #39600, Cell Signaling Technology), followed by incubation with specific secondary antibodies labeled with Alexa Fluor® 488 and 647, respectively.

    Article Title: Maladaptive Sexual Behavior Following Concurrent Methamphetamine and Sexual Experience in Male Rats is Associated with Altered Neural Activity in Frontal Cortex
    Article Snippet: .. One parallel series of sections from experiment 2 containing mPFC and orbitofrontal cortex (OFC) were incubated overnight with either mouse anti-CaMKII (1:10 000; Cat# MA1-048, Pierce Biotechnology, Rockford, IL) or mouse anti-GAD67 (1:200; Cat#MAB5406, lot 2188494, Millipore, Temecula, CA) and Alexa Fluor 555-conjugated goat anti-mouse secondary (1 : 100; 30 min; Cat# , Molecular Probes, Eugene, OR). .. Next, sections were incubated overnight with rabbit anti-pERK (1:4000; overnight; Cell Signaling, Cat# 9101) and with biotinylated goat anti-rabbit for one hour (1:500; cat. # BA-1000, Vector Laboratories, Burlingame, CA, USA), avidin-horseradish peroxidase complex (ABC-elite, 1:1000 in PBS; 1 h; cat. # PK-6100, Vector), biotinylated tyramine/tissue sample amplification (TSA; 1:250 in PBS containing 1 ul/ml of 3% H2O2 for 10 min; NEL700/700A; PerkinElmer Life Sciences, Boston, MA, USA), and Dylight 488 conjugated streptavidin (1:100 in PBS, 30 min; cat. #21832, lot PF204220, Pierce Biotech., Rockford, IL, USA).

    Activity Assay:

    Article Title: Development of viral nanoparticles for efficient intracellular delivery †
    Article Snippet: .. The biotin was detected using horseradish peroxidase-conjugated streptavidin (1 : 1000 in TBS containing 0.05% (v/v) Tween 20), and the horseradish peroxidase activity was detected using SuperSignal West Pico Chemiluminescent Substrate (Pierce). .. The electrochemiluminescent signal was visualized and the number of biotin labels was calculated using biotin standard solutions on the BioRad ChemiDoc XRS system.

    Labeling:

    Article Title: ER stress-induced mediator C/EBP homologous protein thwarts effector T cell activity in tumors through T-bet repression
    Article Snippet: .. After de-paraffinization and antigen retrieval, sections were blocked in 5% goat serum and incubated overnight with mouse monoclonal anti-CD8 (1:100, IgG1, C8/144B, #108M-98, Cell Marque) and mouse monoclonal anti-Chop (1:100, IgG2b, 9C8, #ab11419, Abcam) or mouse monoclonal anti-CD8 and rabbit polyclonal anti-CHOP/GADD153 (1:500, R-20, #sc-793, Santa Cruz Biotechnologies), followed by washes in PBS and incubation in secondary goat anti-mouse IgG1 and IgG2b or goat anti-mouse IgG1 and anti-rabbit IgG labeled with Alexa Fluor® 488 and 647 (all 1:200, ThermoFisher Scientific), respectively. .. For isotype control staining, tissues were similarly incubated with mouse IgG1 isotype (1:100, MG1-45, #401402, Biolegend) and mouse IgG2b isotype (1:100, MPC-11, #400376, Biolegend) or mouse IgG1 (1:100) and rabbit IgG (1:500, DA1E, #39600, Cell Signaling Technology), followed by incubation with specific secondary antibodies labeled with Alexa Fluor® 488 and 647, respectively.

    Expressing:

    Article Title: Plectin protects podocytes from adriamycin‐induced apoptosis and F‐actin cytoskeletal disruption through the integrin α6β4/ FAK/p38 MAPK pathway, et al. Plectin protects podocytes from adriamycin‐induced apoptosis and F‐actin cytoskeletal disruption through the integrin α6β4/FAK/p38 MAPK pathway
    Article Snippet: .. To silence plectin expression, we transiently transfected the podocytes with siPlectin or scrambled siRNA at a final concentration of 20 nmol/L for 72 hours using Lipofectamine 2000 (Invitrogen: Waltham, MA). .. We monitored gene silencing efficiency by Western blot and selected the cells with the most efficient gene silencing for subsequent experiments.

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Maladaptive Sexual Behavior Following Concurrent Methamphetamine and Sexual Experience in Male Rats is Associated with Altered Neural Activity in Frontal Cortex
    Article Snippet: .. One parallel series of sections from experiment 2 containing mPFC and orbitofrontal cortex (OFC) were incubated overnight with either mouse anti-CaMKII (1:10 000; Cat# MA1-048, Pierce Biotechnology, Rockford, IL) or mouse anti-GAD67 (1:200; CatMAB5406, lot 2188494, Millipore, Temecula, CA) and Alexa Fluor 555-conjugated goat anti-mouse secondary (1 : 100; 30 min; Cat# , Molecular Probes, Eugene, OR). .. Next, sections were incubated overnight with rabbit anti-pERK (1:4000; overnight; Cell Signaling, Cat# 9101) and with biotinylated goat anti-rabbit for one hour (1:500; cat. # BA-1000, Vector Laboratories, Burlingame, CA, USA), avidin-horseradish peroxidase complex (ABC-elite, 1:1000 in PBS; 1 h; cat. # PK-6100, Vector), biotinylated tyramine/tissue sample amplification (TSA; 1:250 in PBS containing 1 ul/ml of 3% H2O2 for 10 min; NEL700/700A; PerkinElmer Life Sciences, Boston, MA, USA), and Dylight 488 conjugated streptavidin (1:100 in PBS, 30 min; cat. #21832, lot PF204220, Pierce Biotech., Rockford, IL, USA).

    Blocking Assay:

    Article Title: Transglutaminase 2 inhibits Rb binding of human papillomavirus E7 by incorporating polyamine
    Article Snippet: .. After blocking and probing with HRP-conjugated streptavidin, the blots were developed by ECL protocol (Pierce). .. The above reaction mixture was dialyzed against PBS and the biotinylated spermine-incorporated E7 was isolated using streptavidin-conjugated magnetic beads (Dynal).

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  • 93
    Thermo Fisher horseradish peroxidase conjugated streptavidin
    Depletion of glypican-1 stimulates the endocytosis of PrP C . SH-SY5Y cells expressing wild type PrP C were treated with either control or glypican-1 siRNA and then incubated for 60 h. Cells were surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Where indicated, cells were treated with trypsin to remove remaining cell surface PrP C . Cells were then lysed and total PrP C immunoprecipitated from the sample using antibody 3F4. ( A ) Samples were subjected to western blot analysis and the biotin-labelled PrP C fraction was detected with peroxidase-conjugated <t>streptavidin.</t> ( B ) Densitometric analysis (mean ± s.e.m.) of multiple blots from three separate experiments in (A) is shown. ( C ) Expression of glypican-1 (in lysate samples treated with heparinase I and heparinase III) and PrP C in the cell lysates from (A). β-actin was used as a loading control. ( D ) SH-SY5Y cells expressing PrP C were treated with either control siRNA or glypican-1 siRNA and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( E ) Densitometric analysis of the proportion of total PrP C present in the detergent soluble fractions of the plasma membrane after siRNA treatment from three independent experiments. ( F ) SH-SY5Y cells expressing PrP C were seeded onto glass coverslips and grown to 50% confluency. Cells were fixed, and then incubated with anti-PrP antibody 3F4 and a glypican-1 polyclonal antibody. Finally, cells were incubated with Alexa488-conjugated rabbit anti-mouse and Alexa594-conjugated goat anti-rabbit antibodies and viewed using a DeltaVision Optical Restoration Microscopy System. Images are representative of three individual experiments. Scale bars equal 10 µm. * P
    Horseradish Peroxidase Conjugated Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/horseradish peroxidase conjugated streptavidin/product/Thermo Fisher
    Average 93 stars, based on 33 article reviews
    Price from $9.99 to $1999.99
    horseradish peroxidase conjugated streptavidin - by Bioz Stars, 2020-07
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    91
    Thermo Fisher streptavidin conjugated hrp
    Photoaffinity labeling of various PrP species. <t>Streptavidin-HRP-probed</t> blots of samples photoaffinity labeled with PA-PBD peptide. (A) Samples containing PrP Int1 or PrP C were incubated with or without PA-PBD and exposed to UV light for varying time periods, as indicated. (B) Samples containing α -helical PrP or PrP Int1 were incubated with PA-PBD and exposed to UV light for 5 min. (C) Samples of PrP Int1 were incubated with varying concentrations of PA-PBD, as indicated, and exposed to UV light for 0 or 5 min, as indicated. (D) Sample containing 7 μ g of PrP Int1 photoaffinity labeled with PA-PBD (PA-PrP Int1 ) is compared to a standard curve of biotinylated AviTag PrP for reference.
    Streptavidin Conjugated Hrp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin conjugated hrp/product/Thermo Fisher
    Average 91 stars, based on 21 article reviews
    Price from $9.99 to $1999.99
    streptavidin conjugated hrp - by Bioz Stars, 2020-07
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    Depletion of glypican-1 stimulates the endocytosis of PrP C . SH-SY5Y cells expressing wild type PrP C were treated with either control or glypican-1 siRNA and then incubated for 60 h. Cells were surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Where indicated, cells were treated with trypsin to remove remaining cell surface PrP C . Cells were then lysed and total PrP C immunoprecipitated from the sample using antibody 3F4. ( A ) Samples were subjected to western blot analysis and the biotin-labelled PrP C fraction was detected with peroxidase-conjugated streptavidin. ( B ) Densitometric analysis (mean ± s.e.m.) of multiple blots from three separate experiments in (A) is shown. ( C ) Expression of glypican-1 (in lysate samples treated with heparinase I and heparinase III) and PrP C in the cell lysates from (A). β-actin was used as a loading control. ( D ) SH-SY5Y cells expressing PrP C were treated with either control siRNA or glypican-1 siRNA and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( E ) Densitometric analysis of the proportion of total PrP C present in the detergent soluble fractions of the plasma membrane after siRNA treatment from three independent experiments. ( F ) SH-SY5Y cells expressing PrP C were seeded onto glass coverslips and grown to 50% confluency. Cells were fixed, and then incubated with anti-PrP antibody 3F4 and a glypican-1 polyclonal antibody. Finally, cells were incubated with Alexa488-conjugated rabbit anti-mouse and Alexa594-conjugated goat anti-rabbit antibodies and viewed using a DeltaVision Optical Restoration Microscopy System. Images are representative of three individual experiments. Scale bars equal 10 µm. * P

    Journal: PLoS Pathogens

    Article Title: Glypican-1 Mediates Both Prion Protein Lipid Raft Association and Disease Isoform Formation

    doi: 10.1371/journal.ppat.1000666

    Figure Lengend Snippet: Depletion of glypican-1 stimulates the endocytosis of PrP C . SH-SY5Y cells expressing wild type PrP C were treated with either control or glypican-1 siRNA and then incubated for 60 h. Cells were surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Where indicated, cells were treated with trypsin to remove remaining cell surface PrP C . Cells were then lysed and total PrP C immunoprecipitated from the sample using antibody 3F4. ( A ) Samples were subjected to western blot analysis and the biotin-labelled PrP C fraction was detected with peroxidase-conjugated streptavidin. ( B ) Densitometric analysis (mean ± s.e.m.) of multiple blots from three separate experiments in (A) is shown. ( C ) Expression of glypican-1 (in lysate samples treated with heparinase I and heparinase III) and PrP C in the cell lysates from (A). β-actin was used as a loading control. ( D ) SH-SY5Y cells expressing PrP C were treated with either control siRNA or glypican-1 siRNA and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( E ) Densitometric analysis of the proportion of total PrP C present in the detergent soluble fractions of the plasma membrane after siRNA treatment from three independent experiments. ( F ) SH-SY5Y cells expressing PrP C were seeded onto glass coverslips and grown to 50% confluency. Cells were fixed, and then incubated with anti-PrP antibody 3F4 and a glypican-1 polyclonal antibody. Finally, cells were incubated with Alexa488-conjugated rabbit anti-mouse and Alexa594-conjugated goat anti-rabbit antibodies and viewed using a DeltaVision Optical Restoration Microscopy System. Images are representative of three individual experiments. Scale bars equal 10 µm. * P

    Article Snippet: Where indicated, biotin-labelled PrP was detected by subsequent immunoprecipitation of epitope-tagged PrP from the individual fractions using antibody 3F4 (Eurogentec Ltd., Southampton, U.K.) and subsequent immunoblotting using horseradish peroxidase-conjugated streptavidin (Thermo Fisher Scientific, Cramlington, U.K.).

    Techniques: Expressing, Incubation, Immunoprecipitation, Western Blot, Gradient Centrifugation, Microscopy

    Depletion of glypican-1 does not affect cell division or surface levels of PrP C . ( A ) ScN2a cells were seeded into 96 well plates and treated with transfection reagent only or incubated with either control siRNA or one of the four siRNAs targeted to glypican-1. Those experiments exceeding 48 h were dosed with a second treatment of the indicated siRNAs. Cells were then rinsed with PBS and fixed with 70% (v/v) ethanol. Plates were allowed to dry, stained with Hoescht 33342 and the fluorescence measured. ( B ) ScN2a cells were treated with control or glypican-1 siRNA. After 96 h, cell monolayers were labelled with a membrane impermeable biotin reagent. Biotin-labelled cell surface PrP was detected by immunoprecipitation using 6D11 and subsequent immunoblotting using HRP-conjugated streptavidin. Total PrP and PK-resistant PrP (PrP Sc ) were detected by immunoblotting using antibody 6D11. ( C ) Densitometric analysis of the proportion of the relative amount of biotinylated cell surface PrP in the absence or presence of glypican-1 siRNA from three independent experiments.

    Journal: PLoS Pathogens

    Article Title: Glypican-1 Mediates Both Prion Protein Lipid Raft Association and Disease Isoform Formation

    doi: 10.1371/journal.ppat.1000666

    Figure Lengend Snippet: Depletion of glypican-1 does not affect cell division or surface levels of PrP C . ( A ) ScN2a cells were seeded into 96 well plates and treated with transfection reagent only or incubated with either control siRNA or one of the four siRNAs targeted to glypican-1. Those experiments exceeding 48 h were dosed with a second treatment of the indicated siRNAs. Cells were then rinsed with PBS and fixed with 70% (v/v) ethanol. Plates were allowed to dry, stained with Hoescht 33342 and the fluorescence measured. ( B ) ScN2a cells were treated with control or glypican-1 siRNA. After 96 h, cell monolayers were labelled with a membrane impermeable biotin reagent. Biotin-labelled cell surface PrP was detected by immunoprecipitation using 6D11 and subsequent immunoblotting using HRP-conjugated streptavidin. Total PrP and PK-resistant PrP (PrP Sc ) were detected by immunoblotting using antibody 6D11. ( C ) Densitometric analysis of the proportion of the relative amount of biotinylated cell surface PrP in the absence or presence of glypican-1 siRNA from three independent experiments.

    Article Snippet: Where indicated, biotin-labelled PrP was detected by subsequent immunoprecipitation of epitope-tagged PrP from the individual fractions using antibody 3F4 (Eurogentec Ltd., Southampton, U.K.) and subsequent immunoblotting using horseradish peroxidase-conjugated streptavidin (Thermo Fisher Scientific, Cramlington, U.K.).

    Techniques: Transfection, Incubation, Staining, Fluorescence, Immunoprecipitation

    Heparin stimulates the endocytosis of PrP C in a dose-dependent manner and displaces it from detergent-resistant lipid rafts. ( A ) SH-SY5Y cells expressing PrP C were surface biotinylated and then incubated for 1 h at 37°C in the absence or presence of various concentrations of heparin diluted in OptiMEM. Prior to lysis cells were, where indicated, incubated with trypsin to digest cell surface PrP C . Cells were then lysed and PrP C immunoprecipitated from the sample using antibody 3F4. Samples were subjected to SDS PAGE and western blot analysis and the biotin-labelled PrP C detected with peroxidase-conjugated streptavidin. ( B ) Densitometric analysis of multiple blots from four separate experiments as described in (A) is shown. ( C ) SH-SY5Y cells expressing PrP C were surface biotinylated and then incubated in the absence or presence of 50 µM heparin prepared in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. PrP C was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and subjected to SDS-PAGE and western blotting. The gradient fractions from both the untreated and heparin treated cells were analysed on the same SDS gel and immunoblotted under identical conditions. The biotin-labelled PrP C was detected with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions, respectively. ( D ) Densitometric analysis of the proportion of total PrP C in the detergent soluble fractions of the plasma membrane. ( E ) Untransfected SH-SY5Y cells and SH-SY5Y cells expressing either PrP C or PrP-TM were grown to confluence and then incubated for 1 h in the presence or absence of 50 µM heparin prepared in OptiMEM. Media samples were collected and concentrated and cells harvested and lysed. Cell lysate samples were immunoblotted for PrP C using antibody 3F4, with β-actin used as a loading control. ( F ) Quantification of PrP C and PrP-TM levels after treatment of cells with heparin as in (E). Experiments were performed in triplicate and repeated on three occasions. * P

    Journal: PLoS Pathogens

    Article Title: Glypican-1 Mediates Both Prion Protein Lipid Raft Association and Disease Isoform Formation

    doi: 10.1371/journal.ppat.1000666

    Figure Lengend Snippet: Heparin stimulates the endocytosis of PrP C in a dose-dependent manner and displaces it from detergent-resistant lipid rafts. ( A ) SH-SY5Y cells expressing PrP C were surface biotinylated and then incubated for 1 h at 37°C in the absence or presence of various concentrations of heparin diluted in OptiMEM. Prior to lysis cells were, where indicated, incubated with trypsin to digest cell surface PrP C . Cells were then lysed and PrP C immunoprecipitated from the sample using antibody 3F4. Samples were subjected to SDS PAGE and western blot analysis and the biotin-labelled PrP C detected with peroxidase-conjugated streptavidin. ( B ) Densitometric analysis of multiple blots from four separate experiments as described in (A) is shown. ( C ) SH-SY5Y cells expressing PrP C were surface biotinylated and then incubated in the absence or presence of 50 µM heparin prepared in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. PrP C was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and subjected to SDS-PAGE and western blotting. The gradient fractions from both the untreated and heparin treated cells were analysed on the same SDS gel and immunoblotted under identical conditions. The biotin-labelled PrP C was detected with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions, respectively. ( D ) Densitometric analysis of the proportion of total PrP C in the detergent soluble fractions of the plasma membrane. ( E ) Untransfected SH-SY5Y cells and SH-SY5Y cells expressing either PrP C or PrP-TM were grown to confluence and then incubated for 1 h in the presence or absence of 50 µM heparin prepared in OptiMEM. Media samples were collected and concentrated and cells harvested and lysed. Cell lysate samples were immunoblotted for PrP C using antibody 3F4, with β-actin used as a loading control. ( F ) Quantification of PrP C and PrP-TM levels after treatment of cells with heparin as in (E). Experiments were performed in triplicate and repeated on three occasions. * P

    Article Snippet: Where indicated, biotin-labelled PrP was detected by subsequent immunoprecipitation of epitope-tagged PrP from the individual fractions using antibody 3F4 (Eurogentec Ltd., Southampton, U.K.) and subsequent immunoblotting using horseradish peroxidase-conjugated streptavidin (Thermo Fisher Scientific, Cramlington, U.K.).

    Techniques: Expressing, Incubation, Lysis, Immunoprecipitation, SDS Page, Western Blot, Gradient Centrifugation, SDS-Gel

    Depletion of glypican-1 inhibits the association of PrP-TM with DRMs. SH-SY5Y cells expressing PrP-TM were treated with either control siRNA or siRNA targeted to glypican-1 and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C in the presence of Tyrphostin A23 to block endocytosis. The media was removed and the cells washed in phosphate-buffered saline prior to homogenisation in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( A ) Quantification of glypican-1 and PrP-TM expression in cell lysates. To detect glypican-1, cell lysate samples were treated with heparinase I and heparinase III prior to electrophoresis as described in the materials and methods section. ( B ) PrP-TM was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and then subjected to western blotting with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions, respectively. ( C ) Densitometric analysis of the proportion of total PrP-TM present in the detergent soluble fractions of the plasma membrane after siRNA treatment from multiple blots from three independent experiments. * P

    Journal: PLoS Pathogens

    Article Title: Glypican-1 Mediates Both Prion Protein Lipid Raft Association and Disease Isoform Formation

    doi: 10.1371/journal.ppat.1000666

    Figure Lengend Snippet: Depletion of glypican-1 inhibits the association of PrP-TM with DRMs. SH-SY5Y cells expressing PrP-TM were treated with either control siRNA or siRNA targeted to glypican-1 and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C in the presence of Tyrphostin A23 to block endocytosis. The media was removed and the cells washed in phosphate-buffered saline prior to homogenisation in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( A ) Quantification of glypican-1 and PrP-TM expression in cell lysates. To detect glypican-1, cell lysate samples were treated with heparinase I and heparinase III prior to electrophoresis as described in the materials and methods section. ( B ) PrP-TM was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and then subjected to western blotting with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions, respectively. ( C ) Densitometric analysis of the proportion of total PrP-TM present in the detergent soluble fractions of the plasma membrane after siRNA treatment from multiple blots from three independent experiments. * P

    Article Snippet: Where indicated, biotin-labelled PrP was detected by subsequent immunoprecipitation of epitope-tagged PrP from the individual fractions using antibody 3F4 (Eurogentec Ltd., Southampton, U.K.) and subsequent immunoblotting using horseradish peroxidase-conjugated streptavidin (Thermo Fisher Scientific, Cramlington, U.K.).

    Techniques: Expressing, Incubation, Blocking Assay, Homogenization, Gradient Centrifugation, Electrophoresis, Immunoprecipitation, Western Blot

    The association of PrP-TM with DRMs is disrupted by treatment of cells with either heparin or bacterial PI-PLC. SH-SY5Y cells expressing PrP-TM were surface biotinylated and then ( A ) incubated in the absence or presence of 50 µM heparin prepared in OptiMEM for 1 h at 37°C or ( B ) incubated in the absence or presence of 1 U/ml bacterial PI-PLC for 1 h at 4°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. PrP-TM was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and subjected to western blotting. The biotin-labelled PrP-TM fraction was detected with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions respectively. ( C ) Densitometric analysis of the proportion of total PrP-TM present in the detergent soluble fractions of the plasma membrane after heparin and PI-PLC treatment. Experiments were performed in triplicate and repeated on three occasions. * P

    Journal: PLoS Pathogens

    Article Title: Glypican-1 Mediates Both Prion Protein Lipid Raft Association and Disease Isoform Formation

    doi: 10.1371/journal.ppat.1000666

    Figure Lengend Snippet: The association of PrP-TM with DRMs is disrupted by treatment of cells with either heparin or bacterial PI-PLC. SH-SY5Y cells expressing PrP-TM were surface biotinylated and then ( A ) incubated in the absence or presence of 50 µM heparin prepared in OptiMEM for 1 h at 37°C or ( B ) incubated in the absence or presence of 1 U/ml bacterial PI-PLC for 1 h at 4°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. PrP-TM was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and subjected to western blotting. The biotin-labelled PrP-TM fraction was detected with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions respectively. ( C ) Densitometric analysis of the proportion of total PrP-TM present in the detergent soluble fractions of the plasma membrane after heparin and PI-PLC treatment. Experiments were performed in triplicate and repeated on three occasions. * P

    Article Snippet: Where indicated, biotin-labelled PrP was detected by subsequent immunoprecipitation of epitope-tagged PrP from the individual fractions using antibody 3F4 (Eurogentec Ltd., Southampton, U.K.) and subsequent immunoblotting using horseradish peroxidase-conjugated streptavidin (Thermo Fisher Scientific, Cramlington, U.K.).

    Techniques: Planar Chromatography, Expressing, Incubation, Gradient Centrifugation, Immunoprecipitation, Western Blot

    Oxidative modification of identified proteins in planta upon H 2 O 2 treatment. Transgenic plants expressing the protein of interest fused with the FLAG tag were vacuum infiltrated with either water (mock) or 5 mM H 2 O 2 . For analysis of AtCIAPIN1, eEF1α, and AtPTP1, free thiols in the total protein were labeled with BIAM during protein extraction. For analysis of AtNAP1;1 and AtPDIL1-1, free thiols in the samples were first alkylated by IAM. Samples were then treated with DTT and newly generated free thiols were labeled by BIAM. After that, FLAG-tagged protein from each sample was affinity purified, separated by SDS-PAGE, and detected by HRP-Conjugated Streptavidin (to determine the amount of BIAM attached to the FLAG-tagged protein) or by the anti-FLAG M2 antibody (to determine the amount of the total recombinant protein).

    Journal: Journal of Proteome Research

    Article Title: Proteomic Analysis of Early-Responsive Redox-Sensitive Proteins in Arabidopsis

    doi: 10.1021/pr200918f

    Figure Lengend Snippet: Oxidative modification of identified proteins in planta upon H 2 O 2 treatment. Transgenic plants expressing the protein of interest fused with the FLAG tag were vacuum infiltrated with either water (mock) or 5 mM H 2 O 2 . For analysis of AtCIAPIN1, eEF1α, and AtPTP1, free thiols in the total protein were labeled with BIAM during protein extraction. For analysis of AtNAP1;1 and AtPDIL1-1, free thiols in the samples were first alkylated by IAM. Samples were then treated with DTT and newly generated free thiols were labeled by BIAM. After that, FLAG-tagged protein from each sample was affinity purified, separated by SDS-PAGE, and detected by HRP-Conjugated Streptavidin (to determine the amount of BIAM attached to the FLAG-tagged protein) or by the anti-FLAG M2 antibody (to determine the amount of the total recombinant protein).

    Article Snippet: Immunoprecipitated protein was separated by SDS-PAGE and immunoblotted with either anti-FLAG M2-Peroxidase (HRP) antibody (Sigma) or Horseradish Peroxidase-Conjugated Streptavidin (Thermo Scientific).

    Techniques: Modification, Transgenic Assay, Expressing, FLAG-tag, Labeling, Protein Extraction, Generated, Affinity Purification, SDS Page, Recombinant

    Photoaffinity labeling of various PrP species. Streptavidin-HRP-probed blots of samples photoaffinity labeled with PA-PBD peptide. (A) Samples containing PrP Int1 or PrP C were incubated with or without PA-PBD and exposed to UV light for varying time periods, as indicated. (B) Samples containing α -helical PrP or PrP Int1 were incubated with PA-PBD and exposed to UV light for 5 min. (C) Samples of PrP Int1 were incubated with varying concentrations of PA-PBD, as indicated, and exposed to UV light for 0 or 5 min, as indicated. (D) Sample containing 7 μ g of PrP Int1 photoaffinity labeled with PA-PBD (PA-PrP Int1 ) is compared to a standard curve of biotinylated AviTag PrP for reference.

    Journal: Biochemistry

    Article Title: Prion Nucleation Site Unmasked by Transient Interaction with Phospholipid Cofactor

    doi: 10.1021/bi4014825

    Figure Lengend Snippet: Photoaffinity labeling of various PrP species. Streptavidin-HRP-probed blots of samples photoaffinity labeled with PA-PBD peptide. (A) Samples containing PrP Int1 or PrP C were incubated with or without PA-PBD and exposed to UV light for varying time periods, as indicated. (B) Samples containing α -helical PrP or PrP Int1 were incubated with PA-PBD and exposed to UV light for 5 min. (C) Samples of PrP Int1 were incubated with varying concentrations of PA-PBD, as indicated, and exposed to UV light for 0 or 5 min, as indicated. (D) Sample containing 7 μ g of PrP Int1 photoaffinity labeled with PA-PBD (PA-PrP Int1 ) is compared to a standard curve of biotinylated AviTag PrP for reference.

    Article Snippet: The resulting photoaffinity-labeled molecules were run on SDS-PAGE, transferred to PVDF, blocked with a 2.5% solution of bovine serum albumin (Fisher Scientific, Pittsburgh, PA), and incubated with streptavidin-conjugated HRP (ThermoFisher Scientific, Rockford, IL) at a 1:10 000 dilution before being washed with TBST and developed with SuperSignal West Femto maximum sensitivity substrate (ThermoFisher Scientific, Rockford, IL).

    Techniques: Labeling, Incubation

    TRAF6-mediated GSK3β ubiquitination at lysine 183 is critical for TLR3-dependent cytokine production. ( a ) BMDMs were stimulated with 10 μg ml −1 poly I:C for 10 min and subjected to immunoprecipitation with an anti-Ub antibody followed by western blotting with an anti-GSK3β antibody. ( b ) HEK293T cells transfected with HA-GSK3β and HA-Ub along with Flag-TRAF6 plasmids were subjected to immunoprecipitation with an anti-GSK3β antibody followed by western blotting with an anti-HA antibody. ( c ) HEK293T cells were transfected with HA-GSK3β and HA-Ub along with TRAF6 (WT) or TRAF6 (C70A) plasmids. These experiments were performed as described in b . ( d ) Traf6 +/+ and Traf6 −/− 3T3 cells stimulated with 10 μg ml −1 poly I:C for 10 min were subjected to immunoprecipitation with an anti-GSK3β antibody followed by western blotting with an anti-Ub antibody. ( e ) GSK3β proteins were incubated with E1, E2 and biotinylated-Ub (Bt-Ub) in the presence or absence of Flag-TRAF6 proteins for in vitro ubiquitination of GSK3β. Ubiquitination of GSK3β was analysed by western blotting with streptavidin-HRP. ( f ) HEK293T cells transfected with Ub and Flag-TRAF6 along with HA-GSK3β WT or various HA-GSK3β mutants were subjected to immunoprecipitation with an anti-HA antibody followed by western blotting with an anti-Ub antibody. ( g ) HEK293-TLR3 cells were transiently transfected with GSK3β (WT) or GSK3β (K183R) plasmids. The levels of IL-6, TNF-α and c-Fos mRNA were determined by real-time PCR analysis (top). GSK3β expression levels were confirmed by western blotting with an anti-HA antibody (bottom). A longer exposure of the HA blot shows the presence of ubiquitin ladder. Data are presented as the mean±s.d. from at least three independent experiments. Statistical analyses were calculated using the Student’s t -test (** P

    Journal: Nature Communications

    Article Title: Glycogen synthase kinase 3β ubiquitination by TRAF6 regulates TLR3-mediated pro-inflammatory cytokine production

    doi: 10.1038/ncomms7765

    Figure Lengend Snippet: TRAF6-mediated GSK3β ubiquitination at lysine 183 is critical for TLR3-dependent cytokine production. ( a ) BMDMs were stimulated with 10 μg ml −1 poly I:C for 10 min and subjected to immunoprecipitation with an anti-Ub antibody followed by western blotting with an anti-GSK3β antibody. ( b ) HEK293T cells transfected with HA-GSK3β and HA-Ub along with Flag-TRAF6 plasmids were subjected to immunoprecipitation with an anti-GSK3β antibody followed by western blotting with an anti-HA antibody. ( c ) HEK293T cells were transfected with HA-GSK3β and HA-Ub along with TRAF6 (WT) or TRAF6 (C70A) plasmids. These experiments were performed as described in b . ( d ) Traf6 +/+ and Traf6 −/− 3T3 cells stimulated with 10 μg ml −1 poly I:C for 10 min were subjected to immunoprecipitation with an anti-GSK3β antibody followed by western blotting with an anti-Ub antibody. ( e ) GSK3β proteins were incubated with E1, E2 and biotinylated-Ub (Bt-Ub) in the presence or absence of Flag-TRAF6 proteins for in vitro ubiquitination of GSK3β. Ubiquitination of GSK3β was analysed by western blotting with streptavidin-HRP. ( f ) HEK293T cells transfected with Ub and Flag-TRAF6 along with HA-GSK3β WT or various HA-GSK3β mutants were subjected to immunoprecipitation with an anti-HA antibody followed by western blotting with an anti-Ub antibody. ( g ) HEK293-TLR3 cells were transiently transfected with GSK3β (WT) or GSK3β (K183R) plasmids. The levels of IL-6, TNF-α and c-Fos mRNA were determined by real-time PCR analysis (top). GSK3β expression levels were confirmed by western blotting with an anti-HA antibody (bottom). A longer exposure of the HA blot shows the presence of ubiquitin ladder. Data are presented as the mean±s.d. from at least three independent experiments. Statistical analyses were calculated using the Student’s t -test (** P

    Article Snippet: Samples were subsequently immunoprecipitated with an anti-GSK3β antibody and separated on SDS–PAGE followed by streptavidin conjugated to HRP (Thermo Fisher Scientific).

    Techniques: Immunoprecipitation, Western Blot, Transfection, Incubation, In Vitro, Real-time Polymerase Chain Reaction, Expressing