biotinylated  (Thermo Fisher)


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    Structured Review

    Thermo Fisher biotinylated
    Enzymatic addition of a single 3′ biotin moiety to unmodified oligos Unmodified oligos were <t>biotinylated</t> with biotin-ddUTP and terminal deoxynucleotidyl transferase (TdT). Biotinylation efficiency was assessed by Urea PAGE. Successful biotinylation causes an upward shift of the oligo in the gel. In each lane, 1 pmol of oligo was loaded. Left: Biotinylation efficiency assessment for single-probe reactions, a representative gel is shown. Right: Biotinylation efficiency assessment for the pool of 15 oligos. L = DNA ladder.
    Biotinylated, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/biotinylated/product/Thermo Fisher
    Average 99 stars, based on 13 article reviews
    Price from $9.99 to $1999.99
    biotinylated - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Ribo-Pop: Simple, cost-effective, and widely applicable ribosomal RNA depletion"

    Article Title: Ribo-Pop: Simple, cost-effective, and widely applicable ribosomal RNA depletion

    Journal: bioRxiv

    doi: 10.1101/2020.05.19.102293

    Enzymatic addition of a single 3′ biotin moiety to unmodified oligos Unmodified oligos were biotinylated with biotin-ddUTP and terminal deoxynucleotidyl transferase (TdT). Biotinylation efficiency was assessed by Urea PAGE. Successful biotinylation causes an upward shift of the oligo in the gel. In each lane, 1 pmol of oligo was loaded. Left: Biotinylation efficiency assessment for single-probe reactions, a representative gel is shown. Right: Biotinylation efficiency assessment for the pool of 15 oligos. L = DNA ladder.
    Figure Legend Snippet: Enzymatic addition of a single 3′ biotin moiety to unmodified oligos Unmodified oligos were biotinylated with biotin-ddUTP and terminal deoxynucleotidyl transferase (TdT). Biotinylation efficiency was assessed by Urea PAGE. Successful biotinylation causes an upward shift of the oligo in the gel. In each lane, 1 pmol of oligo was loaded. Left: Biotinylation efficiency assessment for single-probe reactions, a representative gel is shown. Right: Biotinylation efficiency assessment for the pool of 15 oligos. L = DNA ladder.

    Techniques Used: Polyacrylamide Gel Electrophoresis

    Short anti-sense oligos effectively deplete ribosomal RNA Oligonucleotides targeting the Drosophila small rRNA (18S) were individually tested for their ability to deplete the 18S transcript from larval total RNA. The percent of remaining 18S was quantified by qPCR. Values are derived from 18S normalized to Act5c , in turn normalized to a non-depleted sample (no probe control). Values are the averages of three replicates of the depletion experiment, each using a different sample of larval RNA. A) Outline of the single-probe depletion assay. A 3′ biotinylated probe targeting a specific site in the 18S is added to total RNA and subjected to hybridization. The target is captured with streptavidin beads and the remaining target is measured from the supernatant. B) The percent of 18S rRNA remaining for all tested oligos of size 26 – 32 nt, arranged 5′ to 3′ by target site. Error bars are standard deviation between separate hybridization experiments, each performed with a different RNA sample. C) Performance comparison between the initial set of low Tm probes (probes #1 – #11, Tm
    Figure Legend Snippet: Short anti-sense oligos effectively deplete ribosomal RNA Oligonucleotides targeting the Drosophila small rRNA (18S) were individually tested for their ability to deplete the 18S transcript from larval total RNA. The percent of remaining 18S was quantified by qPCR. Values are derived from 18S normalized to Act5c , in turn normalized to a non-depleted sample (no probe control). Values are the averages of three replicates of the depletion experiment, each using a different sample of larval RNA. A) Outline of the single-probe depletion assay. A 3′ biotinylated probe targeting a specific site in the 18S is added to total RNA and subjected to hybridization. The target is captured with streptavidin beads and the remaining target is measured from the supernatant. B) The percent of 18S rRNA remaining for all tested oligos of size 26 – 32 nt, arranged 5′ to 3′ by target site. Error bars are standard deviation between separate hybridization experiments, each performed with a different RNA sample. C) Performance comparison between the initial set of low Tm probes (probes #1 – #11, Tm

    Techniques Used: Real-time Polymerase Chain Reaction, Derivative Assay, Depletion Assay, Hybridization, Standard Deviation

    2) Product Images from "Binding of the Methyl Donor S-Adenosyl-l-Methionine to Middle East Respiratory Syndrome Coronavirus 2′-O-Methyltransferase nsp16 Promotes Recruitment of the Allosteric Activator nsp10"

    Article Title: Binding of the Methyl Donor S-Adenosyl-l-Methionine to Middle East Respiratory Syndrome Coronavirus 2′-O-Methyltransferase nsp16 Promotes Recruitment of the Allosteric Activator nsp10

    Journal: Journal of Virology

    doi: 10.1128/JVI.02217-16

    SAM and SAH modulate nsp10-nsp16 interaction. (A) nsp10/nsp16 steady-state assembly: real-time nsp16 binding to biotinylated nsp10 was measured by Octet biolayer interferometry. Streptavidin biosensors coated with 100 nM biotinylated nsp10 were used to
    Figure Legend Snippet: SAM and SAH modulate nsp10-nsp16 interaction. (A) nsp10/nsp16 steady-state assembly: real-time nsp16 binding to biotinylated nsp10 was measured by Octet biolayer interferometry. Streptavidin biosensors coated with 100 nM biotinylated nsp10 were used to

    Techniques Used: Binding Assay

    3) Product Images from "Combinatorial roles for zebrafish retinoic acid receptors in the hindbrain, limbs and pharyngeal arches"

    Article Title: Combinatorial roles for zebrafish retinoic acid receptors in the hindbrain, limbs and pharyngeal arches

    Journal: Developmental biology

    doi: 10.1016/j.ydbio.2008.09.022

    Grafts of wild-type mesoderm rescue tbx5 expression in rarab morphants. (A) Cell transplantation technique. Donor embryos were injected at the one-cell stage with both a fixable biotinylated dextran and a fluorescent rhodamine-dextran (red). Hosts were
    Figure Legend Snippet: Grafts of wild-type mesoderm rescue tbx5 expression in rarab morphants. (A) Cell transplantation technique. Donor embryos were injected at the one-cell stage with both a fixable biotinylated dextran and a fluorescent rhodamine-dextran (red). Hosts were

    Techniques Used: Expressing, Transplantation Assay, Injection

    4) Product Images from "Staphylococcal acid phosphatase binds to endothelial cells via charge interaction; a pathogenic role in Wegener's granulomatosis?"

    Article Title: Staphylococcal acid phosphatase binds to endothelial cells via charge interaction; a pathogenic role in Wegener's granulomatosis?

    Journal: Clinical and Experimental Immunology

    doi: 10.1046/j.1365-2249.2000.01172.x

    Binding of staphylococcal acid phosphatase (SAcP) to human umbilical vein endothelial cells (HUVEC) (a) measured by incubating HUVEC with biotinylated SAcP followed by peroxidase-labelled streptavidin. Results represent mean ± s.d. (* P
    Figure Legend Snippet: Binding of staphylococcal acid phosphatase (SAcP) to human umbilical vein endothelial cells (HUVEC) (a) measured by incubating HUVEC with biotinylated SAcP followed by peroxidase-labelled streptavidin. Results represent mean ± s.d. (* P

    Techniques Used: Binding Assay

    5) Product Images from "Dual targeting improves microbubble contrast agent adhesion to VCAM-1 and P-selectin under flow"

    Article Title: Dual targeting improves microbubble contrast agent adhesion to VCAM-1 and P-selectin under flow

    Journal: Journal of controlled release : official journal of the Controlled Release Society

    doi: 10.1016/j.jconrel.2009.08.001

    Perfluorocarbon-filled microbubble targeted with MVCAM.A(429) against VCAM-1 (Y-shaped on bubble) and polymeric Sle x (branch-shaped), which binds selectins. A biotin-streptavidin coupling system is used to graft biotinylated mAbs and carbohydrates on
    Figure Legend Snippet: Perfluorocarbon-filled microbubble targeted with MVCAM.A(429) against VCAM-1 (Y-shaped on bubble) and polymeric Sle x (branch-shaped), which binds selectins. A biotin-streptavidin coupling system is used to graft biotinylated mAbs and carbohydrates on

    Techniques Used:

    6) Product Images from "Identification of two distinct peptide-binding pockets in the SH3 domain of human mixed-lineage kinase 3"

    Article Title: Identification of two distinct peptide-binding pockets in the SH3 domain of human mixed-lineage kinase 3

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA117.000262

    Binding properties of synthetic MIP. To determine the IC 50 value of MIP, a GST-MLK3 SH3(43–104) domain fusion protein was preincubated with increasing concentration of unlabeled MIP (AIRINPNGTWSRQAETVES) as competitor and then allowed to interact with biotinylated MIP (AIRINPNGTWSRQAETVES-K-biotin) immobilized on a NeutrAvidin-coated 96-well ELISA plate. Binding of SH3 domain was detected with anti-GST antibody conjugated to HRP, and the levels are presented as percentage of binding in the absence of competitor. Experiments were performed in triplicate, and the results are an averaged value; error bars reflect the standard deviation of each point. The curve fitting was performed with GraphPad Prism 6.0 software.
    Figure Legend Snippet: Binding properties of synthetic MIP. To determine the IC 50 value of MIP, a GST-MLK3 SH3(43–104) domain fusion protein was preincubated with increasing concentration of unlabeled MIP (AIRINPNGTWSRQAETVES) as competitor and then allowed to interact with biotinylated MIP (AIRINPNGTWSRQAETVES-K-biotin) immobilized on a NeutrAvidin-coated 96-well ELISA plate. Binding of SH3 domain was detected with anti-GST antibody conjugated to HRP, and the levels are presented as percentage of binding in the absence of competitor. Experiments were performed in triplicate, and the results are an averaged value; error bars reflect the standard deviation of each point. The curve fitting was performed with GraphPad Prism 6.0 software.

    Techniques Used: Binding Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation, Software

    Competition ELISA between MIP and NS5A peptide. To determine whether the MIP and NS5A peptides competitively bind the SH3 domain of MLK3, a GST-MLK3 SH3(43–104) fusion protein was preincubated with increasing concentrations of unlabeled MIP peptide or unlabeled NS5A peptide as competitor and then allowed to interact with biotinylated MIP ( A ) or biotinylated NS5A ( B ) peptide probes immobilized on a NeutrAvidin-coated 96-well ELISA plate. Binding of the SH3 domain was detected with an anti-GST antibody conjugated to HRP, and the signal levels are presented as a percentage of binding in the absence of competitor. Experiments were performed in triplicate, and the results are an averaged value; error bars reflect the standard deviation of each point. Curve fitting was performed with OriginPro 2017.
    Figure Legend Snippet: Competition ELISA between MIP and NS5A peptide. To determine whether the MIP and NS5A peptides competitively bind the SH3 domain of MLK3, a GST-MLK3 SH3(43–104) fusion protein was preincubated with increasing concentrations of unlabeled MIP peptide or unlabeled NS5A peptide as competitor and then allowed to interact with biotinylated MIP ( A ) or biotinylated NS5A ( B ) peptide probes immobilized on a NeutrAvidin-coated 96-well ELISA plate. Binding of the SH3 domain was detected with an anti-GST antibody conjugated to HRP, and the signal levels are presented as a percentage of binding in the absence of competitor. Experiments were performed in triplicate, and the results are an averaged value; error bars reflect the standard deviation of each point. Curve fitting was performed with OriginPro 2017.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Binding Assay, Standard Deviation

    Binding of MIP and NS5A peptides to SH3 domain of other members of MLK subfamily (MLK1–4). To determine whether MIP and NS5A can interact with SH3 domains of MLK1–4 proteins, the purified GST-MLK1–4 SH3 domains were incubated with biotinylated peptides, NS5A peptide (KKAPTPPPRRRR-GGG-K-biotin), MIP (AIRINPNGTWSRQAETVES-K-biotin), and MIP-R12A (AIRINPNGTWSAQAETVES-K-biotin), immobilized on a NeutrAvidin-coated 96-well ELISA plate. Binding of SH3 domain was detected with anti-GST antibody conjugated to HRP. Experiments were performed in triplicate, and the results are an averaged value; error bars reflect the standard deviation of each point. See also Fig. S6 .
    Figure Legend Snippet: Binding of MIP and NS5A peptides to SH3 domain of other members of MLK subfamily (MLK1–4). To determine whether MIP and NS5A can interact with SH3 domains of MLK1–4 proteins, the purified GST-MLK1–4 SH3 domains were incubated with biotinylated peptides, NS5A peptide (KKAPTPPPRRRR-GGG-K-biotin), MIP (AIRINPNGTWSRQAETVES-K-biotin), and MIP-R12A (AIRINPNGTWSAQAETVES-K-biotin), immobilized on a NeutrAvidin-coated 96-well ELISA plate. Binding of SH3 domain was detected with anti-GST antibody conjugated to HRP. Experiments were performed in triplicate, and the results are an averaged value; error bars reflect the standard deviation of each point. See also Fig. S6 .

    Techniques Used: Binding Assay, Purification, Incubation, Enzyme-linked Immunosorbent Assay, Standard Deviation

    7) Product Images from "Immunoglobulin M Capture Assay for Serologic Confirmation of Early Lyme Disease: Analysis of Immune Complexes with Biotinylated Borrelia burgdorferi Sonicate Enhanced with Flagellin Peptide Epitope"

    Article Title: Immunoglobulin M Capture Assay for Serologic Confirmation of Early Lyme Disease: Analysis of Immune Complexes with Biotinylated Borrelia burgdorferi Sonicate Enhanced with Flagellin Peptide Epitope

    Journal: Journal of Clinical Microbiology

    doi:

    Influence of synthetic epitope flagellin antigen on free and IC-derived L antibodies to B. burgdorferi . Serum samples from patients containing IC-derived antibody (precipitated with PEG and resuspended in PEG) assayed with biotinylated whole-cell sonicate (Bb-bio) alone (a), serum samples containing IC-derived antibody assayed with biotinylated whole-cell sonicate (Bb-bio) plus biotinylated flagellin synthetic epitope-BSA conjugate (Fla-bio) (b), serum samples containing free (untreated) antibody assayed with biotinylated whole-cell sonicate (Bb-bio) alone (c), and serum samples containing free (untreated) antibody assayed with biotinylated whole-cell sonicate (Bb-bio) plus biotinylated flagellin synthetic epitope-BSA conjugate (Fla-bio) (d) as the antigen source were used to detect LD-specific IgM antibodies in M-capture ELISA. Results are expressed as an index value, as defined in Materials and Methods.
    Figure Legend Snippet: Influence of synthetic epitope flagellin antigen on free and IC-derived L antibodies to B. burgdorferi . Serum samples from patients containing IC-derived antibody (precipitated with PEG and resuspended in PEG) assayed with biotinylated whole-cell sonicate (Bb-bio) alone (a), serum samples containing IC-derived antibody assayed with biotinylated whole-cell sonicate (Bb-bio) plus biotinylated flagellin synthetic epitope-BSA conjugate (Fla-bio) (b), serum samples containing free (untreated) antibody assayed with biotinylated whole-cell sonicate (Bb-bio) alone (c), and serum samples containing free (untreated) antibody assayed with biotinylated whole-cell sonicate (Bb-bio) plus biotinylated flagellin synthetic epitope-BSA conjugate (Fla-bio) (d) as the antigen source were used to detect LD-specific IgM antibodies in M-capture ELISA. Results are expressed as an index value, as defined in Materials and Methods.

    Techniques Used: Derivative Assay, Enzyme-linked Immunosorbent Assay

    Influence of synthetic flagellin epitope (unprocessed serum). Serum samples from patients were assayed for LD (Lyme specific IgM antibodies) by M-capture ELISA. Either the biotinylated whole-cell sonicate (Bb-bio) alone (a), the biotinylated whole-cell sonicate (Bb-bio) plus biotinylated flagellin synthetic epitope-BSA conjugate (Fla-bio) (b), or the biotinylated flagellin synthetic epitope-BSA conjugate (Fla-bio) alone (c) was used as the antigen source. Results are expressed as net mean OD 450 , as defined in Materials and Methods. The sets of bars represent data for samples from patients 7, 1 (22 May 1997), 1 (23 May 1997), 2 to 5 and 95-11891 from left to right, respectively.
    Figure Legend Snippet: Influence of synthetic flagellin epitope (unprocessed serum). Serum samples from patients were assayed for LD (Lyme specific IgM antibodies) by M-capture ELISA. Either the biotinylated whole-cell sonicate (Bb-bio) alone (a), the biotinylated whole-cell sonicate (Bb-bio) plus biotinylated flagellin synthetic epitope-BSA conjugate (Fla-bio) (b), or the biotinylated flagellin synthetic epitope-BSA conjugate (Fla-bio) alone (c) was used as the antigen source. Results are expressed as net mean OD 450 , as defined in Materials and Methods. The sets of bars represent data for samples from patients 7, 1 (22 May 1997), 1 (23 May 1997), 2 to 5 and 95-11891 from left to right, respectively.

    Techniques Used: Enzyme-linked Immunosorbent Assay

    8) Product Images from "Integrin-?3 mediates binding of Chordin to the cell surface and promotes its endocytosis"

    Article Title: Integrin-?3 mediates binding of Chordin to the cell surface and promotes its endocytosis

    Journal: EMBO Reports

    doi: 10.1038/sj.embor.embor902

    Chordin affinity columns bind biotinylated cell-surface Integrin-α3. ( A ) A Chordin (Chd)–Fc affinity matrix binds two distinct cell-surface proteins in COS-7 cells. Cell extracts containing biotinylated surface proteins from COS-7 cells were bound to secreted Fc (s-Fc; lane 2) or Chd–Fc (lane 3) columns, or were immunoprecipitated using an anti-Integrin-α3 (Int-α3) antibody (lane 5). Proteins bound to the columns were analysed by immunoblotting with streptavidin–horseradish-peroxidase (SA–HRP; Pierce). Lane 1 shows loading of 1% of the total biotinylated cell lysate. ( B ) A Chordin affinity matrix binds Integrin-α3. COS-7 cell extracts were bound to s-Fc (lane 2) or Chd–Fc affinity columns (lane 3), eluted, and analysed by immunoblotting with anti-Integrin-α3. ( C – G ) Chordin and Integrin-α3 are co-expressed during embryonic development. In situ hybridization analysis of chordin ( C , D ) and integrin-a3 ( E , F ) expression at stages 10.5 and 32. ( G ) RT–PCR (PCR after reverse transcription) analysis of chordin and integrin-a3 messenger RNA levels in control and LiCl-treated embryos. Elongation factor-1α (EF-1α) was used as a loading control.
    Figure Legend Snippet: Chordin affinity columns bind biotinylated cell-surface Integrin-α3. ( A ) A Chordin (Chd)–Fc affinity matrix binds two distinct cell-surface proteins in COS-7 cells. Cell extracts containing biotinylated surface proteins from COS-7 cells were bound to secreted Fc (s-Fc; lane 2) or Chd–Fc (lane 3) columns, or were immunoprecipitated using an anti-Integrin-α3 (Int-α3) antibody (lane 5). Proteins bound to the columns were analysed by immunoblotting with streptavidin–horseradish-peroxidase (SA–HRP; Pierce). Lane 1 shows loading of 1% of the total biotinylated cell lysate. ( B ) A Chordin affinity matrix binds Integrin-α3. COS-7 cell extracts were bound to s-Fc (lane 2) or Chd–Fc affinity columns (lane 3), eluted, and analysed by immunoblotting with anti-Integrin-α3. ( C – G ) Chordin and Integrin-α3 are co-expressed during embryonic development. In situ hybridization analysis of chordin ( C , D ) and integrin-a3 ( E , F ) expression at stages 10.5 and 32. ( G ) RT–PCR (PCR after reverse transcription) analysis of chordin and integrin-a3 messenger RNA levels in control and LiCl-treated embryos. Elongation factor-1α (EF-1α) was used as a loading control.

    Techniques Used: Immunoprecipitation, In Situ Hybridization, Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    9) Product Images from "Isolation of breast cancer and gastric cancer circulating tumor cells by use of an anti HER2-based microfluidic device"

    Article Title: Isolation of breast cancer and gastric cancer circulating tumor cells by use of an anti HER2-based microfluidic device

    Journal: Lab on a chip

    doi: 10.1039/c3lc51039e

    Adsorption test for biotinylated anti-HER2 clone 9G6.10. Increasingconcentrations of biotinylated mouse anti-HER2 antibody clone 9G6.10 wereincubated on a neutravidin-coated plate. A fluorescently labeled secondary antimouse antibody was used to detect
    Figure Legend Snippet: Adsorption test for biotinylated anti-HER2 clone 9G6.10. Increasingconcentrations of biotinylated mouse anti-HER2 antibody clone 9G6.10 wereincubated on a neutravidin-coated plate. A fluorescently labeled secondary antimouse antibody was used to detect

    Techniques Used: Adsorption, Labeling

    10) Product Images from "Structural Based Screening of Antiandrogen Targeting Activation Function-2 Binding Site"

    Article Title: Structural Based Screening of Antiandrogen Targeting Activation Function-2 Binding Site

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2018.01419

    IMB-A6 inhibits AR activity through binding to AF2 site. (A) The Octet binding assay using biotinylated AR-LBD on superstreptavidin sensors and IMB-A6 indicated IMB-A6 bound directly to AR. (B) Competitive binding of IMB-A6 to the AR-LBD evaluated by AR fluorescence polarization (FP) assay showed that IMB-A6 did not bind to the HBP site. (C) Western blot analysis indicated that IMB-A6 did not suppress AR protein expression in LNCaP cells at 1 and 10 μM. (D) IMB-A6 significantly disrupted the interaction between AR and PELP-1 at 10 μM as a result of Co-immunoprecipitation assay.
    Figure Legend Snippet: IMB-A6 inhibits AR activity through binding to AF2 site. (A) The Octet binding assay using biotinylated AR-LBD on superstreptavidin sensors and IMB-A6 indicated IMB-A6 bound directly to AR. (B) Competitive binding of IMB-A6 to the AR-LBD evaluated by AR fluorescence polarization (FP) assay showed that IMB-A6 did not bind to the HBP site. (C) Western blot analysis indicated that IMB-A6 did not suppress AR protein expression in LNCaP cells at 1 and 10 μM. (D) IMB-A6 significantly disrupted the interaction between AR and PELP-1 at 10 μM as a result of Co-immunoprecipitation assay.

    Techniques Used: Activity Assay, Binding Assay, Fluorescence, FP Assay, Western Blot, Expressing, Co-Immunoprecipitation Assay

    11) Product Images from "Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin"

    Article Title: Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0103094

    Analysis of affinity matured ERBB2-binders. ( A ). ERBB2-binding at high concentrations of HSA. Four variants of ADAPT ERBB2-FACS-12 (A8) were displayed at the bacterial surface and incubated with labeled ERBB2 (10 nM) in the presence of varying concentrations of HSA. All mutated variants show a retained ability to bind ERBB2 at very high concentrations of albumin, which is in contrast to the parental ADAPT ERBB2-FACS-12 (A8) . Increasing streptavidin-R-phycoerythrin signals were observed at high concentrations of HSA. This effect was only seen for molecules with retained ERBB2-binding and was also observed for an ERBB2-binding Affibody molecule used as control ( Figure S5 ). Hence, this experimental artifact was not ADAPT-specific. All measurements were done in duplicate on different days and the bars indicate the standard deviation. The albumin concentration is shown on a logarithmic scale, the two highest concentrations were 300 and 600 µM, respectively. ( B ). Competition experiment using ERBB2-binding proteins expressed on staphylococcal cells and flow cytometry. ERBB2-binding (5 nM) to cell-displayed binders was always blocked by a 10-fold excess of the same protein in a soluble form. Only ADAPTs and trastuzumab competed with each other for ERBB2-binding, no competition with the ERBB2-binding Affibody molecule Z ERBB2 was observed. All measurements were done in duplicate on different days and the bars indicate the standard deviation. ( C ). Binding to native ERBB2 on SKOV-3 cells. Cells were incubated with 100 nM biotinylated ADAPT ERBB2-1 or ADAPT ERBB2-FACS-12 (A8) and detected with streptavidin-R-phycoerythrin conjugate. Pre-incubating the cells with an excess of either non-labeled ADAPT ERBB2-FACS-12 (A8) or trastuzumab scFv blocked binding of ADAPT ERBB2-FACS-12 (A8) . A two-sample two-tailed t-test demonstrated that the binding for ADAPT ERBB2-1 was significantly stronger than the signal from the scaffold control (p = 0.0067). All measurements were done in duplicate on different days and the bars indicate the standard deviation. Analyses of non-treated cells were included in all experiments (background).
    Figure Legend Snippet: Analysis of affinity matured ERBB2-binders. ( A ). ERBB2-binding at high concentrations of HSA. Four variants of ADAPT ERBB2-FACS-12 (A8) were displayed at the bacterial surface and incubated with labeled ERBB2 (10 nM) in the presence of varying concentrations of HSA. All mutated variants show a retained ability to bind ERBB2 at very high concentrations of albumin, which is in contrast to the parental ADAPT ERBB2-FACS-12 (A8) . Increasing streptavidin-R-phycoerythrin signals were observed at high concentrations of HSA. This effect was only seen for molecules with retained ERBB2-binding and was also observed for an ERBB2-binding Affibody molecule used as control ( Figure S5 ). Hence, this experimental artifact was not ADAPT-specific. All measurements were done in duplicate on different days and the bars indicate the standard deviation. The albumin concentration is shown on a logarithmic scale, the two highest concentrations were 300 and 600 µM, respectively. ( B ). Competition experiment using ERBB2-binding proteins expressed on staphylococcal cells and flow cytometry. ERBB2-binding (5 nM) to cell-displayed binders was always blocked by a 10-fold excess of the same protein in a soluble form. Only ADAPTs and trastuzumab competed with each other for ERBB2-binding, no competition with the ERBB2-binding Affibody molecule Z ERBB2 was observed. All measurements were done in duplicate on different days and the bars indicate the standard deviation. ( C ). Binding to native ERBB2 on SKOV-3 cells. Cells were incubated with 100 nM biotinylated ADAPT ERBB2-1 or ADAPT ERBB2-FACS-12 (A8) and detected with streptavidin-R-phycoerythrin conjugate. Pre-incubating the cells with an excess of either non-labeled ADAPT ERBB2-FACS-12 (A8) or trastuzumab scFv blocked binding of ADAPT ERBB2-FACS-12 (A8) . A two-sample two-tailed t-test demonstrated that the binding for ADAPT ERBB2-1 was significantly stronger than the signal from the scaffold control (p = 0.0067). All measurements were done in duplicate on different days and the bars indicate the standard deviation. Analyses of non-treated cells were included in all experiments (background).

    Techniques Used: Binding Assay, FACS, Incubation, Labeling, Standard Deviation, Concentration Assay, Flow Cytometry, Cytometry, Two Tailed Test

    12) Product Images from "Regulation of mitochondrion-associated cytosolic ribosomes by mammalian mitochondrial ribonuclease T2 (RNASET2)"

    Article Title: Regulation of mitochondrion-associated cytosolic ribosomes by mammalian mitochondrial ribonuclease T2 (RNASET2)

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.005433

    Mitochondrial RNASET2 regulates cytosolic rRNA levels and nuclear transcription in vivo . A , in vivo RNA synthesis in HEK and RNASET2-overexpressing cells ( T2 ). The lower panel on the left is an immunoblot of tubulin. The right panel shows the quantification of biotinylated RNAs. nt , nucleotides. B , qRT-PCR results of mitochondrial and cytosolic 28S rRNA in HEK, and RNASET2-overexpressing ( T2 ) cells. C , in vivo RNA synthesis in HEK and RNASET2 knockdown cells ( KD ). D , qRT-PCR results of mitochondrial and cytosolic 28S rRNA in HEK, and RNASET2 knockdown ( KD ) cells. E , in vivo RNA synthesis in HEK cells and cells expressing C1-ΔN-RNASET2 ( C1 Δ NT2 ). F , qRT-PCR results of mitochondrial and cytosolic 28S rRNA in HEK and C1-ΔN-RNASET2 expressing ( C1 Δ NT2 ) cells. Statistical comparisons are performed using unpaired t tests ( n = 3 if not specified). *, p
    Figure Legend Snippet: Mitochondrial RNASET2 regulates cytosolic rRNA levels and nuclear transcription in vivo . A , in vivo RNA synthesis in HEK and RNASET2-overexpressing cells ( T2 ). The lower panel on the left is an immunoblot of tubulin. The right panel shows the quantification of biotinylated RNAs. nt , nucleotides. B , qRT-PCR results of mitochondrial and cytosolic 28S rRNA in HEK, and RNASET2-overexpressing ( T2 ) cells. C , in vivo RNA synthesis in HEK and RNASET2 knockdown cells ( KD ). D , qRT-PCR results of mitochondrial and cytosolic 28S rRNA in HEK, and RNASET2 knockdown ( KD ) cells. E , in vivo RNA synthesis in HEK cells and cells expressing C1-ΔN-RNASET2 ( C1 Δ NT2 ). F , qRT-PCR results of mitochondrial and cytosolic 28S rRNA in HEK and C1-ΔN-RNASET2 expressing ( C1 Δ NT2 ) cells. Statistical comparisons are performed using unpaired t tests ( n = 3 if not specified). *, p

    Techniques Used: In Vivo, Quantitative RT-PCR, Expressing

    Mitochondrion-associated cytosolic rRNAs are not degraded by a cytosolic nuclease. A , no RNase activity resides at the outer surface of mitochondrial outer membrane. Isolated mitochondria were resuspended in MitoPrep buffer ( Isotonic ) or hypotonic buffer that ruptures the mitochondrial outer membrane. Biotinylated 28S r RNA was added to the mixture and incubated at 37 °C for 1 or 30 min before the reaction was terminated. The bottom panel shows the mortalin immunoblot of the samples. nt , nucleotides. B , immunoblotting of a mitochondrial IMS protein DDP2 and matrix protein mortalin in the in organello degradation mitochondrial samples at 0, 30, and 60 min and the 0-min sample in the hypotonic buffer ( Hypo ). C , purified mitochondrial IMS fraction was tested for nuclease activity with purified cytosolic RNAs as substrates. The bottom panel shows the immunoblot of mitochondrial IMS protein DDP2. D , in organello decay of mitochondrion-associated cytosolic rRNAs with or without the addition of purified mitochondrial IMS fraction. Statistical comparisons are performed using unpaired t tests ( n = 3 if not specified). *, p
    Figure Legend Snippet: Mitochondrion-associated cytosolic rRNAs are not degraded by a cytosolic nuclease. A , no RNase activity resides at the outer surface of mitochondrial outer membrane. Isolated mitochondria were resuspended in MitoPrep buffer ( Isotonic ) or hypotonic buffer that ruptures the mitochondrial outer membrane. Biotinylated 28S r RNA was added to the mixture and incubated at 37 °C for 1 or 30 min before the reaction was terminated. The bottom panel shows the mortalin immunoblot of the samples. nt , nucleotides. B , immunoblotting of a mitochondrial IMS protein DDP2 and matrix protein mortalin in the in organello degradation mitochondrial samples at 0, 30, and 60 min and the 0-min sample in the hypotonic buffer ( Hypo ). C , purified mitochondrial IMS fraction was tested for nuclease activity with purified cytosolic RNAs as substrates. The bottom panel shows the immunoblot of mitochondrial IMS protein DDP2. D , in organello decay of mitochondrion-associated cytosolic rRNAs with or without the addition of purified mitochondrial IMS fraction. Statistical comparisons are performed using unpaired t tests ( n = 3 if not specified). *, p

    Techniques Used: Activity Assay, Isolation, Incubation, Purification

    13) Product Images from "Dual Resistance to Bacillus thuringiensis Cry1Ac and Cry2Aa Toxins in Heliothis virescens Suggests Multiple Mechanisms of Resistance"

    Article Title: Dual Resistance to Bacillus thuringiensis Cry1Ac and Cry2Aa Toxins in Heliothis virescens Suggests Multiple Mechanisms of Resistance

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.69.10.5898-5906.2003

    Binding of biotinylated Cry1Ac (A) and Cry1Fa (B) toxins to BBMV from YDK, YHD2, and KCBhyb larvae. Toxins (12 nM) were incubated with BBMV proteins (20 μg) for 1 h. Binding reactions were stopped by centrifugation, and washed pellets were separated by SDS-10% PAGE and transferred to polyvinylidene difluoride filters. Biotinylated toxins were detected with streptavidin-peroxidase conjugate and enhanced chemiluminescence.
    Figure Legend Snippet: Binding of biotinylated Cry1Ac (A) and Cry1Fa (B) toxins to BBMV from YDK, YHD2, and KCBhyb larvae. Toxins (12 nM) were incubated with BBMV proteins (20 μg) for 1 h. Binding reactions were stopped by centrifugation, and washed pellets were separated by SDS-10% PAGE and transferred to polyvinylidene difluoride filters. Biotinylated toxins were detected with streptavidin-peroxidase conjugate and enhanced chemiluminescence.

    Techniques Used: Binding Assay, Incubation, Centrifugation, Polyacrylamide Gel Electrophoresis

    14) Product Images from "Antigenicity and Immunogenicity of a Trimeric Envelope Protein from an Indian Clade C HIV-1 Isolate *"

    Article Title: Antigenicity and Immunogenicity of a Trimeric Envelope Protein from an Indian Clade C HIV-1 Isolate *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.621185

    Receptor and co-receptor binding site analysis using Octet RED biolayer interferometry. A , Octet sensorgrams generated by binding of serial dilutions of purified envelope gp120 and gp145 in solution to biotinylated sCD4 immobilized on streptavidin sensors.
    Figure Legend Snippet: Receptor and co-receptor binding site analysis using Octet RED biolayer interferometry. A , Octet sensorgrams generated by binding of serial dilutions of purified envelope gp120 and gp145 in solution to biotinylated sCD4 immobilized on streptavidin sensors.

    Techniques Used: Binding Assay, Generated, Purification

    15) Product Images from "The preclinical pharmacology of the high affinity anti-IL-6R Nanobody® ALX-0061 supports its clinical development in rheumatoid arthritis"

    Article Title: The preclinical pharmacology of the high affinity anti-IL-6R Nanobody® ALX-0061 supports its clinical development in rheumatoid arthritis

    Journal: Arthritis Research & Therapy

    doi: 10.1186/s13075-015-0651-0

    Inhibition profiles of ALX-0061 in an sIL-6R-based ( a ) and an mIL-6R-based ( b ) assay. ALX-0061 was pre-incubated with recombinant hIL-6 and recombinant hIL-6R, in the presence or absence of HSA, followed by the capture of hIL-6R on ELISA plates coated with a non-neutralizing anti-IL-6R mAb. IL-6 that remained in complex with IL-6R was detected using a biotinylated anti-IL-6 tool, and subsequent visualized with streptavidin-HRP. An example experiment for a neutralization experiment is presented in ( a ). Human TF-1 cells were pre-incubated with a dilution series of ALX-0061 in the presence of HSA, after which proliferation was induced with 2 ng/mL of IL-6. After 72 hours of incubation, cell proliferation was assessed by incorporation of 3 H-thymidine. ALX-0061 did not induce proliferation in the absence of IL-6 ( b ). Symbols depict mean responses; error bars represent ± SD of triplicate samples within the experiment. ELISA: enzyme-linked immunesorbent assay; h: human; HRP: horseradish peroxidase; HSA: human serum albumin; IL-6: interleukin-6; IL-6R: IL-6 receptor; mAb: monoclonal antibody; mIL-6R: membrane IL-6R; OD: optical density; SD: standard deviation; sIL-6R: soluble IL-6R
    Figure Legend Snippet: Inhibition profiles of ALX-0061 in an sIL-6R-based ( a ) and an mIL-6R-based ( b ) assay. ALX-0061 was pre-incubated with recombinant hIL-6 and recombinant hIL-6R, in the presence or absence of HSA, followed by the capture of hIL-6R on ELISA plates coated with a non-neutralizing anti-IL-6R mAb. IL-6 that remained in complex with IL-6R was detected using a biotinylated anti-IL-6 tool, and subsequent visualized with streptavidin-HRP. An example experiment for a neutralization experiment is presented in ( a ). Human TF-1 cells were pre-incubated with a dilution series of ALX-0061 in the presence of HSA, after which proliferation was induced with 2 ng/mL of IL-6. After 72 hours of incubation, cell proliferation was assessed by incorporation of 3 H-thymidine. ALX-0061 did not induce proliferation in the absence of IL-6 ( b ). Symbols depict mean responses; error bars represent ± SD of triplicate samples within the experiment. ELISA: enzyme-linked immunesorbent assay; h: human; HRP: horseradish peroxidase; HSA: human serum albumin; IL-6: interleukin-6; IL-6R: IL-6 receptor; mAb: monoclonal antibody; mIL-6R: membrane IL-6R; OD: optical density; SD: standard deviation; sIL-6R: soluble IL-6R

    Techniques Used: Inhibition, Incubation, Recombinant, Enzyme-linked Immunosorbent Assay, Neutralization, Standard Deviation

    16) Product Images from "Identification of HIV gp41-specific antibodies that mediate killing of infected cells"

    Article Title: Identification of HIV gp41-specific antibodies that mediate killing of infected cells

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007572

    Results of competition ELISAs with mAbs that target known epitopes in gp41. ( A) The mAbs used for competition experiments and their epitope targets are shown with a schematic of gp41 below. (B-E) Results of competition experiments reported as biotinylated mAb (B) QA255.006, (C) QA255.016, (D) QA255.067, (E) QA255.072 binding in the presence versus absence of the competitor mAbs. The tested Bt-Ab is indicated at the top of each panel, and the competitor mAb is defined by separate colors as specified in the legend to the right of the graphs. The results shown represent the mean relative binding (+/- SD), are from technical duplicates in the same experiment and are representative of at least two biological replicates.
    Figure Legend Snippet: Results of competition ELISAs with mAbs that target known epitopes in gp41. ( A) The mAbs used for competition experiments and their epitope targets are shown with a schematic of gp41 below. (B-E) Results of competition experiments reported as biotinylated mAb (B) QA255.006, (C) QA255.016, (D) QA255.067, (E) QA255.072 binding in the presence versus absence of the competitor mAbs. The tested Bt-Ab is indicated at the top of each panel, and the competitor mAb is defined by separate colors as specified in the legend to the right of the graphs. The results shown represent the mean relative binding (+/- SD), are from technical duplicates in the same experiment and are representative of at least two biological replicates.

    Techniques Used: Binding Assay

    17) Product Images from "Protein sorting by lipid phase-like domains supports emergent signaling function in B lymphocyte plasma membranes"

    Article Title: Protein sorting by lipid phase-like domains supports emergent signaling function in B lymphocyte plasma membranes

    Journal: eLife

    doi: 10.7554/eLife.19891

    CTxB clusters are not highly correlated with BCR. Average cross-correlation between clustered CTxB and BCR indicates that these two proteins are not colocalized. Biotinylated CTxB was clustered by streptavidin conjugated to Atto 655 and cells were fixed prior to labeling BCR with a f(Ab) 1 fragment conjugated to Alexa 532. The lack of pronounced cross-correlation between BCR and clustered CTxB indicates that CTxB is not forcing BCR to be clustered nor was it strongly recruiting BCR. Interestingly, the weak enrichment of BCR within CTxB clusters is similar to the enrichment of membrane anchored probes around clustered CTxB and BCR, suggesting that the enrichment stems from domain partitioning. The curve is an average of 6 cells with errorbars showing the standard error of the mean. DOI: http://dx.doi.org/10.7554/eLife.19891.027
    Figure Legend Snippet: CTxB clusters are not highly correlated with BCR. Average cross-correlation between clustered CTxB and BCR indicates that these two proteins are not colocalized. Biotinylated CTxB was clustered by streptavidin conjugated to Atto 655 and cells were fixed prior to labeling BCR with a f(Ab) 1 fragment conjugated to Alexa 532. The lack of pronounced cross-correlation between BCR and clustered CTxB indicates that CTxB is not forcing BCR to be clustered nor was it strongly recruiting BCR. Interestingly, the weak enrichment of BCR within CTxB clusters is similar to the enrichment of membrane anchored probes around clustered CTxB and BCR, suggesting that the enrichment stems from domain partitioning. The curve is an average of 6 cells with errorbars showing the standard error of the mean. DOI: http://dx.doi.org/10.7554/eLife.19891.027

    Techniques Used: Labeling

    Representative images from Figure 1 . Representative images from conditions included in average curves but not shown in Figure 1 . Scale bars are 5 µm. (left) Cells expressing mEos3.2-TM were labeled with CTxB-biotin that was then clustered with streptavidin-Atto655. (right) Cells expressing both mEos3.2-GG and YFP-TM. TM was clustered using a biotinylated anti-GFP antibody followed by streptavidin-Atto655. DOI: http://dx.doi.org/10.7554/eLife.19891.010
    Figure Legend Snippet: Representative images from Figure 1 . Representative images from conditions included in average curves but not shown in Figure 1 . Scale bars are 5 µm. (left) Cells expressing mEos3.2-TM were labeled with CTxB-biotin that was then clustered with streptavidin-Atto655. (right) Cells expressing both mEos3.2-GG and YFP-TM. TM was clustered using a biotinylated anti-GFP antibody followed by streptavidin-Atto655. DOI: http://dx.doi.org/10.7554/eLife.19891.010

    Techniques Used: Expressing, Labeling

    Cell surface clustering of cholera toxin subunit B elicits calcium mobilization in B cells. Cytosolic calcium levels were monitored in CH27 B cells both before and after biotinylated CTxB was clustered with streptavidin using the calcium indicator Fluo-4 as described in Methods. Colored curves represent raw fluorescence intensity traces for single cells and the average response of 40 cells is shown in black. The blue shaded region denotes +/- one standard deviation between the averaged cells. One reason for the broad width of this distribution is that individual cells oscillate between high and low fluorescent states, as apparent in the single cell traces. DOI: http://dx.doi.org/10.7554/eLife.19891.026
    Figure Legend Snippet: Cell surface clustering of cholera toxin subunit B elicits calcium mobilization in B cells. Cytosolic calcium levels were monitored in CH27 B cells both before and after biotinylated CTxB was clustered with streptavidin using the calcium indicator Fluo-4 as described in Methods. Colored curves represent raw fluorescence intensity traces for single cells and the average response of 40 cells is shown in black. The blue shaded region denotes +/- one standard deviation between the averaged cells. One reason for the broad width of this distribution is that individual cells oscillate between high and low fluorescent states, as apparent in the single cell traces. DOI: http://dx.doi.org/10.7554/eLife.19891.026

    Techniques Used: Fluorescence, Standard Deviation

    18) Product Images from "Chemokines and galectins form heterodimers to modulate inflammation"

    Article Title: Chemokines and galectins form heterodimers to modulate inflammation

    Journal: EMBO Reports

    doi: 10.15252/embr.201947852

    Physical interaction of Gal‐3 and Gal‐1 with CC and CXC chemokines Chemokines were immobilized on a nitrocellulose membrane and incubated with (A) TBS or (B) TBS containing biotinylated Gal‐1. The membranes were then stepwise incubated in solutions with SA‐HRP and chemiluminescence reagents. (C, D) The blots were subjected to densitometric analysis. Binding of galectins to chemokines was further assessed by immobilizing (E) Gal‐3, (F) Gal‐3 CRD, and (G) Gal‐1 on sensor chips at a density of 700 RU and detecting signals of human chemokines under flow. Signals of the solid‐phase assays (Fig 1 E) are depicted in light blue ( n = 3). Data represent mean ± SD from three independent experiments.
    Figure Legend Snippet: Physical interaction of Gal‐3 and Gal‐1 with CC and CXC chemokines Chemokines were immobilized on a nitrocellulose membrane and incubated with (A) TBS or (B) TBS containing biotinylated Gal‐1. The membranes were then stepwise incubated in solutions with SA‐HRP and chemiluminescence reagents. (C, D) The blots were subjected to densitometric analysis. Binding of galectins to chemokines was further assessed by immobilizing (E) Gal‐3, (F) Gal‐3 CRD, and (G) Gal‐1 on sensor chips at a density of 700 RU and detecting signals of human chemokines under flow. Signals of the solid‐phase assays (Fig 1 E) are depicted in light blue ( n = 3). Data represent mean ± SD from three independent experiments.

    Techniques Used: Incubation, Binding Assay

    Physical interaction of Gal‐3 and Gal‐1 with CC and CXC chemokines Chemokine–galectin interactions were detected by using a solid‐phase immunoassay. For this, 46 human CC and CXC chemokines were adsorbed on nitrocellulose membranes and the stripes incubated in parallel with (A) TBS or (B) TBS containing biotinylated galectins (the representative image shows a processed membrane tested with labeled Gal‐3). Signals had been generated by using SA‐HRP and chemiluminescence reagents. (C, D) The blots were subjected to densitometric analysis, and (E) all independent experiments were combined (binding chemokines in light blue, Gal‐3: n = 5, Gal‐3 CRD: n = 5, Gal‐1: n = 4). For SPR‐based experiments, (F) Gal‐3 (density 650 RU), (G) Gal‐3 CRD (density 1180 RU), and (H) Gal‐1 (density 130 RU) were immobilized and increasing concentrations of CXCL12 were passed over the flow cells. The red curve represents a single‐site fit to the data. Insets are representative sensorgrams of CXCL12 testing on immobilized galectin. Data represent the mean ± SD from six (F) or three (G and H) independent experiments.
    Figure Legend Snippet: Physical interaction of Gal‐3 and Gal‐1 with CC and CXC chemokines Chemokine–galectin interactions were detected by using a solid‐phase immunoassay. For this, 46 human CC and CXC chemokines were adsorbed on nitrocellulose membranes and the stripes incubated in parallel with (A) TBS or (B) TBS containing biotinylated galectins (the representative image shows a processed membrane tested with labeled Gal‐3). Signals had been generated by using SA‐HRP and chemiluminescence reagents. (C, D) The blots were subjected to densitometric analysis, and (E) all independent experiments were combined (binding chemokines in light blue, Gal‐3: n = 5, Gal‐3 CRD: n = 5, Gal‐1: n = 4). For SPR‐based experiments, (F) Gal‐3 (density 650 RU), (G) Gal‐3 CRD (density 1180 RU), and (H) Gal‐1 (density 130 RU) were immobilized and increasing concentrations of CXCL12 were passed over the flow cells. The red curve represents a single‐site fit to the data. Insets are representative sensorgrams of CXCL12 testing on immobilized galectin. Data represent the mean ± SD from six (F) or three (G and H) independent experiments.

    Techniques Used: Incubation, Labeling, Generated, Binding Assay, SPR Assay

    19) Product Images from "Long intronic GAA repeats causing Friedreich ataxia impede transcription elongation"

    Article Title: Long intronic GAA repeats causing Friedreich ataxia impede transcription elongation

    Journal: EMBO Molecular Medicine

    doi: 10.1002/emmm.201000064

    Synthesis but not decay rate of FXN mRNA is different in wild type and FRDA cells Decay kinetics of c-myc mRNA. FXN mRNA decay rate is similar in WT (GM15851) and FRDA (GM15850) cells. (A, B) WT (GM15851) and FRDA (GM15850) cells were treated with Actinomycin D (10 µg/ml) and total RNA was isolated at the indicated time-points. Relative changes in c-myc or FXN mRNA levels were analysed by qRT-PCR and the data were normalized to 18S rRNA. 4-thiouridine (4sU) labelling scheme. Synthesis rates for FXN mRNA in 4sU labelled wild type and FRDA cells are different. The values for biotinylated RNAs were normalized to total RNA Similar decay rates for FXN mRNA in wild type and FRDA cells. The values for unlabelled RNAs were normalized to total RNA.
    Figure Legend Snippet: Synthesis but not decay rate of FXN mRNA is different in wild type and FRDA cells Decay kinetics of c-myc mRNA. FXN mRNA decay rate is similar in WT (GM15851) and FRDA (GM15850) cells. (A, B) WT (GM15851) and FRDA (GM15850) cells were treated with Actinomycin D (10 µg/ml) and total RNA was isolated at the indicated time-points. Relative changes in c-myc or FXN mRNA levels were analysed by qRT-PCR and the data were normalized to 18S rRNA. 4-thiouridine (4sU) labelling scheme. Synthesis rates for FXN mRNA in 4sU labelled wild type and FRDA cells are different. The values for biotinylated RNAs were normalized to total RNA Similar decay rates for FXN mRNA in wild type and FRDA cells. The values for unlabelled RNAs were normalized to total RNA.

    Techniques Used: Isolation, Quantitative RT-PCR

    20) Product Images from "Identification and assessment of markers of biotin status in healthy adults"

    Article Title: Identification and assessment of markers of biotin status in healthy adults

    Journal: The British journal of nutrition

    doi: 10.1017/S0007114512005065

    Abundance of biotinylated (holo-)pyruvate carboxylase (PC), 3-methylcrotonyl-CoA carboxylase (MCC) and propionyl-CoA carboxylase (PCC) in lymphocytes from biotin-deficient, biotin-sufficient and biotin-supplemented subjects at the end of adjustment and
    Figure Legend Snippet: Abundance of biotinylated (holo-)pyruvate carboxylase (PC), 3-methylcrotonyl-CoA carboxylase (MCC) and propionyl-CoA carboxylase (PCC) in lymphocytes from biotin-deficient, biotin-sufficient and biotin-supplemented subjects at the end of adjustment and

    Techniques Used: Periodic Counter-current Chromatography

    (A) Abundance of biotinylated (holo-)pyruvate carboxylase (PC), 3-methylcrotonyl-CoA carboxylase (MCC) and propionyl-CoA carboxylase (PCC) in lymphocytes from biotin-deficient (DEF), biotin-sufficient (SUF) and biotin-supplemented (SUP) healthy adults
    Figure Legend Snippet: (A) Abundance of biotinylated (holo-)pyruvate carboxylase (PC), 3-methylcrotonyl-CoA carboxylase (MCC) and propionyl-CoA carboxylase (PCC) in lymphocytes from biotin-deficient (DEF), biotin-sufficient (SUF) and biotin-supplemented (SUP) healthy adults

    Techniques Used: Periodic Counter-current Chromatography

    21) Product Images from "S-Nitrosylation of Cyclin-Dependent Kinase 5 (Cdk5) Regulates Its Kinase Activity and Dendrite Growth During Neuronal Development"

    Article Title: S-Nitrosylation of Cyclin-Dependent Kinase 5 (Cdk5) Regulates Its Kinase Activity and Dendrite Growth During Neuronal Development

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.3899-10.2010

    Cys83 residue of Cdk5 is the target site for S-nitrosylation. a , Amino acid sequence of mouse Cdk5 protein. The eight cysteine residues, which are the potential S-nitrosylation sites, were denoted in red color. b , HEK 293T cells were transfected with expression constructs encoding the WT or specific cysteine mutants of Cdk5 as indicated. The cell lysate was then incubated with GSH or GSNO for 30 min and then subjected to biotin switch assay. The biotinylated proteins immunoprecipitated (IP) with Neutravidin-agarose were subjected to Western blot analysis for Cdk5.
    Figure Legend Snippet: Cys83 residue of Cdk5 is the target site for S-nitrosylation. a , Amino acid sequence of mouse Cdk5 protein. The eight cysteine residues, which are the potential S-nitrosylation sites, were denoted in red color. b , HEK 293T cells were transfected with expression constructs encoding the WT or specific cysteine mutants of Cdk5 as indicated. The cell lysate was then incubated with GSH or GSNO for 30 min and then subjected to biotin switch assay. The biotinylated proteins immunoprecipitated (IP) with Neutravidin-agarose were subjected to Western blot analysis for Cdk5.

    Techniques Used: Sequencing, Transfection, Expressing, Construct, Incubation, Biotin Switch Assay, Immunoprecipitation, Western Blot

    Cdk5 protein is S-nitrosylated in vitro and in vivo . a , NO S-nitrosylates Cdk5 in vitro . Lysate of HEK 293T cells overexpressing Cdk5 was incubated with NO donor GSNO or NOC12 (GSH or depleted NOC12 as control) for 30 min, and then subjected to biotin switch assay. b , Cdk5 was S-nitrosylated in adult rat brain. Adult mouse brain lysate was subjected to the biotin switch assay in the presence of biotin-HPDP or ascorbate as indicated. The biotinylated proteins were immunoprecipitated (IP) with Neutravidin-agarose, followed by Western blot analysis for Cdk5.
    Figure Legend Snippet: Cdk5 protein is S-nitrosylated in vitro and in vivo . a , NO S-nitrosylates Cdk5 in vitro . Lysate of HEK 293T cells overexpressing Cdk5 was incubated with NO donor GSNO or NOC12 (GSH or depleted NOC12 as control) for 30 min, and then subjected to biotin switch assay. b , Cdk5 was S-nitrosylated in adult rat brain. Adult mouse brain lysate was subjected to the biotin switch assay in the presence of biotin-HPDP or ascorbate as indicated. The biotinylated proteins were immunoprecipitated (IP) with Neutravidin-agarose, followed by Western blot analysis for Cdk5.

    Techniques Used: In Vitro, In Vivo, Incubation, Biotin Switch Assay, Immunoprecipitation, Western Blot

    22) Product Images from "The human 2B4 and NTB-A receptors bind the influenza viral hemagglutinin and co-stimulate NK cell cytotoxicity"

    Article Title: The human 2B4 and NTB-A receptors bind the influenza viral hemagglutinin and co-stimulate NK cell cytotoxicity

    Journal: Oncotarget

    doi: 10.18632/oncotarget.7597

    2B4 and NTB-A directly interacts with viral HA A. JEG-3 cells were incubated or not with PR8 influenza virus and stained with anti-HA mAb. The black empty histogram represents the staining of JEG-3 cells. The gray empty histograms represent the staining of JEG-3 cells in the presence of influenza. The filled histograms represent the staining of JEG-3 cells with secondary mAb only. The background staining of the JEG-3 cells in the presence of influenza with secondary antibodies was similar to that of the JEG-3 cells and is therefore not shown in the figure. B. JEG-3 cells were incubated with influenza A/Puerto Rico/8/34 H1N1 and then incubated with or without anti-HA antibody, followed by staining with 2B4-Ig, NTB-A-Ig and NKp46-Ig (indicated on the x axis of the histograms). The filled histograms represent the staining of JEG-3 cells with the various fusion proteins, the black empty histograms represent the staining of the influenza-JEG-3 cells and the gray empty histograms represent the staining of the influenza-JEG-3 cells with the indicated fusion proteins, following blocking of the HA. Figure shows one representative experiment out of 3 performed. C. Various fusion proteins (indicated on the X axis) were coated on an ELISA plate and then biotinylated HA-Ig was added. Shown are mean values and SD derived from triplicates, ** p
    Figure Legend Snippet: 2B4 and NTB-A directly interacts with viral HA A. JEG-3 cells were incubated or not with PR8 influenza virus and stained with anti-HA mAb. The black empty histogram represents the staining of JEG-3 cells. The gray empty histograms represent the staining of JEG-3 cells in the presence of influenza. The filled histograms represent the staining of JEG-3 cells with secondary mAb only. The background staining of the JEG-3 cells in the presence of influenza with secondary antibodies was similar to that of the JEG-3 cells and is therefore not shown in the figure. B. JEG-3 cells were incubated with influenza A/Puerto Rico/8/34 H1N1 and then incubated with or without anti-HA antibody, followed by staining with 2B4-Ig, NTB-A-Ig and NKp46-Ig (indicated on the x axis of the histograms). The filled histograms represent the staining of JEG-3 cells with the various fusion proteins, the black empty histograms represent the staining of the influenza-JEG-3 cells and the gray empty histograms represent the staining of the influenza-JEG-3 cells with the indicated fusion proteins, following blocking of the HA. Figure shows one representative experiment out of 3 performed. C. Various fusion proteins (indicated on the X axis) were coated on an ELISA plate and then biotinylated HA-Ig was added. Shown are mean values and SD derived from triplicates, ** p

    Techniques Used: Incubation, Staining, Blocking Assay, Enzyme-linked Immunosorbent Assay, Derivative Assay

    23) Product Images from "Neutral sphingomyelinases control extracellular vesicles budding from the plasma membrane"

    Article Title: Neutral sphingomyelinases control extracellular vesicles budding from the plasma membrane

    Journal: Journal of Extracellular Vesicles

    doi: 10.1080/20013078.2017.1378056

    Increase of EV originates from the plasma membrane. (a) Scheme of biotinylation flow cytometry assay. After biotinylation, cells were treated with DMSO, 5 µM GW4869, 80 µM Dynasore for 16 hours before FACS measurement of stained P14-EVs or cells. (b) Representative fluorescence blot of Streptavidin-FITC labeled P14 measured for 30 seconds. (c) Quantification of biotinylated P14-EVs from DMSO, 5 µM GW4869, 80 µM Dynasore treated cells. Unbiotinylated EVs were used as negative controls, as well as Ig-FITC labeled antibodies. n = 4 (d) Flow cytometry: Cell surface staining of CD147-APC, IgG-APC was used as control staining. (e) Quantification of cell surface staining of CD147-APC n = 3 (f) Quantification of P14 surface staining with CD147-APC n = 3 (g) CD81 and Tsg101 immunofluorescence labeling of SKBR3 cells treated with DMSO or GW4869 for 2 hours. (h) Quantification of cell surface staining and (i) P14-EV surface staining with CD81-FITC from DMSO and 5 µM GW4869 treated cells (16h) n = 3. Significance level: ***
    Figure Legend Snippet: Increase of EV originates from the plasma membrane. (a) Scheme of biotinylation flow cytometry assay. After biotinylation, cells were treated with DMSO, 5 µM GW4869, 80 µM Dynasore for 16 hours before FACS measurement of stained P14-EVs or cells. (b) Representative fluorescence blot of Streptavidin-FITC labeled P14 measured for 30 seconds. (c) Quantification of biotinylated P14-EVs from DMSO, 5 µM GW4869, 80 µM Dynasore treated cells. Unbiotinylated EVs were used as negative controls, as well as Ig-FITC labeled antibodies. n = 4 (d) Flow cytometry: Cell surface staining of CD147-APC, IgG-APC was used as control staining. (e) Quantification of cell surface staining of CD147-APC n = 3 (f) Quantification of P14 surface staining with CD147-APC n = 3 (g) CD81 and Tsg101 immunofluorescence labeling of SKBR3 cells treated with DMSO or GW4869 for 2 hours. (h) Quantification of cell surface staining and (i) P14-EV surface staining with CD81-FITC from DMSO and 5 µM GW4869 treated cells (16h) n = 3. Significance level: ***

    Techniques Used: Flow Cytometry, Cytometry, FACS, Staining, Fluorescence, Labeling, Immunofluorescence

    24) Product Images from "ILDR2 Is a Novel B7-like Protein That Negatively Regulates T Cell Responses"

    Article Title: ILDR2 Is a Novel B7-like Protein That Negatively Regulates T Cell Responses

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

    doi: 10.4049/jimmunol.1700325

    ILDR2-Fc binds to activated T cells. Binding of ILDR2-hFc was analyzed on resting and activated human CD4 + ( A ) and CD8 + ( B ) T cells. Isolated human CD4 + or CD8 + . Cells were incubated with ILDR2-hFc or isotype control (human IgG1; Synagis) at 100 μg/ml, followed by staining with PE-conjugated anti–human IgG Fc, and evaluated by flow cytometry. The results, shown as line graphs of ILDR2-hFc and isotype control (human IgG1) binding, are representative of two independent experiments. ( C ) Binding kinetics of ILDR2-mFc to resting and activated mouse CD4 + T cells. CD4 + CD25 − . Binding of biotinylated ILDR2-mFc and isotype control (mIgG2a) was tested at different time points of T cell activation and evaluated by flow cytometry. Results are shown as line graphs of ILDR2-mFc and isotype control (mIgG2a) binding.
    Figure Legend Snippet: ILDR2-Fc binds to activated T cells. Binding of ILDR2-hFc was analyzed on resting and activated human CD4 + ( A ) and CD8 + ( B ) T cells. Isolated human CD4 + or CD8 + . Cells were incubated with ILDR2-hFc or isotype control (human IgG1; Synagis) at 100 μg/ml, followed by staining with PE-conjugated anti–human IgG Fc, and evaluated by flow cytometry. The results, shown as line graphs of ILDR2-hFc and isotype control (human IgG1) binding, are representative of two independent experiments. ( C ) Binding kinetics of ILDR2-mFc to resting and activated mouse CD4 + T cells. CD4 + CD25 − . Binding of biotinylated ILDR2-mFc and isotype control (mIgG2a) was tested at different time points of T cell activation and evaluated by flow cytometry. Results are shown as line graphs of ILDR2-mFc and isotype control (mIgG2a) binding.

    Techniques Used: Binding Assay, Isolation, Incubation, Staining, Flow Cytometry, Cytometry, Activation Assay

    25) Product Images from "FUBP3 interacts with FGF9 3? microsatellite and positively regulates FGF9 translation"

    Article Title: FUBP3 interacts with FGF9 3? microsatellite and positively regulates FGF9 translation

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq1295

    Riboprotein complex formation by UG repeats of FGF9 3′-UTR. ( A ) UV-cross-linking was conducted using biotinylated (UG) 15 oligoribonucleotide and protein extracts (cytoplasmic extract, CE; nuclear extract, NE) prepared from HEK293 (left) and NT2D1 (right) cells. Excess molar ratio of unlabeled cold probe was added as indicated in the competition assay. ( B ) The same labeled probe, cold probe and protein extracts as in (A), were used in RNA-EMSA and showed similar results in HEK293 (left) and NT2D1 (right) cells. C1–C6: complexes 1–6. ( C ) (UG) 15 binding proteins identified by LC–MS. Proteins pulled down in the absence (left lane) or the presence of riboprobe (UG) 15 (right lane) were assayed by silver stain. Differentially expressed bands (marked by 1–8) were excised for protein identification by LC–MS.
    Figure Legend Snippet: Riboprotein complex formation by UG repeats of FGF9 3′-UTR. ( A ) UV-cross-linking was conducted using biotinylated (UG) 15 oligoribonucleotide and protein extracts (cytoplasmic extract, CE; nuclear extract, NE) prepared from HEK293 (left) and NT2D1 (right) cells. Excess molar ratio of unlabeled cold probe was added as indicated in the competition assay. ( B ) The same labeled probe, cold probe and protein extracts as in (A), were used in RNA-EMSA and showed similar results in HEK293 (left) and NT2D1 (right) cells. C1–C6: complexes 1–6. ( C ) (UG) 15 binding proteins identified by LC–MS. Proteins pulled down in the absence (left lane) or the presence of riboprobe (UG) 15 (right lane) were assayed by silver stain. Differentially expressed bands (marked by 1–8) were excised for protein identification by LC–MS.

    Techniques Used: Competitive Binding Assay, Labeling, Binding Assay, Liquid Chromatography with Mass Spectroscopy, Silver Staining

    Identification of FUBP3 as UG-repeat binding protein. ( A ) The RNA–protein complex was detected using specific antibodies as indicated in RNA-EMSA in the protein extracts from NT2D1 (left) and HEK293 (right) cells. CE, cytoplasmic extract; NE, nuclear extract. IgG was used as a control. ( B ) Pull-down assays were performed using biotinylated (UG) 15 riboprobe and in vitro -transcribed FGF9 3′ - UTR containing (UG) 15 repeats in the cytoplasmic extract from HEK293 cells. Specific antibodies to FUBP3, GAPDH and AUF1 were used to detect the pulled-down proteins. H 2 O and in vitro -transcribed GAPDH probe were used as negative controls for pull-down assays. ( C ) Immunoprecipitated RNA–protein complexes using anti-FUBP3 antibody showed the presence of endogenous FGF9 , but not GAPDH mRNA in HEK293 cells (lane 2). Goat normal IgG was used as a control for the immunoprecipitation reaction. The enrichment of FGF9 mRNA precipitated by anti-FUBP3 antibody is shown relative to goat normal IgG. Data were normalized to GAPDH .
    Figure Legend Snippet: Identification of FUBP3 as UG-repeat binding protein. ( A ) The RNA–protein complex was detected using specific antibodies as indicated in RNA-EMSA in the protein extracts from NT2D1 (left) and HEK293 (right) cells. CE, cytoplasmic extract; NE, nuclear extract. IgG was used as a control. ( B ) Pull-down assays were performed using biotinylated (UG) 15 riboprobe and in vitro -transcribed FGF9 3′ - UTR containing (UG) 15 repeats in the cytoplasmic extract from HEK293 cells. Specific antibodies to FUBP3, GAPDH and AUF1 were used to detect the pulled-down proteins. H 2 O and in vitro -transcribed GAPDH probe were used as negative controls for pull-down assays. ( C ) Immunoprecipitated RNA–protein complexes using anti-FUBP3 antibody showed the presence of endogenous FGF9 , but not GAPDH mRNA in HEK293 cells (lane 2). Goat normal IgG was used as a control for the immunoprecipitation reaction. The enrichment of FGF9 mRNA precipitated by anti-FUBP3 antibody is shown relative to goat normal IgG. Data were normalized to GAPDH .

    Techniques Used: Binding Assay, In Vitro, Immunoprecipitation

    26) Product Images from "Identification of prothymosin-?1, the necrosis-apoptosis switch molecule in cortical neuronal cultures"

    Article Title: Identification of prothymosin-?1, the necrosis-apoptosis switch molecule in cortical neuronal cultures

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200608022

    ProTα induced inhibition of necrosis through PKCβ II . (a and b) ProTα induced reversal of the decreases in the cellular ATP level (a) and [ 3 H]2-DG uptake (b) after serum-free stress. [ 3 H]2-DG uptake was performed for 2 h immediately after the start of the culture. (c) Time course of the ProTα-induced reversal of the decrease in the cellular ATP level after LOG stress. 80 nM ProTα was added to the culture from the time of LOG stress to the end of experiments. (d and e) Decreased translocation of GLUT1 and -4 to the plasma membrane at 2 h after LOG stress and its reversal by ProTα. All the proteins on the outer surface of cortical neurons (LD cultures) were biotinylated and subjected to immunoprecipitation by streptavidin (e). (f) ProTα induced PKC activation in terms of phosphorylation of PKCα (p-PKCα) and translocation of PKCβ I and -β II at 10 min after ProTα treatment in serum-free culture. No substantial ProTα-induced activation of other PKC isoforms was observed in experiments using a rabbit anti-PKCγ antibody (1:100), goat anti-PKCɛ antibody (1:100), rabbit anti–phosphorylated PKCδ antibody (1:100), or rabbit anti–phosphorylated PKCζ antibody (1:100; not depicted). (g) Immunoblot analysis of the protein expression of phosphorylated or total PKCα at 2 h after LOG stress. (h) Signal transduction for ProTα-induced reversal of the LOG stress–induced decrease in GLUT1/4 membrane translocation ( n = 3). The method for quantifying the GLUT1/4 membrane localization is described in Fig. S2 (available at http://www.jcb.org/cgi/content/full/jcb.200608022/DC1 ). Cells were treated with 1 μM U73122, 1 μM U73343, or 100 ng/ml pertussis toxin (PTX) from 30 min before the LOG treatment. Treatment of LD cultures with AS-ODNs for PKCα, PKCβ I , PKCβ II , or Gα q/11 was started 3 d before the LOG treatment. Selective down-regulation of Gα q/11 by its AS-ODN was confirmed by Western blot analysis (Fig. S3). (i) Selective down-regulation of PKCβ II by its AS-ODN. The specificities of the other AS-ODNs are shown in Fig. S3. (j) ProTα induced inhibition of necrotic cell death through PKCβ II activation. PI was added to the cells at 12 h after reperfusion and incubated for 30 min. 80 nM ProTα was added to the culture from the time of LOG stress. *, P
    Figure Legend Snippet: ProTα induced inhibition of necrosis through PKCβ II . (a and b) ProTα induced reversal of the decreases in the cellular ATP level (a) and [ 3 H]2-DG uptake (b) after serum-free stress. [ 3 H]2-DG uptake was performed for 2 h immediately after the start of the culture. (c) Time course of the ProTα-induced reversal of the decrease in the cellular ATP level after LOG stress. 80 nM ProTα was added to the culture from the time of LOG stress to the end of experiments. (d and e) Decreased translocation of GLUT1 and -4 to the plasma membrane at 2 h after LOG stress and its reversal by ProTα. All the proteins on the outer surface of cortical neurons (LD cultures) were biotinylated and subjected to immunoprecipitation by streptavidin (e). (f) ProTα induced PKC activation in terms of phosphorylation of PKCα (p-PKCα) and translocation of PKCβ I and -β II at 10 min after ProTα treatment in serum-free culture. No substantial ProTα-induced activation of other PKC isoforms was observed in experiments using a rabbit anti-PKCγ antibody (1:100), goat anti-PKCɛ antibody (1:100), rabbit anti–phosphorylated PKCδ antibody (1:100), or rabbit anti–phosphorylated PKCζ antibody (1:100; not depicted). (g) Immunoblot analysis of the protein expression of phosphorylated or total PKCα at 2 h after LOG stress. (h) Signal transduction for ProTα-induced reversal of the LOG stress–induced decrease in GLUT1/4 membrane translocation ( n = 3). The method for quantifying the GLUT1/4 membrane localization is described in Fig. S2 (available at http://www.jcb.org/cgi/content/full/jcb.200608022/DC1 ). Cells were treated with 1 μM U73122, 1 μM U73343, or 100 ng/ml pertussis toxin (PTX) from 30 min before the LOG treatment. Treatment of LD cultures with AS-ODNs for PKCα, PKCβ I , PKCβ II , or Gα q/11 was started 3 d before the LOG treatment. Selective down-regulation of Gα q/11 by its AS-ODN was confirmed by Western blot analysis (Fig. S3). (i) Selective down-regulation of PKCβ II by its AS-ODN. The specificities of the other AS-ODNs are shown in Fig. S3. (j) ProTα induced inhibition of necrotic cell death through PKCβ II activation. PI was added to the cells at 12 h after reperfusion and incubated for 30 min. 80 nM ProTα was added to the culture from the time of LOG stress. *, P

    Techniques Used: Inhibition, Translocation Assay, Immunoprecipitation, Activation Assay, Expressing, Transduction, Western Blot, Incubation

    27) Product Images from "Reprogramming the antigen specificity of B cells using genome-editing technologies"

    Article Title: Reprogramming the antigen specificity of B cells using genome-editing technologies

    Journal: eLife

    doi: 10.7554/eLife.42995

    Example of gating strategy for the selection of higher affinity B cell receptor variants in V781(PG9)-engineered and C108 SOSIP-enriched cells. After determining the EC10 concentrations in preliminary experiments, biotinylated MGRM8 or WITO SOSIP bound to APC labeled streptavidin tetramers were incubated with cells along with FITC labeled anti-lambda LC antibody to normalize for cell variants with higher levels of surface expressed BCR. 5–10% of the live single cell gate that showed the highest FITC normalized fluorescence was sorted for further culture and two subsequent rounds of enrichment. Top left is forward scatter (X-axis) side scatter (Y-axis) plot to gate on live lymphocytes. Top left plot is forwards scatter-H (X-axis) vs. forward scatter-W (Y-axis) to select single cells. Large plot is lambda chain FITC (X-axis) vs. MGRM8 SOSIP-streptavidin-APC.
    Figure Legend Snippet: Example of gating strategy for the selection of higher affinity B cell receptor variants in V781(PG9)-engineered and C108 SOSIP-enriched cells. After determining the EC10 concentrations in preliminary experiments, biotinylated MGRM8 or WITO SOSIP bound to APC labeled streptavidin tetramers were incubated with cells along with FITC labeled anti-lambda LC antibody to normalize for cell variants with higher levels of surface expressed BCR. 5–10% of the live single cell gate that showed the highest FITC normalized fluorescence was sorted for further culture and two subsequent rounds of enrichment. Top left is forward scatter (X-axis) side scatter (Y-axis) plot to gate on live lymphocytes. Top left plot is forwards scatter-H (X-axis) vs. forward scatter-W (Y-axis) to select single cells. Large plot is lambda chain FITC (X-axis) vs. MGRM8 SOSIP-streptavidin-APC.

    Techniques Used: Selection, Labeling, Incubation, Fluorescence

    28) Product Images from "Single-molecule FRET method to investigate the dynamics of transcription elongation through the nucleosome by RNA polymerase II"

    Article Title: Single-molecule FRET method to investigate the dynamics of transcription elongation through the nucleosome by RNA polymerase II

    Journal: Methods (San Diego, Calif.)

    doi: 10.1016/j.ymeth.2019.01.009

    The schematics of the single-molecule FRET system to investigate the dynamics of the nucleosome structure during transcription elongation. (A) Nucleosome assembled on the shown template is complexed with RNAPII (yeast Pol II). Rpb1 CTD antibody is immobilized on a microscope slide surface via biotinylated Protein A that is conjugated to streptavidin coated on the slide. A FRET pair (Cy3 and Atto647N) is labeled at the +34 th and +112 th nucleotide. This FRET pair location is sensitive to the nucleosomal dynamics at the proximal-dimer/DNA contact region. (B) The sequence of the upper and lower strands of the transcription template illustrated in A .
    Figure Legend Snippet: The schematics of the single-molecule FRET system to investigate the dynamics of the nucleosome structure during transcription elongation. (A) Nucleosome assembled on the shown template is complexed with RNAPII (yeast Pol II). Rpb1 CTD antibody is immobilized on a microscope slide surface via biotinylated Protein A that is conjugated to streptavidin coated on the slide. A FRET pair (Cy3 and Atto647N) is labeled at the +34 th and +112 th nucleotide. This FRET pair location is sensitive to the nucleosomal dynamics at the proximal-dimer/DNA contact region. (B) The sequence of the upper and lower strands of the transcription template illustrated in A .

    Techniques Used: Microscopy, Labeling, Sequencing

    29) Product Images from "Transcytotic Efflux from Early Endosomes Is Dependent on Cholesterol and Glycosphingolipids in Polarized Hepatic Cells"

    Article Title: Transcytotic Efflux from Early Endosomes Is Dependent on Cholesterol and Glycosphingolipids in Polarized Hepatic Cells

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E02-12-0816

    Apical residents are internalized in mβCD-treated cells and found in early endosomes. (A) To measure internalization, WIF-B cells were continuously labeled with biotinylated antibodies diluted in LPDM in the absence or presence of 5 mM mβCD for the indicated times at 37°C. The remaining PM-associated antibodies were eluted with isoglycine for 5 min at room temperature and the cells lysed. Aliquots of the eluate and lysate (the internalized population) were assayed for amounts of biotinylated antibodies using streptavidin-coated 96-well plates and colorimetric detection of HRP-conjugated secondary antibodies. The total amount (in nanograms) of antibody internalized is plotted relative to the maximum observed at 60 min in control cells, which was set to 100%. Values are expressed as the mean ± SD. Measurements were done on at least three experiments each performed in duplicate. (B) WIF-B cells were pretreated for 5 min in LPDM in the absence (a, b, e, f, i, j, m, and n) or presence of 5 mM mβCD (c, d, g, h, k, l, o, and p). The indicated apical residents or recycling receptors present at the basolateral PM were continuously labeled with specific antibodies for 60 min at 37°C. The cells were fixed, permeabilized, and the trafficked antibody–antigen complexes visualized with secondary antibodies. Arrows are pointing to intracellular clusters enlarged in the insets approximately twofold. In c, d, g, and o, images were intentionally overexposed to highlight the intracellular population of the transcytosing proteins. Thus, the apparent apical labeling is exaggerated. The insets in m and n are highlighting a region where Tf-R and ASGP-R do not significantly overlap. Arrowheads are pointing to colocalized structures. Bar, 10 μm.
    Figure Legend Snippet: Apical residents are internalized in mβCD-treated cells and found in early endosomes. (A) To measure internalization, WIF-B cells were continuously labeled with biotinylated antibodies diluted in LPDM in the absence or presence of 5 mM mβCD for the indicated times at 37°C. The remaining PM-associated antibodies were eluted with isoglycine for 5 min at room temperature and the cells lysed. Aliquots of the eluate and lysate (the internalized population) were assayed for amounts of biotinylated antibodies using streptavidin-coated 96-well plates and colorimetric detection of HRP-conjugated secondary antibodies. The total amount (in nanograms) of antibody internalized is plotted relative to the maximum observed at 60 min in control cells, which was set to 100%. Values are expressed as the mean ± SD. Measurements were done on at least three experiments each performed in duplicate. (B) WIF-B cells were pretreated for 5 min in LPDM in the absence (a, b, e, f, i, j, m, and n) or presence of 5 mM mβCD (c, d, g, h, k, l, o, and p). The indicated apical residents or recycling receptors present at the basolateral PM were continuously labeled with specific antibodies for 60 min at 37°C. The cells were fixed, permeabilized, and the trafficked antibody–antigen complexes visualized with secondary antibodies. Arrows are pointing to intracellular clusters enlarged in the insets approximately twofold. In c, d, g, and o, images were intentionally overexposed to highlight the intracellular population of the transcytosing proteins. Thus, the apparent apical labeling is exaggerated. The insets in m and n are highlighting a region where Tf-R and ASGP-R do not significantly overlap. Arrowheads are pointing to colocalized structures. Bar, 10 μm.

    Techniques Used: Labeling

    30) Product Images from "Lipid mediator-induced expression of bactericidal/ permeability-increasing protein (BPI) in human mucosal epithelia"

    Article Title: Lipid mediator-induced expression of bactericidal/ permeability-increasing protein (BPI) in human mucosal epithelia

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

    doi: 10.1073/pnas.052533799

    Localization of BPI to the cell surface. ( A ) BPI was localized by confocal microscopy in nonpermeabilized Caco2 cells exposed to vehicle ( Top ) or ATLa (1 μM, 24 h, Middle and Bottom ). BPI adsorbed antisera was used as a control ( Bottom ). Shown here are confocal sections through the mid-zone, subjunctional portion of epithelial monolayers. Representative experiment from n = 2. ( B) T84 cells were preexposed to ATLa (1 μM) for indicated periods of time. Cell surface proteins were nonspecifically labeled with biotin, BPI was immunoprecipitated from cell lysates, resolved by SDS/PAGE, and Western blots were probed with avidin peroxidase. Also shown is the immunoprecipitation control (omission of primary Ab) as well as a biotinylated BPI standard. Representative experiment from n = 3.
    Figure Legend Snippet: Localization of BPI to the cell surface. ( A ) BPI was localized by confocal microscopy in nonpermeabilized Caco2 cells exposed to vehicle ( Top ) or ATLa (1 μM, 24 h, Middle and Bottom ). BPI adsorbed antisera was used as a control ( Bottom ). Shown here are confocal sections through the mid-zone, subjunctional portion of epithelial monolayers. Representative experiment from n = 2. ( B) T84 cells were preexposed to ATLa (1 μM) for indicated periods of time. Cell surface proteins were nonspecifically labeled with biotin, BPI was immunoprecipitated from cell lysates, resolved by SDS/PAGE, and Western blots were probed with avidin peroxidase. Also shown is the immunoprecipitation control (omission of primary Ab) as well as a biotinylated BPI standard. Representative experiment from n = 3.

    Techniques Used: Confocal Microscopy, Labeling, Immunoprecipitation, SDS Page, Western Blot, Avidin-Biotin Assay

    31) Product Images from "Real-time PCR mapping of DNaseI-hypersensitive sites using a novel ligation-mediated amplification technique"

    Article Title: Real-time PCR mapping of DNaseI-hypersensitive sites using a novel ligation-mediated amplification technique

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm108

    ( A ) Nuclei are isolated from cells, and aliquots are digested with a range of DNaseI as detailed in ‘Materials and methods’ section. The DNA is extracted and run on 1% agarose gels using gel electrophoresis (right panel). With the example shown, the 40-units sample gave maximal enrichment at a housekeeping promoter using the complete protocol. ( B ) The digested DNA is blunt-ended with T4 polymerase and ligated to the LP21–25 linker as detailed in ‘Materials and methods’ section. Following amplification with the biotinylated LP25 primer, the extracted DNA template represents a library of whole-genome DHS. When samples of this library are visualised using gel electrophoresis (bottom panel), the majority of products are between 300 and 500 bp in size.
    Figure Legend Snippet: ( A ) Nuclei are isolated from cells, and aliquots are digested with a range of DNaseI as detailed in ‘Materials and methods’ section. The DNA is extracted and run on 1% agarose gels using gel electrophoresis (right panel). With the example shown, the 40-units sample gave maximal enrichment at a housekeeping promoter using the complete protocol. ( B ) The digested DNA is blunt-ended with T4 polymerase and ligated to the LP21–25 linker as detailed in ‘Materials and methods’ section. Following amplification with the biotinylated LP25 primer, the extracted DNA template represents a library of whole-genome DHS. When samples of this library are visualised using gel electrophoresis (bottom panel), the majority of products are between 300 and 500 bp in size.

    Techniques Used: Isolation, Nucleic Acid Electrophoresis, Amplification

    32) Product Images from "Directed Evolution of a Highly Specific FN3 Monobody to the SH3 Domain of Human Lyn Tyrosine Kinase"

    Article Title: Directed Evolution of a Highly Specific FN3 Monobody to the SH3 Domain of Human Lyn Tyrosine Kinase

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0145872

    Pull-down of endogenous Lyn from human cells. Ramos cells were cultured and the clarified lysate was incubated with biotinylated 2H7 or wild-type FN3 (WT-FN3) monobody. Protein complex was then captured with streptavidin-coated magnetic beads and analyzed by Western blot. The Western blot included a recombinant Lyn protein as a positive control, along with the input and output samples of the pull-down experiment. The Lyn proteins were detected on the blot with a commercial anti-Lyn antibody.
    Figure Legend Snippet: Pull-down of endogenous Lyn from human cells. Ramos cells were cultured and the clarified lysate was incubated with biotinylated 2H7 or wild-type FN3 (WT-FN3) monobody. Protein complex was then captured with streptavidin-coated magnetic beads and analyzed by Western blot. The Western blot included a recombinant Lyn protein as a positive control, along with the input and output samples of the pull-down experiment. The Lyn proteins were detected on the blot with a commercial anti-Lyn antibody.

    Techniques Used: Cell Culture, Incubation, Magnetic Beads, Western Blot, Recombinant, Positive Control

    33) Product Images from "Fungal Virulence and Development Is Regulated by Alternative Pre-mRNA 3?End Processing in Magnaporthe oryzae"

    Article Title: Fungal Virulence and Development Is Regulated by Alternative Pre-mRNA 3?End Processing in Magnaporthe oryzae

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1002441

    RBP35 isoforms bind poly(G) RNA homopolymers in vitro and show a steady-state nuclear localisation. ( A ) His-tagged RBP35 binds biotinylated poly(G) 30 RNA homopolymers. The single- (ssDNA) and double-stranded (dsDNA) DNA derived from calf thymus was attached to cellulose beads. wt, total protein extracts from wild type. ( B ) RBP35 recognises with low affinity biotinylated single-stranded sense and anti-sense telomeric DNA repeats and not poly(dG) DNA. ( C ) Steady-state nuclear localisation of RBP35. Δrbp35 was constructed with a cytoplasmic GFP (green fluorescent protein) to visualise its growth in planta . RBP35-mRFP variants colocalise in the nucleus with GFP-tagged histone H3 (H3-gfp). VE: vesicle; IH: invasive hyphae; AP: appressorium; CO: conidium; PS: polystyrene; PHIL: hydrophilic; PHOB: hydrophobic; HY: hyphopodia. Scale bar:15 µm. ( D ) RBP35-mRFP-N is excluded from the nucleolus. Scale bar: 5 µm.
    Figure Legend Snippet: RBP35 isoforms bind poly(G) RNA homopolymers in vitro and show a steady-state nuclear localisation. ( A ) His-tagged RBP35 binds biotinylated poly(G) 30 RNA homopolymers. The single- (ssDNA) and double-stranded (dsDNA) DNA derived from calf thymus was attached to cellulose beads. wt, total protein extracts from wild type. ( B ) RBP35 recognises with low affinity biotinylated single-stranded sense and anti-sense telomeric DNA repeats and not poly(dG) DNA. ( C ) Steady-state nuclear localisation of RBP35. Δrbp35 was constructed with a cytoplasmic GFP (green fluorescent protein) to visualise its growth in planta . RBP35-mRFP variants colocalise in the nucleus with GFP-tagged histone H3 (H3-gfp). VE: vesicle; IH: invasive hyphae; AP: appressorium; CO: conidium; PS: polystyrene; PHIL: hydrophilic; PHOB: hydrophobic; HY: hyphopodia. Scale bar:15 µm. ( D ) RBP35-mRFP-N is excluded from the nucleolus. Scale bar: 5 µm.

    Techniques Used: In Vitro, Derivative Assay, Construct

    34) Product Images from "ILDR2 Is a Novel B7-like Protein That Negatively Regulates T Cell Responses"

    Article Title: ILDR2 Is a Novel B7-like Protein That Negatively Regulates T Cell Responses

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

    doi: 10.4049/jimmunol.1700325

    ILDR2-Fc binds to activated T cells. Binding of ILDR2-hFc was analyzed on resting and activated human CD4 + ( A ) and CD8 + ( B ) T cells. Isolated human CD4 + or CD8 + . Cells were incubated with ILDR2-hFc or isotype control (human IgG1; Synagis) at 100 μg/ml, followed by staining with PE-conjugated anti–human IgG Fc, and evaluated by flow cytometry. The results, shown as line graphs of ILDR2-hFc and isotype control (human IgG1) binding, are representative of two independent experiments. ( C ) Binding kinetics of ILDR2-mFc to resting and activated mouse CD4 + T cells. CD4 + CD25 − . Binding of biotinylated ILDR2-mFc and isotype control (mIgG2a) was tested at different time points of T cell activation and evaluated by flow cytometry. Results are shown as line graphs of ILDR2-mFc and isotype control (mIgG2a) binding.
    Figure Legend Snippet: ILDR2-Fc binds to activated T cells. Binding of ILDR2-hFc was analyzed on resting and activated human CD4 + ( A ) and CD8 + ( B ) T cells. Isolated human CD4 + or CD8 + . Cells were incubated with ILDR2-hFc or isotype control (human IgG1; Synagis) at 100 μg/ml, followed by staining with PE-conjugated anti–human IgG Fc, and evaluated by flow cytometry. The results, shown as line graphs of ILDR2-hFc and isotype control (human IgG1) binding, are representative of two independent experiments. ( C ) Binding kinetics of ILDR2-mFc to resting and activated mouse CD4 + T cells. CD4 + CD25 − . Binding of biotinylated ILDR2-mFc and isotype control (mIgG2a) was tested at different time points of T cell activation and evaluated by flow cytometry. Results are shown as line graphs of ILDR2-mFc and isotype control (mIgG2a) binding.

    Techniques Used: Binding Assay, Isolation, Incubation, Staining, Flow Cytometry, Cytometry, Activation Assay

    35) Product Images from "Mammalian mitochondrial RNAs are degraded in the mitochondrial intermembrane space by RNASET2"

    Article Title: Mammalian mitochondrial RNAs are degraded in the mitochondrial intermembrane space by RNASET2

    Journal: Protein & Cell

    doi: 10.1007/s13238-017-0448-9

    Characterization of a ribonuclease activity in the mitochondrial IMS . (A) Immunoblots of total mitochondria and fractions. Mitochondria were separated into four fractions: total soluble (including IMS and Matrix), total membrane, IMS and matrix. Immunoblotting was performed using antibodies for Mortalin (Matrix), TIM23 (Inner membrane), DDP2 (IMS), and TOM40 (Outer membrane). (B) Four mitochondrial fractions were examined for ribonuclease activity using biotinylated UCP2 mRNA as a substrate. (C) No ribonuclease activity localizes at the outer surface of mitochondrial outer membrane. Isolated mitochondria were resuspended in mitoprep buffer (M buffer) or hypotonic buffer (H buffer) that ruptures the mitochondrial outer membrane. Biotinylated UCP2 RNA was added to the mixture and incubated at 37°C for 1 min or 5 min before the reaction was terminated. (D) IMS ribonuclease activity was tested for its sensitivity to EDTA (2 mmol/L) and Cu 2+ (0.5 mmol/L) using biotinylated UCP2 mRNA as a substrate. (E) IMS ribonuclease activity was tested for its sensitivity to different concentrations of Cu 2+ (0.5 mmol/L and 5 mmol/L), and Mg 2+ (0.5 mmol/L and 10 mmol/L) using RNAs purified from isolated mitochondria as substrates. (F) IMS ribonuclease activity and RNaseI were tested for sensitivity to different concentrations of ATP (0 mmol/L, 5 mmol/L, and 10 mmol/L). (G) IMS ribonuclease activity was tested for sensitivity to Proteinase K (ProK), EDTA (2 mmol/L), Cu 2+ (0.5 mmol/L), and heat (90°C 10 min)
    Figure Legend Snippet: Characterization of a ribonuclease activity in the mitochondrial IMS . (A) Immunoblots of total mitochondria and fractions. Mitochondria were separated into four fractions: total soluble (including IMS and Matrix), total membrane, IMS and matrix. Immunoblotting was performed using antibodies for Mortalin (Matrix), TIM23 (Inner membrane), DDP2 (IMS), and TOM40 (Outer membrane). (B) Four mitochondrial fractions were examined for ribonuclease activity using biotinylated UCP2 mRNA as a substrate. (C) No ribonuclease activity localizes at the outer surface of mitochondrial outer membrane. Isolated mitochondria were resuspended in mitoprep buffer (M buffer) or hypotonic buffer (H buffer) that ruptures the mitochondrial outer membrane. Biotinylated UCP2 RNA was added to the mixture and incubated at 37°C for 1 min or 5 min before the reaction was terminated. (D) IMS ribonuclease activity was tested for its sensitivity to EDTA (2 mmol/L) and Cu 2+ (0.5 mmol/L) using biotinylated UCP2 mRNA as a substrate. (E) IMS ribonuclease activity was tested for its sensitivity to different concentrations of Cu 2+ (0.5 mmol/L and 5 mmol/L), and Mg 2+ (0.5 mmol/L and 10 mmol/L) using RNAs purified from isolated mitochondria as substrates. (F) IMS ribonuclease activity and RNaseI were tested for sensitivity to different concentrations of ATP (0 mmol/L, 5 mmol/L, and 10 mmol/L). (G) IMS ribonuclease activity was tested for sensitivity to Proteinase K (ProK), EDTA (2 mmol/L), Cu 2+ (0.5 mmol/L), and heat (90°C 10 min)

    Techniques Used: Activity Assay, Western Blot, Isolation, Incubation, Purification

    Characterization of mtRNA degradation using an in organello system . (A) Coomassie staining of human PNPASE samples purified from HEK293 mitochondria (HEK mito) or E . coli . For PNPASE purified from HEK mitochondria, two concentrations of samples were loaded. Con: same volume of eluate from cells harboring the empty vector; PNP: eluate from PNPASE-HisPC expressing cells. (B) PNPASE purified from E . coli or HEK mitochondria was incubated with Biotin-labeled UCP2 RNA. The lower panel shows immunoblotting of PNPASE. (C) PNPASE purified from E . coli or HEK mitochondria was incubated with total cytosolic RNA and the samples were resolved on an agarose gel. The lower panel shows immunoblotting of PNPASE. (D) Mitochondria were separated into total soluble and total membrane and the two fractions were examined for ribonuclease activity using biotinylated UCP2 mRNA as a substrate. Lower panels show Immunoblotting of PNPASE (Membrane) and DDP2 (Soluble). (E) Effect of temperature on in organello mtRNA degradation. Degradation was performed at 37°C (the temperature used for the other experiments if not specified) or 25°C. The three numbers (1, 2, and 3) represent three time points (5 min, 25 min, and 45 min). Top panel on the left shows the remaining labeled mtRNAs. Bottom panel is an immunoblot of mitochondrial protein Mortalin showing the amount of mitochondria taken out at each time point. Right panel shows the quantification of labeled mtRNAs ( n = 3). (F) Effect of pH on in organello mtRNA degradation. Degradation was performed at pH 7.4 (the pH used for the other experiments if not specified) or pH 6.5. (G) Effect of Cu 2+ on in organello mtRNA degradation. Two concentrations (0 mmol/L and 0.5 mmol/L) of Cu 2+ were used. (H) Effect of Mg 2+ on in organello mtRNA degradation. Three concentrations (0 mmol/L, 0.5 mmol/L, and 20 mmol/L) of Mg 2+ were used. (I) Effect of ATP on in organello mtRNA degradation. Three concentrations (0 mmol/L, 0.5 mmol/L, and 8 mmol/L) of ATP were used. Statistical comparisons are performed using unpaired t -tests ( n = 3 if not specified); * P
    Figure Legend Snippet: Characterization of mtRNA degradation using an in organello system . (A) Coomassie staining of human PNPASE samples purified from HEK293 mitochondria (HEK mito) or E . coli . For PNPASE purified from HEK mitochondria, two concentrations of samples were loaded. Con: same volume of eluate from cells harboring the empty vector; PNP: eluate from PNPASE-HisPC expressing cells. (B) PNPASE purified from E . coli or HEK mitochondria was incubated with Biotin-labeled UCP2 RNA. The lower panel shows immunoblotting of PNPASE. (C) PNPASE purified from E . coli or HEK mitochondria was incubated with total cytosolic RNA and the samples were resolved on an agarose gel. The lower panel shows immunoblotting of PNPASE. (D) Mitochondria were separated into total soluble and total membrane and the two fractions were examined for ribonuclease activity using biotinylated UCP2 mRNA as a substrate. Lower panels show Immunoblotting of PNPASE (Membrane) and DDP2 (Soluble). (E) Effect of temperature on in organello mtRNA degradation. Degradation was performed at 37°C (the temperature used for the other experiments if not specified) or 25°C. The three numbers (1, 2, and 3) represent three time points (5 min, 25 min, and 45 min). Top panel on the left shows the remaining labeled mtRNAs. Bottom panel is an immunoblot of mitochondrial protein Mortalin showing the amount of mitochondria taken out at each time point. Right panel shows the quantification of labeled mtRNAs ( n = 3). (F) Effect of pH on in organello mtRNA degradation. Degradation was performed at pH 7.4 (the pH used for the other experiments if not specified) or pH 6.5. (G) Effect of Cu 2+ on in organello mtRNA degradation. Two concentrations (0 mmol/L and 0.5 mmol/L) of Cu 2+ were used. (H) Effect of Mg 2+ on in organello mtRNA degradation. Three concentrations (0 mmol/L, 0.5 mmol/L, and 20 mmol/L) of Mg 2+ were used. (I) Effect of ATP on in organello mtRNA degradation. Three concentrations (0 mmol/L, 0.5 mmol/L, and 8 mmol/L) of ATP were used. Statistical comparisons are performed using unpaired t -tests ( n = 3 if not specified); * P

    Techniques Used: Staining, Purification, Plasmid Preparation, Expressing, Incubation, Labeling, Agarose Gel Electrophoresis, Activity Assay

    Characterization of RNASET2 purified from HEK mitochondria . (A) Dual-tag purification of RNASET2 (His and HA). Purification was performed using IMS from control HEK cells (C) or RNASET2-overexpressing cells (T2) under native condition. (B) Ribonuclease activity were examined in IMS samples and the purification samples (Eluate) from control HEK cells (C) or RNASET2-overexpressing cells (T2) using biotinylated UCP2 mRNA as a substrate. The sensitivity of these activities to Cu 2+ (0.5 mmol/L) and proteinase K (ProK) was also tested. (C) RNASET2 was purified from RNASET2-overexpressing mitochondria under denaturing condition and checked for ribonuclease activity using RNA purified from isolated mitochondria as substrates; C (control pulldown from HEK mitochondria) and T2 (RNASET2). (D) RNASET2s purified under native conditions and denaturing conditions had the same responses to proteinase K (ProK) and Cu 2+ treatment. Lower panel is a coomassie staining gel of mitochondrial lysates as a positive control for proteinase K treatment. (E) Effect of temperature on RNASET2 purified from HEK mitochondria, RNaseI, and RNaseA (50 ng). Degradation was performed at 37°C (the temperature used for the other experiments if not specified) or 25°C. RNAs purified from isolated mitochondria were used as substrates. Con (control pulldown from HEK mitochondria). (F) Effect of pH on RNASET2 purified from HEK mitochondria, RNaseI, and RNaseA. Degradation was performed at pH 7.4 (the pH used for the other experiments if not specified), pH 6.5 or pH 5.5. (G) Effect of ATP on RNASET2 purified from HEK mitochondria, RNaseI and RNaseA. (H) Effect of Mg 2+ and Cu 2+ on RNASET2 purified from HEK mitochondria, RNaseI and RNaseA
    Figure Legend Snippet: Characterization of RNASET2 purified from HEK mitochondria . (A) Dual-tag purification of RNASET2 (His and HA). Purification was performed using IMS from control HEK cells (C) or RNASET2-overexpressing cells (T2) under native condition. (B) Ribonuclease activity were examined in IMS samples and the purification samples (Eluate) from control HEK cells (C) or RNASET2-overexpressing cells (T2) using biotinylated UCP2 mRNA as a substrate. The sensitivity of these activities to Cu 2+ (0.5 mmol/L) and proteinase K (ProK) was also tested. (C) RNASET2 was purified from RNASET2-overexpressing mitochondria under denaturing condition and checked for ribonuclease activity using RNA purified from isolated mitochondria as substrates; C (control pulldown from HEK mitochondria) and T2 (RNASET2). (D) RNASET2s purified under native conditions and denaturing conditions had the same responses to proteinase K (ProK) and Cu 2+ treatment. Lower panel is a coomassie staining gel of mitochondrial lysates as a positive control for proteinase K treatment. (E) Effect of temperature on RNASET2 purified from HEK mitochondria, RNaseI, and RNaseA (50 ng). Degradation was performed at 37°C (the temperature used for the other experiments if not specified) or 25°C. RNAs purified from isolated mitochondria were used as substrates. Con (control pulldown from HEK mitochondria). (F) Effect of pH on RNASET2 purified from HEK mitochondria, RNaseI, and RNaseA. Degradation was performed at pH 7.4 (the pH used for the other experiments if not specified), pH 6.5 or pH 5.5. (G) Effect of ATP on RNASET2 purified from HEK mitochondria, RNaseI and RNaseA. (H) Effect of Mg 2+ and Cu 2+ on RNASET2 purified from HEK mitochondria, RNaseI and RNaseA

    Techniques Used: Purification, Activity Assay, Isolation, Staining, Positive Control

    36) Product Images from "Synthetic Antibodies with a Human Framework That Protect Mice from Lethal Sudan Ebolavirus Challenge"

    Article Title: Synthetic Antibodies with a Human Framework That Protect Mice from Lethal Sudan Ebolavirus Challenge

    Journal: ACS Chemical Biology

    doi: 10.1021/cb5006454

    Competition ELISA of 16F6, F4, and E10. Binding of biotinylated 16F6 (b16F6) to immobilized GP SUDV was competed with unbiotinylated 16F6, E10, or F4 yielding IC 50 s below 0.2 μM for 16F6 and F4 and ∼0.6 μM for E10.
    Figure Legend Snippet: Competition ELISA of 16F6, F4, and E10. Binding of biotinylated 16F6 (b16F6) to immobilized GP SUDV was competed with unbiotinylated 16F6, E10, or F4 yielding IC 50 s below 0.2 μM for 16F6 and F4 and ∼0.6 μM for E10.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Binding Assay

    37) Product Images from "Two Synthetic Antibodies that Recognize and Neutralize Distinct Proteolytic Forms of the Ebola Virus Envelope Glycoprotein"

    Article Title: Two Synthetic Antibodies that Recognize and Neutralize Distinct Proteolytic Forms of the Ebola Virus Envelope Glycoprotein

    Journal: Chembiochem : a European journal of chemical biology

    doi: 10.1002/cbic.201200493

    Competition ELISAs. A) Biotinylated Fab CL (bFab CL ) versus four unbiotinylated competitors: Fab CL , Fab UNCL ] . Fab CL competed with bFab CL ; the data were fit to a four-parameter
    Figure Legend Snippet: Competition ELISAs. A) Biotinylated Fab CL (bFab CL ) versus four unbiotinylated competitors: Fab CL , Fab UNCL ] . Fab CL competed with bFab CL ; the data were fit to a four-parameter

    Techniques Used:

    38) Product Images from "Immune targeting of fibroblast activation protein triggers recognition of multipotent bone marrow stromal cells and cachexia"

    Article Title: Immune targeting of fibroblast activation protein triggers recognition of multipotent bone marrow stromal cells and cachexia

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20130110

    FAP expression in mouse tumors, and in vivo activity of FAP5-CAR–transduced T cells against various murine tumors. In vitro cultured B16, MC38, MC17-51, 4T1, CT26, and Renca murine tumors were assessed for FAP expression by flow cytometry with the FAP-specific antibody FAP5 (A). Solid line is isotype control and filled histogram is FAP5 stained. Results are representative of at least two independent experiments. Established (∼11–16 d) subcutaneously implanted B16 (B), MC38 (C), MC17-51 (D), 4T1 (E), CT26 (F), and Renca (G) tumors were harvested from mice (irradiated before harvest) and assessed for FAP expression by IHC using biotinylated-FAP5 antibody. Bars, 400 µm. Representative of at least two independent experiments. C57BL/6 mice bearing established B16 (H), MC38 (I), MC17-51 (J) tumors, and BALB/c mice bearing established 4T1 (K), CT26 (L), or Renca (M) tumors were left untreated (No Tx) or treated with 10 7 UnTd or 10 7 FAP5-CAR Td T cells, and the perpendicular diameters of the tumors were measured over time. Mean ± SEM. Results are representative of at least two independent experiments for H–J and one experiment for K–M with initially five mice per group. *, P
    Figure Legend Snippet: FAP expression in mouse tumors, and in vivo activity of FAP5-CAR–transduced T cells against various murine tumors. In vitro cultured B16, MC38, MC17-51, 4T1, CT26, and Renca murine tumors were assessed for FAP expression by flow cytometry with the FAP-specific antibody FAP5 (A). Solid line is isotype control and filled histogram is FAP5 stained. Results are representative of at least two independent experiments. Established (∼11–16 d) subcutaneously implanted B16 (B), MC38 (C), MC17-51 (D), 4T1 (E), CT26 (F), and Renca (G) tumors were harvested from mice (irradiated before harvest) and assessed for FAP expression by IHC using biotinylated-FAP5 antibody. Bars, 400 µm. Representative of at least two independent experiments. C57BL/6 mice bearing established B16 (H), MC38 (I), MC17-51 (J) tumors, and BALB/c mice bearing established 4T1 (K), CT26 (L), or Renca (M) tumors were left untreated (No Tx) or treated with 10 7 UnTd or 10 7 FAP5-CAR Td T cells, and the perpendicular diameters of the tumors were measured over time. Mean ± SEM. Results are representative of at least two independent experiments for H–J and one experiment for K–M with initially five mice per group. *, P

    Techniques Used: Expressing, In Vivo, Activity Assay, In Vitro, Cell Culture, Flow Cytometry, Cytometry, Staining, Mouse Assay, Irradiation, Immunohistochemistry

    39) Product Images from "Selection of Antibodies with Tailored Properties by Application of High-Throughput Multiparameter Fluorescence-Activated Cell Sorting of Yeast-Displayed Immune Libraries"

    Article Title: Selection of Antibodies with Tailored Properties by Application of High-Throughput Multiparameter Fluorescence-Activated Cell Sorting of Yeast-Displayed Immune Libraries

    Journal: Molecular Biotechnology

    doi: 10.1007/s12033-018-0109-0

    Selection process of libraries generated from lymph node cells of h-RTK-immunized kappa OmniRats™. Bivariate plots of yeast-cells labeled with biotinylated h-RTK–ECD–His, SA-Alexa Fluor 647 (antigen binding) and anti-kappa R-PE for detection of surface expression (display) during round I and II. After round II, cells were double labeled with h-RTK–ECD–His and m-RTK–ECD–His to identify clones exhibiting simultaneous binding to both proteins. After round III, h-RTK subdomain-specific cells were selected by applying two different gates (R1: h-RTK-Domain-A; R2: h-RTK-Domain-B). Yeast cells were coincubated with RTK-Domain-AB (biotinylated) and RTK-Domain-BC (Alexa Fluor 647) subdomains of h-RTK, followed by secondary labeling with SA-PE
    Figure Legend Snippet: Selection process of libraries generated from lymph node cells of h-RTK-immunized kappa OmniRats™. Bivariate plots of yeast-cells labeled with biotinylated h-RTK–ECD–His, SA-Alexa Fluor 647 (antigen binding) and anti-kappa R-PE for detection of surface expression (display) during round I and II. After round II, cells were double labeled with h-RTK–ECD–His and m-RTK–ECD–His to identify clones exhibiting simultaneous binding to both proteins. After round III, h-RTK subdomain-specific cells were selected by applying two different gates (R1: h-RTK-Domain-A; R2: h-RTK-Domain-B). Yeast cells were coincubated with RTK-Domain-AB (biotinylated) and RTK-Domain-BC (Alexa Fluor 647) subdomains of h-RTK, followed by secondary labeling with SA-PE

    Techniques Used: Selection, Generated, Labeling, Binding Assay, Expressing, Clone Assay

    40) Product Images from "Microfluidic Preparation of Polymer-Nucleic Acid Nanocomplexes Improves Nonviral Gene Transfer"

    Article Title: Microfluidic Preparation of Polymer-Nucleic Acid Nanocomplexes Improves Nonviral Gene Transfer

    Journal: Scientific Reports

    doi: 10.1038/srep03155

    Cellular internalization and intracellular unpacking. (A) Labeling scheme to detect uptake and unpacking using QD-FRET. Biotinylated pDNA was labeled with QD energy donors, while the polymer was functionalized with the Cy5 QD-FRET acceptor. QD-FRET emission is detected while polyplexes are intact. Following unpacking, QD-FRET signal is lost and separate donor and acceptor emissions are recovered. (B), (C) Cellular internalization was quantified by measuring the fluorescence signal of QD-labeled pDNA in cells at different time points using flow cytometry. No significant difference was observed in the rates of uptake by cell number of normalized geometric mean fluorescence. (D) Intracellular unpacking was quantified by measuring the QD-FRET signal in cells at different time points using flow cytometry. Bulk controls unpacked more rapidly than MAC polyplexes (p
    Figure Legend Snippet: Cellular internalization and intracellular unpacking. (A) Labeling scheme to detect uptake and unpacking using QD-FRET. Biotinylated pDNA was labeled with QD energy donors, while the polymer was functionalized with the Cy5 QD-FRET acceptor. QD-FRET emission is detected while polyplexes are intact. Following unpacking, QD-FRET signal is lost and separate donor and acceptor emissions are recovered. (B), (C) Cellular internalization was quantified by measuring the fluorescence signal of QD-labeled pDNA in cells at different time points using flow cytometry. No significant difference was observed in the rates of uptake by cell number of normalized geometric mean fluorescence. (D) Intracellular unpacking was quantified by measuring the QD-FRET signal in cells at different time points using flow cytometry. Bulk controls unpacked more rapidly than MAC polyplexes (p

    Techniques Used: Labeling, Fluorescence, Flow Cytometry, Cytometry

    Related Articles

    Flow Cytometry:

    Article Title: Capture and display of antibodies secreted by hybridoma cells enables fluorescent on-cell screening
    Article Snippet: .. Secondary antibodies and labeling reagents AB1: Alexa Fluor 488® AffiniPure F(ab’)2 Fragment Goat Anti-Human IgG, F(ab’)2 fragment specific (109-546-097; Jackson ImmunoResearch, West Grove, PA) RRID: AB_2337849 AB2: Alexa Fluor® 488 AffiniPure F(ab’)₂ Fragment Goat Anti-Human IgG, Fcγ fragment specific (109-546-098; Jackson ImmunoResearch) RRID: AB_2337850 AB3: Alexa Fluor 488® Streptavidin (016-540-084; Jackson ImmunoResearch) RRID: AB_2337249 AB4: APC Streptavidin (016-130-084; Jackson ImmunoResearch) RRID: AB_2337342 AB5: APC AffiniPure F(ab’)2 fragment Goat anti-rabbit IgG (H + L) (111-136-144; Jackson ImmunoResearch) RRID: AB_2337987 AB6: CellTrace™ CFSE Cell Proliferation Kit, for flow cytometry (C3455 4; Thermo Fisher, Waltham, MA) AB7: CY™5 AffiniPure Goat Anti-Mouse IgG (H + L) (115-175-146; Jackson ImmunoResearch) RRID: AB_2338713 AB8: EZ-Link™ Sulfo-NHS-LC-Biotinylation Kit (21327; Thermo Fisher) AB9: Goat anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (A-11029; Thermo Fisher) RRID: AB2534088 AB10: Goat anti-Human IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 (A-A21433; Thermo Fisher) RRID: AB_2534088 AB11: gp130 mAb (AN-H2) (sc-9994; Santa Cruz Biotechnology, Dallas, TX) RRID: AB_627685 AB12: Pierce™ Protein A, Biotinylated (29989; Thermo Fisher). .. Nickname biotinylated Protein A AB13: Rabbit IgG-BIOT (0111-08; SouthernBiotech, Birmingham, AL) RRID: AB _627685 AB14: (RAH) rabbit mAb [H169-1-5] anti-Human IgG Fc (ab125909; Abcam, Cambridge, MA) AB15: Rabbit F(ab’)2 Anti-Human IgG(H + L)-UNLB (6000-01; SouthernBiotech) AB16: Rabbit Anti-Human IgG(H + L)-UNLB (6140-01; SouthernBiotech) AB17: Rabbit Fab Anti-human IgG (H & L) (809-4102; Rockland Immunochemicals, Pottstown, PA) AB18: Streptavidin, Alexa Fluor™ 555 conjugate (S21381; Thermo Fisher) RRID: AB_2307336 AB19: Anti-NMDAR1 Antibody, clone 54.1 (MAB363; Millipore Sigma, St. Louis, MO) RRID: AB_94946 AB20:Anti-NMDAR1 Antibody, (all splice variants), clone R1JHL (MAB1586; Millipore Sigma)

    Article Title: Specific amino acids in the N-terminus of the gp41 ectodomain contribute to the stabilization of a soluble, cleaved gp140 envelope glycoprotein from human immunodeficiency virus type 1
    Article Snippet: .. A BIAcore streptavidin (SA) chip was used for capturing ~1000 response units (RU) of biotinylated protein G (Pierce) in both the experimental and the control flow-cells. .. Biotin was then used to block the uncoated streptavidin surface on both flow-cells.

    Purification:

    Article Title: Fabrication of a Dual Substrate Display to Test Roles of Cell Adhesion Proteins in Vesicle Targeting to Plasma Membrane Domains
    Article Snippet: .. Coverslips were sequentially incubated with Neutravidin (5mg/ml in PBS; Pierce), biotinylated protein A (0.3mg/ml in PBS; Pierce), D-Biotin (15mM in DMSO; Pierce), and Cy5.5-labeled purified Fc-E-cadherin fusion protein (200μg/ml; [ ]) for 1hr each at room temperature in the dark, with 3x PBS washes between each incubation. .. Where appropriate, purified IgG heavy chain (Fc; 200μg/ml; Sigma) was substituted in the final step in place of Fc-E-cadherin protein as a negative (non-adhesive) control.

    Cytometry:

    Article Title: Capture and display of antibodies secreted by hybridoma cells enables fluorescent on-cell screening
    Article Snippet: .. Secondary antibodies and labeling reagents AB1: Alexa Fluor 488® AffiniPure F(ab’)2 Fragment Goat Anti-Human IgG, F(ab’)2 fragment specific (109-546-097; Jackson ImmunoResearch, West Grove, PA) RRID: AB_2337849 AB2: Alexa Fluor® 488 AffiniPure F(ab’)₂ Fragment Goat Anti-Human IgG, Fcγ fragment specific (109-546-098; Jackson ImmunoResearch) RRID: AB_2337850 AB3: Alexa Fluor 488® Streptavidin (016-540-084; Jackson ImmunoResearch) RRID: AB_2337249 AB4: APC Streptavidin (016-130-084; Jackson ImmunoResearch) RRID: AB_2337342 AB5: APC AffiniPure F(ab’)2 fragment Goat anti-rabbit IgG (H + L) (111-136-144; Jackson ImmunoResearch) RRID: AB_2337987 AB6: CellTrace™ CFSE Cell Proliferation Kit, for flow cytometry (C3455 4; Thermo Fisher, Waltham, MA) AB7: CY™5 AffiniPure Goat Anti-Mouse IgG (H + L) (115-175-146; Jackson ImmunoResearch) RRID: AB_2338713 AB8: EZ-Link™ Sulfo-NHS-LC-Biotinylation Kit (21327; Thermo Fisher) AB9: Goat anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (A-11029; Thermo Fisher) RRID: AB2534088 AB10: Goat anti-Human IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 (A-A21433; Thermo Fisher) RRID: AB_2534088 AB11: gp130 mAb (AN-H2) (sc-9994; Santa Cruz Biotechnology, Dallas, TX) RRID: AB_627685 AB12: Pierce™ Protein A, Biotinylated (29989; Thermo Fisher). .. Nickname biotinylated Protein A AB13: Rabbit IgG-BIOT (0111-08; SouthernBiotech, Birmingham, AL) RRID: AB _627685 AB14: (RAH) rabbit mAb [H169-1-5] anti-Human IgG Fc (ab125909; Abcam, Cambridge, MA) AB15: Rabbit F(ab’)2 Anti-Human IgG(H + L)-UNLB (6000-01; SouthernBiotech) AB16: Rabbit Anti-Human IgG(H + L)-UNLB (6140-01; SouthernBiotech) AB17: Rabbit Fab Anti-human IgG (H & L) (809-4102; Rockland Immunochemicals, Pottstown, PA) AB18: Streptavidin, Alexa Fluor™ 555 conjugate (S21381; Thermo Fisher) RRID: AB_2307336 AB19: Anti-NMDAR1 Antibody, clone 54.1 (MAB363; Millipore Sigma, St. Louis, MO) RRID: AB_94946 AB20:Anti-NMDAR1 Antibody, (all splice variants), clone R1JHL (MAB1586; Millipore Sigma)

    Labeling:

    Article Title: Capture and display of antibodies secreted by hybridoma cells enables fluorescent on-cell screening
    Article Snippet: .. Secondary antibodies and labeling reagents AB1: Alexa Fluor 488® AffiniPure F(ab’)2 Fragment Goat Anti-Human IgG, F(ab’)2 fragment specific (109-546-097; Jackson ImmunoResearch, West Grove, PA) RRID: AB_2337849 AB2: Alexa Fluor® 488 AffiniPure F(ab’)₂ Fragment Goat Anti-Human IgG, Fcγ fragment specific (109-546-098; Jackson ImmunoResearch) RRID: AB_2337850 AB3: Alexa Fluor 488® Streptavidin (016-540-084; Jackson ImmunoResearch) RRID: AB_2337249 AB4: APC Streptavidin (016-130-084; Jackson ImmunoResearch) RRID: AB_2337342 AB5: APC AffiniPure F(ab’)2 fragment Goat anti-rabbit IgG (H + L) (111-136-144; Jackson ImmunoResearch) RRID: AB_2337987 AB6: CellTrace™ CFSE Cell Proliferation Kit, for flow cytometry (C3455 4; Thermo Fisher, Waltham, MA) AB7: CY™5 AffiniPure Goat Anti-Mouse IgG (H + L) (115-175-146; Jackson ImmunoResearch) RRID: AB_2338713 AB8: EZ-Link™ Sulfo-NHS-LC-Biotinylation Kit (21327; Thermo Fisher) AB9: Goat anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (A-11029; Thermo Fisher) RRID: AB2534088 AB10: Goat anti-Human IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 (A-A21433; Thermo Fisher) RRID: AB_2534088 AB11: gp130 mAb (AN-H2) (sc-9994; Santa Cruz Biotechnology, Dallas, TX) RRID: AB_627685 AB12: Pierce™ Protein A, Biotinylated (29989; Thermo Fisher). .. Nickname biotinylated Protein A AB13: Rabbit IgG-BIOT (0111-08; SouthernBiotech, Birmingham, AL) RRID: AB _627685 AB14: (RAH) rabbit mAb [H169-1-5] anti-Human IgG Fc (ab125909; Abcam, Cambridge, MA) AB15: Rabbit F(ab’)2 Anti-Human IgG(H + L)-UNLB (6000-01; SouthernBiotech) AB16: Rabbit Anti-Human IgG(H + L)-UNLB (6140-01; SouthernBiotech) AB17: Rabbit Fab Anti-human IgG (H & L) (809-4102; Rockland Immunochemicals, Pottstown, PA) AB18: Streptavidin, Alexa Fluor™ 555 conjugate (S21381; Thermo Fisher) RRID: AB_2307336 AB19: Anti-NMDAR1 Antibody, clone 54.1 (MAB363; Millipore Sigma, St. Louis, MO) RRID: AB_94946 AB20:Anti-NMDAR1 Antibody, (all splice variants), clone R1JHL (MAB1586; Millipore Sigma)

    Avidin-Biotin Assay:

    Article Title: Link Protein N-terminal Peptide Binds to Bone Morphogenetic Protein (BMP) Type II Receptor and Drives Matrix Protein Expression in Rabbit Intervertebral Disc Cells *
    Article Snippet: .. To capture biotinylated LPP-bound protein, the supernatant containing biotinylated peptide-protein conjugates was passed through a monomeric avidin-Sepharose column (Pierce). .. Eluted peptide-protein conjugates were subjected to SDS-PAGE and transferred to PVDF membranes, and proteins were detected using peroxidase-conjugated streptavidin.

    Enzyme-linked Immunosorbent Assay:

    Article Title: Immunization Against Oxidized Elastin Exacerbates Structural and Functional Damage in Mouse Model of Smoke-Induced Ocular Injury
    Article Snippet: .. The plates were again washed and incubated with biotinylated anti-mouse secondary antibodies (anti-IgG and anti-IgM) followed by streptavidin (1:1000) and color development using Turbo-TMB ELISA (Pierce; Thermo Scientific, Rockford, IL, USA). .. Western AnalysisMouse RPE/choroid/sclera (hereafter referred to as RPE/choroid fraction) preparations were prepared from eyecups and protein was extracted by solubilizing in RIPA buffer (10 mM Tris-HCl [pH 7.5], 300 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% SDS, and 0.1% sodium deoxycholate; ThermoFisher Scientific, Waltham, MA, USA) containing protease inhibitor cocktail (Sigma-Aldrich).

    Concentration Assay:

    Article Title: Translationally controlled tumor protein of Brugia malayi functions as an antioxidant protein
    Article Snippet: .. Concentration of the biotinylated protein was then estimated by a BCA method (Pierce Biotechnology), and the biotinylated rBmTCTP was stored at 4°C until further use. .. The interaction of Trx and rBmTCTP were analyzed by an in vitro binding assay.

    Incubation:

    Article Title: Immunization Against Oxidized Elastin Exacerbates Structural and Functional Damage in Mouse Model of Smoke-Induced Ocular Injury
    Article Snippet: .. The plates were again washed and incubated with biotinylated anti-mouse secondary antibodies (anti-IgG and anti-IgM) followed by streptavidin (1:1000) and color development using Turbo-TMB ELISA (Pierce; Thermo Scientific, Rockford, IL, USA). .. Western AnalysisMouse RPE/choroid/sclera (hereafter referred to as RPE/choroid fraction) preparations were prepared from eyecups and protein was extracted by solubilizing in RIPA buffer (10 mM Tris-HCl [pH 7.5], 300 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% SDS, and 0.1% sodium deoxycholate; ThermoFisher Scientific, Waltham, MA, USA) containing protease inhibitor cocktail (Sigma-Aldrich).

    Article Title: Repeated Cycles of Rapid Actin Assembly and Disassembly on Epithelial Cell Phagosomes V⃞
    Article Snippet: .. To coat polystyrene beads with E-cadherin.Fc , 1-μm polystyrene beads (Polysciences, Warrington, PA) were incubated in 1 mg/ml biotinylated protein A (Pierce Chemical, Rockford, IL) at room temperature with rotation for 3 h. Beads were washed once in PBS, resuspended in E-cadherin.Fc (0.2 mg/ml), and incubated for 1 h at room temperature and then overnight at 4°C with rotation. .. Beads were washed once in 137 mM NaCl, 3 mM KCl, 50 μM CaCl2 , 5 mM HEPES, pH 7.4, and resuspended in this buffer to a final concentration of ∼2 × 106 beads/μl.

    Article Title: Fabrication of a Dual Substrate Display to Test Roles of Cell Adhesion Proteins in Vesicle Targeting to Plasma Membrane Domains
    Article Snippet: .. Coverslips were sequentially incubated with Neutravidin (5mg/ml in PBS; Pierce), biotinylated protein A (0.3mg/ml in PBS; Pierce), D-Biotin (15mM in DMSO; Pierce), and Cy5.5-labeled purified Fc-E-cadherin fusion protein (200μg/ml; [ ]) for 1hr each at room temperature in the dark, with 3x PBS washes between each incubation. .. Where appropriate, purified IgG heavy chain (Fc; 200μg/ml; Sigma) was substituted in the final step in place of Fc-E-cadherin protein as a negative (non-adhesive) control.

    BIA-KA:

    Article Title: Translationally controlled tumor protein of Brugia malayi functions as an antioxidant protein
    Article Snippet: .. Concentration of the biotinylated protein was then estimated by a BCA method (Pierce Biotechnology), and the biotinylated rBmTCTP was stored at 4°C until further use. .. The interaction of Trx and rBmTCTP were analyzed by an in vitro binding assay.

    Binding Assay:

    Article Title: Capture and display of antibodies secreted by hybridoma cells enables fluorescent on-cell screening
    Article Snippet: .. Human and rabbit IgG binding to the PV OCMS™ cell lines ( )) were assessed by incubating 5 × 105 cells overnight with 1 µg/ml rabbit F(ab’)2 Anti-Human IgG (H + L) (6000-01; SouthernBiotech) (AB15), followed by washing and then 1 µg/ml Pierce™ Protein A, Biotinylated (29989; Thermo Fisher) (AB12) for 1 hour. .. Cells were washed and incubated with 1:200 Alexa Fluor 488® Streptavidin (016-540-084; Jackson ImmunoResearch) (AB3) and 1:200 APC AffiniPure F(ab’)2 fragment Goat anti-rabbit IgG (H + L) (111-136-144; Jackson ImmunoResearch) (AB5) for one hour, and then analyzed by flow cytometry.

    Chromatin Immunoprecipitation:

    Article Title: Specific amino acids in the N-terminus of the gp41 ectodomain contribute to the stabilization of a soluble, cleaved gp140 envelope glycoprotein from human immunodeficiency virus type 1
    Article Snippet: .. A BIAcore streptavidin (SA) chip was used for capturing ~1000 response units (RU) of biotinylated protein G (Pierce) in both the experimental and the control flow-cells. .. Biotin was then used to block the uncoated streptavidin surface on both flow-cells.

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  • 93
    Thermo Fisher biotinylated anti β1 mab ts2 16
    Cell surface ADAM-17 is more efficiently co-immunoprecipitated with integrin α5β1 from Colo320-CD9 cells than from Colo320 cells. (A) Only cell surface integrin α 5 β 1 molecules were selectively immunoprecipitated by incubating the cells with mAb Lia1/2 (anti-β 1 ) in the presence of Ca 2+ +Mg 2+ (500 μM each) and washing the excess non-bound antibody prior to cell lysis and immunoprecipitation. Immunoprecipitated integrin α 5 β 1 and co-immunoprecipitated ADAM17 were detected by immunoblotting with the <t>anti-β1</t> <t>(TS2/16)</t> and anti ADAM17 (A300D) mAbs, respectively. The gel shown is representative of five different experiments. The graph below shows the densitometric quantitation of the amount of precipitated integrin and co-immunoprecipitated ADAM17 (means ± SEM) from five different experiments, normalized to β 1 precipitated in Colo320 cells in each experiment. (B) Integrin α5β1 and ADAM17 are coimmunoprecipitated with CD9 from cell surface TEMs. CD9 was immunoprecipitated with mAb PAINS10 as described for integrin β1 in (A) but under two different extracellular cation conditions: in the presence of Ca 2+ +Mg 2+ (500 μM each) or Mn 2+ (200 μM). Immunoprecipitated CD9 and co-immunoprecipitated β 1 and ADAM17 were detected by immunoblotting with mAbs PAINS10 (anti-CD9), TS2/16 (anti-β1), and A300D (anti ADAM17), respectively. The gel shown is representative of four different experiments. The graph below represents the densitometric quantitation of the amount of precipitated CD9 and co-immunoprecipitated integrin β 1 and ADAM17 (means ± SEM) normalized to the immunoprecipitated CD9 in each of the four independent experiments. Statistical analysis was carried out using two-tailed paired T -test. * p
    Biotinylated Anti β1 Mab Ts2 16, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher biotinylated rna pull down assays t98g
    Determination of the EV71 IRES sequences required for the binding of PTB. A Prediction of the <t>RNA</t> secondary structure of the EV71 5′UTR by the mfold web serve. The modified schematic representation of the secondary structure of poliovirus (PV) 5′UTR (modified from Hellen et al . 1994 ) is shown in the top right corner. B Plasmids carrying different deletions in the five stem-loops of EV71 IRES: pGEM-3zf-(SL II, 121–181), (SL III, 190–230), (SL IV, 241–450), (SL V, 451–463), (SL VI, 564–742). C Analysis of the regions responsible for the interaction in the EV71 IRES region using various truncated RNA forms, transcribed in vitro and <t>biotinylated.</t> <t>T98G</t> cell lysate were incubated with these biotin-labeled RNAs and the non-biotinylated IRES RNA probes were used as controls. After being pulled down by streptavidin, the protein complex was separated by SDS-PAGE and Western blot was carried out to detect PTB in the pulled-down complex (lanes 1–7).
    Biotinylated Rna Pull Down Assays T98g, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/biotinylated rna pull down assays t98g/product/Thermo Fisher
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    biotinylated rna pull down assays t98g - by Bioz Stars, 2020-09
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    89
    Thermo Fisher biotinylated cell surface proteins cell surface proteins
    Dopamine reduced turnover and solubility of PrP C . Cell surface proteins was <t>biotinylated</t> and chased at time zero (input) or after 1 h incubation in the presence or absence of 50 μM of dopamine. After 1 h, dopamine treated cells showed higher amount of remaining PrP C compared to untreated cells (A) . After dopamine treatment, insoluble proteins in sarkosyl 1% were recovered by ultracentrifugation. Higher amounts of PrP C were recovered in pellet fractions and lower amount in supernatants (SN), indicating lower solubility (B) . Mean band intensity of three independent experiments was plotted with respective CI95. Dark gray bars represent SN and light gray bars represent pellet quantification (B) . * no overlap of CI95 between indicated group and control group (0 μM). rPrP (10 μg) was incubated with designated concentration of dopamine (DA) for 24 h or with 400 μM of dopamine for indicated times (C) . When 400 μM of metabisulfite (#) was added, the oligomerization was prevented (C) . rPrP monomer migrated close to 26 kDa marker. Three independent experiments were performed and representative images are shown.
    Biotinylated Cell Surface Proteins Cell Surface Proteins, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/biotinylated cell surface proteins cell surface proteins/product/Thermo Fisher
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    90
    Thermo Fisher biotinylated at8 antibody
    Anti-tau antibody decreased phospho-tau staining in the hippocampal CA1 cell layer. (A) Representative coronal sections of <t>biotinylated</t> <t>AT8</t> antibody staining of phosphorylated tau in the hippocampal CA1 cellular region of 9-month-old P301S mice treated for 3 months with vehicle and HJ8.5 at 50 mg/kg. The lower images are higher power views of the CA1 region in the uppers panels. Red arrows indicate the area magnified in the lower image. Black arrows indicate the hippocampal CA1 cell layer. (B) Quantification of biotinylated AT8 antibody staining of abnormally phosphorylated tau revealed a significant decrease in AT8 staining in mice treated with HJ8.5 at 50 mg/kg in the hippocampal CA1 cellular layer compared to vehicle-treated mice ( P = 0.035). HJ8.5 at 10 mg/kg treated mice also showed decreased AT8 staining compared to the vehicle-treated group, but this was not statistically significant ( P > 0.05). Values represent mean ± SEM. * P
    Biotinylated At8 Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Cell surface ADAM-17 is more efficiently co-immunoprecipitated with integrin α5β1 from Colo320-CD9 cells than from Colo320 cells. (A) Only cell surface integrin α 5 β 1 molecules were selectively immunoprecipitated by incubating the cells with mAb Lia1/2 (anti-β 1 ) in the presence of Ca 2+ +Mg 2+ (500 μM each) and washing the excess non-bound antibody prior to cell lysis and immunoprecipitation. Immunoprecipitated integrin α 5 β 1 and co-immunoprecipitated ADAM17 were detected by immunoblotting with the anti-β1 (TS2/16) and anti ADAM17 (A300D) mAbs, respectively. The gel shown is representative of five different experiments. The graph below shows the densitometric quantitation of the amount of precipitated integrin and co-immunoprecipitated ADAM17 (means ± SEM) from five different experiments, normalized to β 1 precipitated in Colo320 cells in each experiment. (B) Integrin α5β1 and ADAM17 are coimmunoprecipitated with CD9 from cell surface TEMs. CD9 was immunoprecipitated with mAb PAINS10 as described for integrin β1 in (A) but under two different extracellular cation conditions: in the presence of Ca 2+ +Mg 2+ (500 μM each) or Mn 2+ (200 μM). Immunoprecipitated CD9 and co-immunoprecipitated β 1 and ADAM17 were detected by immunoblotting with mAbs PAINS10 (anti-CD9), TS2/16 (anti-β1), and A300D (anti ADAM17), respectively. The gel shown is representative of four different experiments. The graph below represents the densitometric quantitation of the amount of precipitated CD9 and co-immunoprecipitated integrin β 1 and ADAM17 (means ± SEM) normalized to the immunoprecipitated CD9 in each of the four independent experiments. Statistical analysis was carried out using two-tailed paired T -test. * p

    Journal: Frontiers in Immunology

    Article Title: CD9 Controls Integrin α5β1-Mediated Cell Adhesion by Modulating Its Association With the Metalloproteinase ADAM17

    doi: 10.3389/fimmu.2018.02474

    Figure Lengend Snippet: Cell surface ADAM-17 is more efficiently co-immunoprecipitated with integrin α5β1 from Colo320-CD9 cells than from Colo320 cells. (A) Only cell surface integrin α 5 β 1 molecules were selectively immunoprecipitated by incubating the cells with mAb Lia1/2 (anti-β 1 ) in the presence of Ca 2+ +Mg 2+ (500 μM each) and washing the excess non-bound antibody prior to cell lysis and immunoprecipitation. Immunoprecipitated integrin α 5 β 1 and co-immunoprecipitated ADAM17 were detected by immunoblotting with the anti-β1 (TS2/16) and anti ADAM17 (A300D) mAbs, respectively. The gel shown is representative of five different experiments. The graph below shows the densitometric quantitation of the amount of precipitated integrin and co-immunoprecipitated ADAM17 (means ± SEM) from five different experiments, normalized to β 1 precipitated in Colo320 cells in each experiment. (B) Integrin α5β1 and ADAM17 are coimmunoprecipitated with CD9 from cell surface TEMs. CD9 was immunoprecipitated with mAb PAINS10 as described for integrin β1 in (A) but under two different extracellular cation conditions: in the presence of Ca 2+ +Mg 2+ (500 μM each) or Mn 2+ (200 μM). Immunoprecipitated CD9 and co-immunoprecipitated β 1 and ADAM17 were detected by immunoblotting with mAbs PAINS10 (anti-CD9), TS2/16 (anti-β1), and A300D (anti ADAM17), respectively. The gel shown is representative of four different experiments. The graph below represents the densitometric quantitation of the amount of precipitated CD9 and co-immunoprecipitated integrin β 1 and ADAM17 (means ± SEM) normalized to the immunoprecipitated CD9 in each of the four independent experiments. Statistical analysis was carried out using two-tailed paired T -test. * p

    Article Snippet: Membranes were blocked with 3% BSA and developed either with biotinylated anti-CD9 mAb PAINS10 or biotinylated anti-β1 mAb TS2/16 or anti-ADAM17 mAb A300D, followed by incubation respectively with streptavidin-HRP (Thermo-Fisher Scientific) or anti-mouse IgG-HRP (Sigma-Aldrich) secondary reagents and ECL-chemiluminescence detection on an ImageQuant LAS4000-mini system.

    Techniques: Immunoprecipitation, Lysis, Quantitation Assay, Two Tailed Test

    Determination of the EV71 IRES sequences required for the binding of PTB. A Prediction of the RNA secondary structure of the EV71 5′UTR by the mfold web serve. The modified schematic representation of the secondary structure of poliovirus (PV) 5′UTR (modified from Hellen et al . 1994 ) is shown in the top right corner. B Plasmids carrying different deletions in the five stem-loops of EV71 IRES: pGEM-3zf-(SL II, 121–181), (SL III, 190–230), (SL IV, 241–450), (SL V, 451–463), (SL VI, 564–742). C Analysis of the regions responsible for the interaction in the EV71 IRES region using various truncated RNA forms, transcribed in vitro and biotinylated. T98G cell lysate were incubated with these biotin-labeled RNAs and the non-biotinylated IRES RNA probes were used as controls. After being pulled down by streptavidin, the protein complex was separated by SDS-PAGE and Western blot was carried out to detect PTB in the pulled-down complex (lanes 1–7).

    Journal: Virologica Sinica

    Article Title: Polypyrimidine Tract-Binding Protein Regulates Enterovirus 71 Translation Through Interaction with the Internal Ribosomal Entry Site

    doi: 10.1007/s12250-019-00089-1

    Figure Lengend Snippet: Determination of the EV71 IRES sequences required for the binding of PTB. A Prediction of the RNA secondary structure of the EV71 5′UTR by the mfold web serve. The modified schematic representation of the secondary structure of poliovirus (PV) 5′UTR (modified from Hellen et al . 1994 ) is shown in the top right corner. B Plasmids carrying different deletions in the five stem-loops of EV71 IRES: pGEM-3zf-(SL II, 121–181), (SL III, 190–230), (SL IV, 241–450), (SL V, 451–463), (SL VI, 564–742). C Analysis of the regions responsible for the interaction in the EV71 IRES region using various truncated RNA forms, transcribed in vitro and biotinylated. T98G cell lysate were incubated with these biotin-labeled RNAs and the non-biotinylated IRES RNA probes were used as controls. After being pulled down by streptavidin, the protein complex was separated by SDS-PAGE and Western blot was carried out to detect PTB in the pulled-down complex (lanes 1–7).

    Article Snippet: Biotinylated RNA Pull-Down Assays T98G, HA, RD, and SH-SY5Y cells were pelleted, and then prepared using nuclear protein extraction reagents (Thermo, USA).

    Techniques: Binding Assay, Modification, In Vitro, Incubation, Labeling, SDS Page, Western Blot

    Interaction of PTB with the EV71 IRES region. A Pull down of proteins with the EV71 IRES in T98G cell lysate. The specific bands interacting with EV71 IRES are shown by silver staining. Lane 1: biotinylated IRES RNA, Lane 2: nonbiotinylated EV71 IRES RNA. B A specific association between PTB and the EV71 IRES region was confirmed by Western blotting and a competition assay. Increasing amounts of unlabelled RNA were added to compete with the biotin-labeled EV71 IRES RNA interacting with PTB. The eluted proteins were separated by 12% SDS-PAGE. Lanes are as follows: lanes 1–4, unlabeled EV71 IRES RNA; lanes 5–8, unlabeled yeast tRNA. C Extracts of RD cells, SH-SY5Y cells, and HA cells were prepared and then incubated without RNA (lane 2), with biotinylated actin RNA (lane 3), non-biotinylated EV71 IRES RNA (lane 4), or biotinylated EV71 IRES RNA (lane 5). After pull-down assay, the bound proteins were eluted, boiled, and subjected to 12% SDS-PAGE. PTB protein was detected by western blot with a rabbit anti-PTB antibody. The inputs were cell extracts of RD, SH-SY5Y and HA (lane 1). D EV71 IRES RNA was pulled down with PTB from EV71-infected T98G cell lysate. T98G cells were infected with EV71 at an MOI of 20 for 6 h and then cell extracts were incubated with rabbit anti-PTB antibody (lanes 2 and 6), normal rabbit IgG (lanes 3 and 7), or without antibody (− Ab) (lanes 4 and 8). Following washing and dissociation, the RNA extract was prepared and subjected to RT-PCR analysis with primers specific for the ribosomal protein S16 (PRS16) RNA (lanes1–4) or for EV71 IRES region RNA (lanes 5–8). Lane 1, cell lysate without immunoprecipitation as a positive RT-PCR control; Lane 2, anti-PTB antibody incubated with 200 mg infected-cell lysate; Lane 3, negative control with rabbit IgG; Lane 4, negative control with no antibody.

    Journal: Virologica Sinica

    Article Title: Polypyrimidine Tract-Binding Protein Regulates Enterovirus 71 Translation Through Interaction with the Internal Ribosomal Entry Site

    doi: 10.1007/s12250-019-00089-1

    Figure Lengend Snippet: Interaction of PTB with the EV71 IRES region. A Pull down of proteins with the EV71 IRES in T98G cell lysate. The specific bands interacting with EV71 IRES are shown by silver staining. Lane 1: biotinylated IRES RNA, Lane 2: nonbiotinylated EV71 IRES RNA. B A specific association between PTB and the EV71 IRES region was confirmed by Western blotting and a competition assay. Increasing amounts of unlabelled RNA were added to compete with the biotin-labeled EV71 IRES RNA interacting with PTB. The eluted proteins were separated by 12% SDS-PAGE. Lanes are as follows: lanes 1–4, unlabeled EV71 IRES RNA; lanes 5–8, unlabeled yeast tRNA. C Extracts of RD cells, SH-SY5Y cells, and HA cells were prepared and then incubated without RNA (lane 2), with biotinylated actin RNA (lane 3), non-biotinylated EV71 IRES RNA (lane 4), or biotinylated EV71 IRES RNA (lane 5). After pull-down assay, the bound proteins were eluted, boiled, and subjected to 12% SDS-PAGE. PTB protein was detected by western blot with a rabbit anti-PTB antibody. The inputs were cell extracts of RD, SH-SY5Y and HA (lane 1). D EV71 IRES RNA was pulled down with PTB from EV71-infected T98G cell lysate. T98G cells were infected with EV71 at an MOI of 20 for 6 h and then cell extracts were incubated with rabbit anti-PTB antibody (lanes 2 and 6), normal rabbit IgG (lanes 3 and 7), or without antibody (− Ab) (lanes 4 and 8). Following washing and dissociation, the RNA extract was prepared and subjected to RT-PCR analysis with primers specific for the ribosomal protein S16 (PRS16) RNA (lanes1–4) or for EV71 IRES region RNA (lanes 5–8). Lane 1, cell lysate without immunoprecipitation as a positive RT-PCR control; Lane 2, anti-PTB antibody incubated with 200 mg infected-cell lysate; Lane 3, negative control with rabbit IgG; Lane 4, negative control with no antibody.

    Article Snippet: Biotinylated RNA Pull-Down Assays T98G, HA, RD, and SH-SY5Y cells were pelleted, and then prepared using nuclear protein extraction reagents (Thermo, USA).

    Techniques: Silver Staining, Western Blot, Competitive Binding Assay, Labeling, SDS Page, Incubation, Pull Down Assay, Infection, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation, Negative Control

    Dopamine reduced turnover and solubility of PrP C . Cell surface proteins was biotinylated and chased at time zero (input) or after 1 h incubation in the presence or absence of 50 μM of dopamine. After 1 h, dopamine treated cells showed higher amount of remaining PrP C compared to untreated cells (A) . After dopamine treatment, insoluble proteins in sarkosyl 1% were recovered by ultracentrifugation. Higher amounts of PrP C were recovered in pellet fractions and lower amount in supernatants (SN), indicating lower solubility (B) . Mean band intensity of three independent experiments was plotted with respective CI95. Dark gray bars represent SN and light gray bars represent pellet quantification (B) . * no overlap of CI95 between indicated group and control group (0 μM). rPrP (10 μg) was incubated with designated concentration of dopamine (DA) for 24 h or with 400 μM of dopamine for indicated times (C) . When 400 μM of metabisulfite (#) was added, the oligomerization was prevented (C) . rPrP monomer migrated close to 26 kDa marker. Three independent experiments were performed and representative images are shown.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Dopamine induces the accumulation of insoluble prion protein and affects autophagic flux

    doi: 10.3389/fncel.2015.00012

    Figure Lengend Snippet: Dopamine reduced turnover and solubility of PrP C . Cell surface proteins was biotinylated and chased at time zero (input) or after 1 h incubation in the presence or absence of 50 μM of dopamine. After 1 h, dopamine treated cells showed higher amount of remaining PrP C compared to untreated cells (A) . After dopamine treatment, insoluble proteins in sarkosyl 1% were recovered by ultracentrifugation. Higher amounts of PrP C were recovered in pellet fractions and lower amount in supernatants (SN), indicating lower solubility (B) . Mean band intensity of three independent experiments was plotted with respective CI95. Dark gray bars represent SN and light gray bars represent pellet quantification (B) . * no overlap of CI95 between indicated group and control group (0 μM). rPrP (10 μg) was incubated with designated concentration of dopamine (DA) for 24 h or with 400 μM of dopamine for indicated times (C) . When 400 μM of metabisulfite (#) was added, the oligomerization was prevented (C) . rPrP monomer migrated close to 26 kDa marker. Three independent experiments were performed and representative images are shown.

    Article Snippet: Purification of biotinylated cell surface proteins Cell surface proteins were biotinylated using Pierce Cell Surface Protein Isolation Kit (Thermo Scientific).

    Techniques: Solubility, Incubation, Concentration Assay, Marker

    Anti-tau antibody decreased phospho-tau staining in the hippocampal CA1 cell layer. (A) Representative coronal sections of biotinylated AT8 antibody staining of phosphorylated tau in the hippocampal CA1 cellular region of 9-month-old P301S mice treated for 3 months with vehicle and HJ8.5 at 50 mg/kg. The lower images are higher power views of the CA1 region in the uppers panels. Red arrows indicate the area magnified in the lower image. Black arrows indicate the hippocampal CA1 cell layer. (B) Quantification of biotinylated AT8 antibody staining of abnormally phosphorylated tau revealed a significant decrease in AT8 staining in mice treated with HJ8.5 at 50 mg/kg in the hippocampal CA1 cellular layer compared to vehicle-treated mice ( P = 0.035). HJ8.5 at 10 mg/kg treated mice also showed decreased AT8 staining compared to the vehicle-treated group, but this was not statistically significant ( P > 0.05). Values represent mean ± SEM. * P

    Journal: Annals of Clinical and Translational Neurology

    Article Title: Anti-tau antibody reduces insoluble tau and decreases brain atrophy

    doi: 10.1002/acn3.176

    Figure Lengend Snippet: Anti-tau antibody decreased phospho-tau staining in the hippocampal CA1 cell layer. (A) Representative coronal sections of biotinylated AT8 antibody staining of phosphorylated tau in the hippocampal CA1 cellular region of 9-month-old P301S mice treated for 3 months with vehicle and HJ8.5 at 50 mg/kg. The lower images are higher power views of the CA1 region in the uppers panels. Red arrows indicate the area magnified in the lower image. Black arrows indicate the hippocampal CA1 cell layer. (B) Quantification of biotinylated AT8 antibody staining of abnormally phosphorylated tau revealed a significant decrease in AT8 staining in mice treated with HJ8.5 at 50 mg/kg in the hippocampal CA1 cellular layer compared to vehicle-treated mice ( P = 0.035). HJ8.5 at 10 mg/kg treated mice also showed decreased AT8 staining compared to the vehicle-treated group, but this was not statistically significant ( P > 0.05). Values represent mean ± SEM. * P

    Article Snippet: Mouse monoclonal BT2 antibody, biotinylated mouse monoclonal anti-tau BT-2 antibody, mouse monoclonal anti-human tau-specific biotinylated HT7 antibody, and biotinylated AT8 antibody were purchased from Thermo Scientific (Asheville, NC).

    Techniques: Staining, Mouse Assay