biotinylated proteins Search Results


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  • 88
    RayBiotech biotinylated proteins
    L1000 antibody arrays (composed of L507 on the left and L493 on the right) identify a number of glutathionylated proteins secreted from LPS-treated THP-1 cells. ( a ) Conditioned media from LPS-treated BioGEE-loaded THP-1 cells were applied to the array. Streptavidin-HRP was used to detect the presence of bound <t>biotinylated</t> proteins. Triplicate positive control spots are indicated by arrows. ( b ) A second aliquot of the same sample was reduced with DTT and applied to a second array.
    Biotinylated Proteins, supplied by RayBiotech, used in various techniques. Bioz Stars score: 88/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher biotinylated proteins
    Fig. 4. ( A ) Western blot detection of cell surface-bound E2. <t>Biotinylated</t> HepG2 cells were incubated in the presence (lanes 1 and 3) or in the absence (lanes 2 and 4) of E2 recombinant protein. Bound E2 was cross-linked with DTSSP and the E2–receptor complexes were immunoprecipitated with an antibody against the His tag of the E2 recombinant protein. Samples eluted under both non-reducing (lanes 1 and 2) and reducing (lanes 3 and 4) conditions were loaded onto a 10% SDS–PAGE gel. E2 protein was detected as a monomer under reducing conditions (lane 3) and at a higher molecular weight under non- reducing conditions (lane 1), by using an anti-E2 rat mAb followed by anti-rat HRP-conjugated. ( B ) Immunoblot detection of biotinylated cell surface proteins interacting with E2. The reactivity with HRP conjugated streptavidin revealed a biotinylated protein band at 82 kDa only under reducing conditions (lane 3).
    Biotinylated Proteins, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 5666 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore avidin biotin complex blotting proteins
    Identification of protein S -nitrosylated by SNAP in the spinal cord as actin . A . S2 and P2 fractions of mouse spinal cords were incubated with the indicated concentrations of SNAP for 1 h and subjected to the biotin-switch assay for S -nitrosylation of proteins. Samples (5 μg of each) were resolved by non-reducing SDS-PAGE and immunobloted with anti-biotin antibody. B . The S2 fraction was incubated with 100 μM SNAP for 1 h and subjected to the biotin-switch assay. The <t>biotinylated</t> proteins were purified by using a streptavidin-agarose gel, and the bound proteins were eluted by SDS-sample buffer containing 2-mercaptoethanol. Eluates were analyzed by SDS-PAGE, and proteins were visualized by silver staining. Three major bands were identified as β-tubulin, β- and γ-actin, and glyceraldehyde-3-phosphate dehydorogenase (GAPDH). C . MALDI-TOF MS spectrum used for identification of actin. Twelve matched mass peaks are indicated. D . Amino acid sequence of mouse γ-actin. The parts of the sequence in red correspond to the peptide fragments obtained by tryptic digestion. The amino acid sequence of β-actin completely matches residues 20-351 of γ-actin except that P at the 31st residue of γ-actin was replaced by S at the 12th residue of β-actin. E . S -Nitrosylation of actin by SNAP in the S2 fraction, but not in the P2 fraction, of the spinal cord. S2 and P2 fractions of spinal cords were incubated with 100 μM SNAP for 1 h and subjected to the biotin-switch assay. After removing free biotin-HPDP, S -nitrosylated proteins were purified on streptavidin-agarose gels. Eluates were immunoblotted with anti-actin antibody as described in
    Avidin Biotin Complex Blotting Proteins, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 88 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Bio-Rad biotinylated marker proteins
    Identification of protein S -nitrosylated by SNAP in the spinal cord as actin . A . S2 and P2 fractions of mouse spinal cords were incubated with the indicated concentrations of SNAP for 1 h and subjected to the biotin-switch assay for S -nitrosylation of proteins. Samples (5 μg of each) were resolved by non-reducing SDS-PAGE and immunobloted with anti-biotin antibody. B . The S2 fraction was incubated with 100 μM SNAP for 1 h and subjected to the biotin-switch assay. The <t>biotinylated</t> proteins were purified by using a streptavidin-agarose gel, and the bound proteins were eluted by SDS-sample buffer containing 2-mercaptoethanol. Eluates were analyzed by SDS-PAGE, and proteins were visualized by silver staining. Three major bands were identified as β-tubulin, β- and γ-actin, and glyceraldehyde-3-phosphate dehydorogenase (GAPDH). C . MALDI-TOF MS spectrum used for identification of actin. Twelve matched mass peaks are indicated. D . Amino acid sequence of mouse γ-actin. The parts of the sequence in red correspond to the peptide fragments obtained by tryptic digestion. The amino acid sequence of β-actin completely matches residues 20-351 of γ-actin except that P at the 31st residue of γ-actin was replaced by S at the 12th residue of β-actin. E . S -Nitrosylation of actin by SNAP in the S2 fraction, but not in the P2 fraction, of the spinal cord. S2 and P2 fractions of spinal cords were incubated with 100 μM SNAP for 1 h and subjected to the biotin-switch assay. After removing free biotin-HPDP, S -nitrosylated proteins were purified on streptavidin-agarose gels. Eluates were immunoblotted with anti-actin antibody as described in
    Biotinylated Marker Proteins, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 91/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Cell Signaling Technology Inc biotinylated proteins
    Identification of protein S -nitrosylated by SNAP in the spinal cord as actin . A . S2 and P2 fractions of mouse spinal cords were incubated with the indicated concentrations of SNAP for 1 h and subjected to the biotin-switch assay for S -nitrosylation of proteins. Samples (5 μg of each) were resolved by non-reducing SDS-PAGE and immunobloted with anti-biotin antibody. B . The S2 fraction was incubated with 100 μM SNAP for 1 h and subjected to the biotin-switch assay. The <t>biotinylated</t> proteins were purified by using a streptavidin-agarose gel, and the bound proteins were eluted by SDS-sample buffer containing 2-mercaptoethanol. Eluates were analyzed by SDS-PAGE, and proteins were visualized by silver staining. Three major bands were identified as β-tubulin, β- and γ-actin, and glyceraldehyde-3-phosphate dehydorogenase (GAPDH). C . MALDI-TOF MS spectrum used for identification of actin. Twelve matched mass peaks are indicated. D . Amino acid sequence of mouse γ-actin. The parts of the sequence in red correspond to the peptide fragments obtained by tryptic digestion. The amino acid sequence of β-actin completely matches residues 20-351 of γ-actin except that P at the 31st residue of γ-actin was replaced by S at the 12th residue of β-actin. E . S -Nitrosylation of actin by SNAP in the S2 fraction, but not in the P2 fraction, of the spinal cord. S2 and P2 fractions of spinal cords were incubated with 100 μM SNAP for 1 h and subjected to the biotin-switch assay. After removing free biotin-HPDP, S -nitrosylated proteins were purified on streptavidin-agarose gels. Eluates were immunoblotted with anti-actin antibody as described in
    Biotinylated Proteins, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 83 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher pgp 9 5 antisera
    Characterization of neural cells in mouse striatal cultures (A-G). (A) Phase contrast photomicrograph of striatal cells at 5-7 days  in vitro . A majority of the cells are neurons; scale bar = 20 μm. (B) Astrocytes were characterized by large nuclei with multiple nucleoli and dispersed heterochromatin (Hoechst 33342 blue immunofluorescence) and glial fibrillary acidic (GFAP) immunofluorescence (Cy3 red fluorochrome); scale bar = 25 μm. (C-E) Subpopulations of striatal neurons possessed μ (C), κ (E), and to a lesser extent δ (not shown), opioid receptor immunoreactivity. D    E show phase contrast and κ opioid receptor immunofluorescent images of the same cells; scale bar = 20 μm. (F-G) Triple-label-identification of neurons, astrocytes and microglia in striatal cultures. Neurons were identified using anti-PGP 9.5 indirect immunofluorescence (Alexa 488, green product), while astrocytes were labeled using anti-GFAP indirect immunofluorescence (Alexa 546, red product) and microglia were detected using IB 4  biotinylated isolectin-conjugated (via avidin) to Alexa 350 (blue fluorescent product) (arrow) (F). A majority of the cells were neurons; microglia comprised 0.7% of the total cells. (G) A phase-contrast photomicrograph of the same cells as in Fig. 1F, some of which were non-viable/degenerating (hatched arrows); PGP 9.5 immunoreactivity is retained by many degenerating neurons; scale bar = 20 μm.
    Pgp 9 5 Antisera, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    R&D Systems biotinylated ephrin fc proteins
    Binding interactions of <t>ephrin</t> Fc fusion proteins with Eph receptors. Apparent dissociation constant (K D ) values were obtained from curves measuring the binding of <t>biotinylated</t> ephrin Fc proteins to Eph receptor Fc proteins immobilized on ELISA plates.
    Biotinylated Ephrin Fc Proteins, supplied by R&D Systems, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    BioLegend avidin biotin blocking system
    Binding interactions of <t>ephrin</t> Fc fusion proteins with Eph receptors. Apparent dissociation constant (K D ) values were obtained from curves measuring the binding of <t>biotinylated</t> ephrin Fc proteins to Eph receptor Fc proteins immobilized on ELISA plates.
    Avidin Biotin Blocking System, supplied by BioLegend, used in various techniques. Bioz Stars score: 95/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    L1000 antibody arrays (composed of L507 on the left and L493 on the right) identify a number of glutathionylated proteins secreted from LPS-treated THP-1 cells. ( a ) Conditioned media from LPS-treated BioGEE-loaded THP-1 cells were applied to the array. Streptavidin-HRP was used to detect the presence of bound biotinylated proteins. Triplicate positive control spots are indicated by arrows. ( b ) A second aliquot of the same sample was reduced with DTT and applied to a second array.

    Journal: Scientific Reports

    Article Title: Development of ‘Redox Arrays’ for identifying novel glutathionylated proteins in the secretome

    doi: 10.1038/srep14630

    Figure Lengend Snippet: L1000 antibody arrays (composed of L507 on the left and L493 on the right) identify a number of glutathionylated proteins secreted from LPS-treated THP-1 cells. ( a ) Conditioned media from LPS-treated BioGEE-loaded THP-1 cells were applied to the array. Streptavidin-HRP was used to detect the presence of bound biotinylated proteins. Triplicate positive control spots are indicated by arrows. ( b ) A second aliquot of the same sample was reduced with DTT and applied to a second array.

    Article Snippet: After washing, biotinylated proteins were detected according to the manufacturer’s instructions (Ray Biotech) using Streptavidin-HRP and ECL reagent.

    Techniques: Positive Control

    Fig. 4. ( A ) Western blot detection of cell surface-bound E2. Biotinylated HepG2 cells were incubated in the presence (lanes 1 and 3) or in the absence (lanes 2 and 4) of E2 recombinant protein. Bound E2 was cross-linked with DTSSP and the E2–receptor complexes were immunoprecipitated with an antibody against the His tag of the E2 recombinant protein. Samples eluted under both non-reducing (lanes 1 and 2) and reducing (lanes 3 and 4) conditions were loaded onto a 10% SDS–PAGE gel. E2 protein was detected as a monomer under reducing conditions (lane 3) and at a higher molecular weight under non- reducing conditions (lane 1), by using an anti-E2 rat mAb followed by anti-rat HRP-conjugated. ( B ) Immunoblot detection of biotinylated cell surface proteins interacting with E2. The reactivity with HRP conjugated streptavidin revealed a biotinylated protein band at 82 kDa only under reducing conditions (lane 3).

    Journal: The EMBO Journal

    Article Title: The human scavenger receptor class B type I is a novel candidate receptor for the hepatitis C virus

    doi: 10.1093/emboj/cdf529

    Figure Lengend Snippet: Fig. 4. ( A ) Western blot detection of cell surface-bound E2. Biotinylated HepG2 cells were incubated in the presence (lanes 1 and 3) or in the absence (lanes 2 and 4) of E2 recombinant protein. Bound E2 was cross-linked with DTSSP and the E2–receptor complexes were immunoprecipitated with an antibody against the His tag of the E2 recombinant protein. Samples eluted under both non-reducing (lanes 1 and 2) and reducing (lanes 3 and 4) conditions were loaded onto a 10% SDS–PAGE gel. E2 protein was detected as a monomer under reducing conditions (lane 3) and at a higher molecular weight under non- reducing conditions (lane 1), by using an anti-E2 rat mAb followed by anti-rat HRP-conjugated. ( B ) Immunoblot detection of biotinylated cell surface proteins interacting with E2. The reactivity with HRP conjugated streptavidin revealed a biotinylated protein band at 82 kDa only under reducing conditions (lane 3).

    Article Snippet: Samples obtained by boiling the beads were loaded on to an SDS–PAGE gel and analyzed by western blot for detection of biotinylated proteins using horseradish peroxidase (HRP)-conjugated streptavidin (Pierce) diluted 1:25 000 in Tris-buffered saline 0.05% Tween 20 (TBST), 2% bovine serum albumin (BSA).

    Techniques: Western Blot, Incubation, Recombinant, Immunoprecipitation, SDS Page, Molecular Weight

    Identification of protein S -nitrosylated by SNAP in the spinal cord as actin . A . S2 and P2 fractions of mouse spinal cords were incubated with the indicated concentrations of SNAP for 1 h and subjected to the biotin-switch assay for S -nitrosylation of proteins. Samples (5 μg of each) were resolved by non-reducing SDS-PAGE and immunobloted with anti-biotin antibody. B . The S2 fraction was incubated with 100 μM SNAP for 1 h and subjected to the biotin-switch assay. The biotinylated proteins were purified by using a streptavidin-agarose gel, and the bound proteins were eluted by SDS-sample buffer containing 2-mercaptoethanol. Eluates were analyzed by SDS-PAGE, and proteins were visualized by silver staining. Three major bands were identified as β-tubulin, β- and γ-actin, and glyceraldehyde-3-phosphate dehydorogenase (GAPDH). C . MALDI-TOF MS spectrum used for identification of actin. Twelve matched mass peaks are indicated. D . Amino acid sequence of mouse γ-actin. The parts of the sequence in red correspond to the peptide fragments obtained by tryptic digestion. The amino acid sequence of β-actin completely matches residues 20-351 of γ-actin except that P at the 31st residue of γ-actin was replaced by S at the 12th residue of β-actin. E . S -Nitrosylation of actin by SNAP in the S2 fraction, but not in the P2 fraction, of the spinal cord. S2 and P2 fractions of spinal cords were incubated with 100 μM SNAP for 1 h and subjected to the biotin-switch assay. After removing free biotin-HPDP, S -nitrosylated proteins were purified on streptavidin-agarose gels. Eluates were immunoblotted with anti-actin antibody as described in

    Journal: Molecular Pain

    Article Title: Involvement of S-nitrosylation of actin in inhibition of neurotransmitter release by nitric oxide

    doi: 10.1186/1744-8069-5-58

    Figure Lengend Snippet: Identification of protein S -nitrosylated by SNAP in the spinal cord as actin . A . S2 and P2 fractions of mouse spinal cords were incubated with the indicated concentrations of SNAP for 1 h and subjected to the biotin-switch assay for S -nitrosylation of proteins. Samples (5 μg of each) were resolved by non-reducing SDS-PAGE and immunobloted with anti-biotin antibody. B . The S2 fraction was incubated with 100 μM SNAP for 1 h and subjected to the biotin-switch assay. The biotinylated proteins were purified by using a streptavidin-agarose gel, and the bound proteins were eluted by SDS-sample buffer containing 2-mercaptoethanol. Eluates were analyzed by SDS-PAGE, and proteins were visualized by silver staining. Three major bands were identified as β-tubulin, β- and γ-actin, and glyceraldehyde-3-phosphate dehydorogenase (GAPDH). C . MALDI-TOF MS spectrum used for identification of actin. Twelve matched mass peaks are indicated. D . Amino acid sequence of mouse γ-actin. The parts of the sequence in red correspond to the peptide fragments obtained by tryptic digestion. The amino acid sequence of β-actin completely matches residues 20-351 of γ-actin except that P at the 31st residue of γ-actin was replaced by S at the 12th residue of β-actin. E . S -Nitrosylation of actin by SNAP in the S2 fraction, but not in the P2 fraction, of the spinal cord. S2 and P2 fractions of spinal cords were incubated with 100 μM SNAP for 1 h and subjected to the biotin-switch assay. After removing free biotin-HPDP, S -nitrosylated proteins were purified on streptavidin-agarose gels. Eluates were immunoblotted with anti-actin antibody as described in "Methods."

    Article Snippet: Biotinylated proteins were resolved by non-reducing SDS-polyacrylamide gel electrophoresis (PAGE), and transferred to a polyvinylidene difluoride membrane, followed by immunoblotting with peroxidase-conjugated anti-biotin antibody (1:1000; Sigma-Aldrich).

    Techniques: Incubation, Biotin Switch Assay, SDS Page, Purification, Agarose Gel Electrophoresis, Silver Staining, Mass Spectrometry, Sequencing

    C362S/C363S mutation abolishes YO-PRO-1 uptake of pdP2X7 in HEK293 cells. ( A ) YO-PRO-1 uptake of full length and the C362S/C363S mutant. Representative trace of eight independent experiments is shown. ( B ) Surface expression of the wildtype and the C362S/C363S pdP2X7 mutant. FLAG tag was attached to the N-terminus and expressed in HEK293 cells. Surface expressed protein was biotinylated and cells were lysed. Biotinylated protein was collected by streptactin resin. Total protein sample after solubilization (total) and eluted sample from resin (biotinylated) were applied to SDS-PAGE and immunoblotting was performed using FLAG antibody.

    Journal: eLife

    Article Title: The P2X7 receptor forms a dye-permeable pore independent of its intracellular domain but dependent on membrane lipid composition

    doi: 10.7554/eLife.31186

    Figure Lengend Snippet: C362S/C363S mutation abolishes YO-PRO-1 uptake of pdP2X7 in HEK293 cells. ( A ) YO-PRO-1 uptake of full length and the C362S/C363S mutant. Representative trace of eight independent experiments is shown. ( B ) Surface expression of the wildtype and the C362S/C363S pdP2X7 mutant. FLAG tag was attached to the N-terminus and expressed in HEK293 cells. Surface expressed protein was biotinylated and cells were lysed. Biotinylated protein was collected by streptactin resin. Total protein sample after solubilization (total) and eluted sample from resin (biotinylated) were applied to SDS-PAGE and immunoblotting was performed using FLAG antibody.

    Article Snippet: The cell lysate prior to Strep-Tactin affinity purification (total) and the pulled down proteins (biotinylated) were applied to 12% SDS-PAGE, transferred onto a nitrocellulose membrane, detected with monoclonal anti-FLAG antibody (1:1,000; Sigma-Aldrich), labeled with an anti-mouse AP-conjugate secondary antibody (1:1,000; Bio-Rad Laboratories, Hercules, CA), and developed using colorimetric AP substrate (Bio-Rad Laboratories).

    Techniques: Mutagenesis, Expressing, FLAG-tag, SDS Page

    The plasma membrane-resident Na,K-ATPase subunits are degraded at a similar rate. A , cell monolayers of non-transfected, YFP-β 1 -expressing, and YFP-β 2 -expressing MDCK cells were biotinylated from the basolateral side using a membrane-impermeable biotinylation reagent and then were chased at 37 °C for the indicated time periods. After cell lysis, the biotinylated proteins were isolated and analyzed by SDS-PAGE followed by immunoblotting using the antibodies against the α 1 and β 1 subunits of the Na,K-ATPase and against YFP. B , densitometry of the results presented in A. Error bars , ±S.D. ( n = 3); PM , plasma membrane; Na,K -α 1 and Na,K- β 1 , the endogenous α 1 and β 1 subunits of the Na,K-ATPase; Non-tr ., non-transfected MDCK cells.

    Journal: The Journal of Biological Chemistry

    Article Title: Diverse Pathways for Maturation of the Na,K-ATPase β1 and β2 Subunits in the Endoplasmic Reticulum of Madin-Darby Canine Kidney Cells

    doi: 10.1074/jbc.M110.172858

    Figure Lengend Snippet: The plasma membrane-resident Na,K-ATPase subunits are degraded at a similar rate. A , cell monolayers of non-transfected, YFP-β 1 -expressing, and YFP-β 2 -expressing MDCK cells were biotinylated from the basolateral side using a membrane-impermeable biotinylation reagent and then were chased at 37 °C for the indicated time periods. After cell lysis, the biotinylated proteins were isolated and analyzed by SDS-PAGE followed by immunoblotting using the antibodies against the α 1 and β 1 subunits of the Na,K-ATPase and against YFP. B , densitometry of the results presented in A. Error bars , ±S.D. ( n = 3); PM , plasma membrane; Na,K -α 1 and Na,K- β 1 , the endogenous α 1 and β 1 subunits of the Na,K-ATPase; Non-tr ., non-transfected MDCK cells.

    Article Snippet: To isolate surface-biotinylated proteins, the cell extract was incubated with 100 μl of streptavidin-agarose beads (Sigma-Aldrich) in a total volume of 1 ml of 0.15 m NaCl in 15 m m Tris, pH 8.0, with 0.5% Triton X-100 and 4 m m EGTA at 4 °C with continuous rotation for 60 min.

    Techniques: Transfection, Expressing, Lysis, Isolation, SDS Page

    Biotinylation of N. meningitidis proteome and confirmation of the presence of biotinylated proteins bound to hBMECs. (A) Lane 1–Protein extract of meningococci prior to biotinylation separated on SDS-PAGE. Lane 2–Biotinylated proteins were incubated with NeutrAvidin capture beads, eluted with 50 mM DTT and separated on SDS-PAGE. (B) Dot blot. S2–Protein extract of hBMECs obtained after incubation with biotinylated proteins of meningococci spotted on the membrane and detected with IRdye®;800 Streptavidin. Negative control–Total protein extract of hBMECs was spotted on the membrane and incubated with IRdye®;800 Streptavidin, Input control–Biotinylated proteins of meningococci were spotted on the membrane and detected with IRdye®;800 Streptavidin.

    Journal: Frontiers in Microbiology

    Article Title: Deciphering the Interactome of Neisseria meningitidis With Human Brain Microvascular Endothelial Cells

    doi: 10.3389/fmicb.2018.02294

    Figure Lengend Snippet: Biotinylation of N. meningitidis proteome and confirmation of the presence of biotinylated proteins bound to hBMECs. (A) Lane 1–Protein extract of meningococci prior to biotinylation separated on SDS-PAGE. Lane 2–Biotinylated proteins were incubated with NeutrAvidin capture beads, eluted with 50 mM DTT and separated on SDS-PAGE. (B) Dot blot. S2–Protein extract of hBMECs obtained after incubation with biotinylated proteins of meningococci spotted on the membrane and detected with IRdye®;800 Streptavidin. Negative control–Total protein extract of hBMECs was spotted on the membrane and incubated with IRdye®;800 Streptavidin, Input control–Biotinylated proteins of meningococci were spotted on the membrane and detected with IRdye®;800 Streptavidin.

    Article Snippet: Cells were washed four times with Dulbecco's-PBS (Sigma) to remove unbound biotinylated proteins.

    Techniques: SDS Page, Incubation, Dot Blot, Negative Control

    Schematic representation of experimental and bioinformatic workflow performed in this study. Proteins of N. meningitidis were isolated 1 and biotinylated 2 . Biotinylated proteome was incubated with human brain microvascular endothelial cells (hBMECs) 3A . Unbound proteins were washed off 3Band3C and interacting proteins bound to hBMECs were recovered from cell lyste 3D with NeutrAvidin capture beads 4aand4b , eluted with 50 mM DTT 4c and identified by SWATH-MS 5 . Combination of bioinformatic tools was used for analyzing the interactome of N. meningitidis 6 . Surface proteins were grouped for selection of potential ligands. First analysis 6−I (Psortb and Cello) segregated proteins according to subcellular location and intracellular proteins were removed. With the second set of analytic tools 6−II , interactome was classified into the outer (group 1), inner (group 2) and secretory (group 3) proteins by applying algorithms to predict transmembrane helices (TMHMM and HMMtop) and type I and II signal peptides (SignalP and LipoP). Gene ontology (Blast2GO), UniProt and literature review 6−III were performed further on proteins grouped above in 6-II. Bioinformatic pipeline enabled us to select most probable protein candidates that may interact with hBMECs and contribute in pathogenesis 7 . Those protein candidates were overexpressed in E.coli 8 and used for validation with ELISA and immunocytochemistry 9 .

    Journal: Frontiers in Microbiology

    Article Title: Deciphering the Interactome of Neisseria meningitidis With Human Brain Microvascular Endothelial Cells

    doi: 10.3389/fmicb.2018.02294

    Figure Lengend Snippet: Schematic representation of experimental and bioinformatic workflow performed in this study. Proteins of N. meningitidis were isolated 1 and biotinylated 2 . Biotinylated proteome was incubated with human brain microvascular endothelial cells (hBMECs) 3A . Unbound proteins were washed off 3Band3C and interacting proteins bound to hBMECs were recovered from cell lyste 3D with NeutrAvidin capture beads 4aand4b , eluted with 50 mM DTT 4c and identified by SWATH-MS 5 . Combination of bioinformatic tools was used for analyzing the interactome of N. meningitidis 6 . Surface proteins were grouped for selection of potential ligands. First analysis 6−I (Psortb and Cello) segregated proteins according to subcellular location and intracellular proteins were removed. With the second set of analytic tools 6−II , interactome was classified into the outer (group 1), inner (group 2) and secretory (group 3) proteins by applying algorithms to predict transmembrane helices (TMHMM and HMMtop) and type I and II signal peptides (SignalP and LipoP). Gene ontology (Blast2GO), UniProt and literature review 6−III were performed further on proteins grouped above in 6-II. Bioinformatic pipeline enabled us to select most probable protein candidates that may interact with hBMECs and contribute in pathogenesis 7 . Those protein candidates were overexpressed in E.coli 8 and used for validation with ELISA and immunocytochemistry 9 .

    Article Snippet: Cells were washed four times with Dulbecco's-PBS (Sigma) to remove unbound biotinylated proteins.

    Techniques: Isolation, Incubation, Mass Spectrometry, Selection, Enzyme-linked Immunosorbent Assay, Immunocytochemistry

    Surface plasmon resonance confirms the P . vivax MSP3.10-MSP7.1 interaction. (A) Recombinant, his-tagged P . vivax MSP7.1 eluted as a main peak with a small shoulder at higher masses than expected after SEC, likely due to oligomerization, and with a main band of the expected size by SDS-PAGE (inset). (B) Increasing concentrations of P . vivax MSP7.1 were injected over immobilized biotinylated P . vivax MSP3.10. Relatively high-affinity binding was observed, although none of the concentrations used reached equilibrium (inset). The binding did not fit a 1:1 model (red dashed line). The increase in response units at the start of the dissociation phase at the higher analyte concentrations of P . vivax MSP7.1 is likely an artefactual buffer effect. SEC, size-exclusion chromatography.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: A Library of Plasmodium vivax Recombinant Merozoite Proteins Reveals New Vaccine Candidates and Protein-Protein Interactions

    doi: 10.1371/journal.pntd.0004264

    Figure Lengend Snippet: Surface plasmon resonance confirms the P . vivax MSP3.10-MSP7.1 interaction. (A) Recombinant, his-tagged P . vivax MSP7.1 eluted as a main peak with a small shoulder at higher masses than expected after SEC, likely due to oligomerization, and with a main band of the expected size by SDS-PAGE (inset). (B) Increasing concentrations of P . vivax MSP7.1 were injected over immobilized biotinylated P . vivax MSP3.10. Relatively high-affinity binding was observed, although none of the concentrations used reached equilibrium (inset). The binding did not fit a 1:1 model (red dashed line). The increase in response units at the start of the dissociation phase at the higher analyte concentrations of P . vivax MSP7.1 is likely an artefactual buffer effect. SEC, size-exclusion chromatography.

    Article Snippet: Biotin binding sites on streptavidin-coated plates (Thermo Scientific Nunc) were saturated with biotinylated bait proteins for 1 h. Plates were then washed 3 times in HBS/0.1% Tween, probed with beta-lactamase-tagged prey proteins for 1 h, washed twice with HBS/0.1% Tween, washed once with HBS, and incubated with nitrocefin (60 μl at 125 μg/ml; Calbiochem, USA).

    Techniques: SPR Assay, Recombinant, Size-exclusion Chromatography, SDS Page, Injection, Binding Assay

    Multiple P . vivax recombinant proteins are immunoreactive and contain conformational epitopes. The immunoreactivity of 34 biotinylated P . vivax recombinant proteins was assessed using diluted (1:600) plasma pools from 14 Cambodian vivax malaria patients (blue bars) and five American malaria-naïve individuals (green bars). The immunoreactivity of heat-treated proteins was assessed in parallel using the Cambodian plasma pool (red bars); reduced responses indicate the presence of heat-labile conformational epitopes. The immunoreactivity of highly reactive proteins (*) was assessed using more-diluted (1:1000) plasma pools. Absorbance (A) at 405 nm was measured at various times, but only the mean value nearest to 1.0 for each antigen is shown. Negative control (–ve) was rat Cd4d3+d4 tag. Bar charts show mean ± SD; n = 3.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: A Library of Plasmodium vivax Recombinant Merozoite Proteins Reveals New Vaccine Candidates and Protein-Protein Interactions

    doi: 10.1371/journal.pntd.0004264

    Figure Lengend Snippet: Multiple P . vivax recombinant proteins are immunoreactive and contain conformational epitopes. The immunoreactivity of 34 biotinylated P . vivax recombinant proteins was assessed using diluted (1:600) plasma pools from 14 Cambodian vivax malaria patients (blue bars) and five American malaria-naïve individuals (green bars). The immunoreactivity of heat-treated proteins was assessed in parallel using the Cambodian plasma pool (red bars); reduced responses indicate the presence of heat-labile conformational epitopes. The immunoreactivity of highly reactive proteins (*) was assessed using more-diluted (1:1000) plasma pools. Absorbance (A) at 405 nm was measured at various times, but only the mean value nearest to 1.0 for each antigen is shown. Negative control (–ve) was rat Cd4d3+d4 tag. Bar charts show mean ± SD; n = 3.

    Article Snippet: Biotin binding sites on streptavidin-coated plates (Thermo Scientific Nunc) were saturated with biotinylated bait proteins for 1 h. Plates were then washed 3 times in HBS/0.1% Tween, probed with beta-lactamase-tagged prey proteins for 1 h, washed twice with HBS/0.1% Tween, washed once with HBS, and incubated with nitrocefin (60 μl at 125 μg/ml; Calbiochem, USA).

    Techniques: Recombinant, Negative Control

    Western blot analysis confirms expression of 34/37 P . vivax recombinant proteins. Biotinylated proteins were resolved by SDS-PAGE under reducing conditions, blotted, and probed using streptavidin-HRP. All proteins contain a ~25-kDa rat Cd4d3+4 tag. (*) indicates proteins that were run with the right ladder; all others were run with the left or both ladders.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: A Library of Plasmodium vivax Recombinant Merozoite Proteins Reveals New Vaccine Candidates and Protein-Protein Interactions

    doi: 10.1371/journal.pntd.0004264

    Figure Lengend Snippet: Western blot analysis confirms expression of 34/37 P . vivax recombinant proteins. Biotinylated proteins were resolved by SDS-PAGE under reducing conditions, blotted, and probed using streptavidin-HRP. All proteins contain a ~25-kDa rat Cd4d3+4 tag. (*) indicates proteins that were run with the right ladder; all others were run with the left or both ladders.

    Article Snippet: Biotin binding sites on streptavidin-coated plates (Thermo Scientific Nunc) were saturated with biotinylated bait proteins for 1 h. Plates were then washed 3 times in HBS/0.1% Tween, probed with beta-lactamase-tagged prey proteins for 1 h, washed twice with HBS/0.1% Tween, washed once with HBS, and incubated with nitrocefin (60 μl at 125 μg/ml; Calbiochem, USA).

    Techniques: Western Blot, Expressing, Recombinant, SDS Page

    Quantification of the P . vivax P12-P41 interaction affinity by surface plasmon resonance. (A) Recombinant, his-tagged P . vivax P12 and P . vivax P41, each eluted as a monodisperse peak after SEC which resolved as a single band of the expected size by SDS-PAGE (insets). (B, C, D) Increasing concentrations of analyte protein were injected over immobilized biotinylated ligand protein. Reference-subtracted binding data were plotted as a binding curve and the equilibrium dissociation constant was calculated using R eq = C R max /(C+ K D ). Experiments included analyte-ligand combinations P . vivax P12-P41 (B), P . vivax P41-P12 (C), and P . vivax P12- P . falciparum P41 (D). Lower concentrations failed to reach equilibrium, which resulted in an overestimated K D . SEC, size-exclusion chromatography.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: A Library of Plasmodium vivax Recombinant Merozoite Proteins Reveals New Vaccine Candidates and Protein-Protein Interactions

    doi: 10.1371/journal.pntd.0004264

    Figure Lengend Snippet: Quantification of the P . vivax P12-P41 interaction affinity by surface plasmon resonance. (A) Recombinant, his-tagged P . vivax P12 and P . vivax P41, each eluted as a monodisperse peak after SEC which resolved as a single band of the expected size by SDS-PAGE (insets). (B, C, D) Increasing concentrations of analyte protein were injected over immobilized biotinylated ligand protein. Reference-subtracted binding data were plotted as a binding curve and the equilibrium dissociation constant was calculated using R eq = C R max /(C+ K D ). Experiments included analyte-ligand combinations P . vivax P12-P41 (B), P . vivax P41-P12 (C), and P . vivax P12- P . falciparum P41 (D). Lower concentrations failed to reach equilibrium, which resulted in an overestimated K D . SEC, size-exclusion chromatography.

    Article Snippet: Biotin binding sites on streptavidin-coated plates (Thermo Scientific Nunc) were saturated with biotinylated bait proteins for 1 h. Plates were then washed 3 times in HBS/0.1% Tween, probed with beta-lactamase-tagged prey proteins for 1 h, washed twice with HBS/0.1% Tween, washed once with HBS, and incubated with nitrocefin (60 μl at 125 μg/ml; Calbiochem, USA).

    Techniques: SPR Assay, Recombinant, Size-exclusion Chromatography, SDS Page, Injection, Binding Assay

    Thr/Val and Ala/Leu mutations reduce cell surface expression of GluK2 and GluK3 subunit proteins. a Transiently transfected HEK293 cells expressing wild type (GluK2-WT, GluK3-WT) or mutant subunits with impaired ligand binding sites (GluK2-T659V, GluK2-A487L, GluK3-T661V; red) or conversion mutants with functional ligand binding sites (GluK2-S689N/N690S, GluK2-A487T, GluK3-N691S; blue) were surface biotinylated 24 h after transfection. Following the solubilisation of membranes (total; T), biotin-labelled (cell surface exposed; S) proteins were separated from non-biotinylated (intracellular; I) proteins using streptavidin coated beads. The different subunit populations were analysed using immunoblotting with a GluK2/3 specific antibody. The β-actin content of the samples was analysed to confirm that biotinylation only labelled cell surface exposed proteins in transfected HEK293 cells. Due to differences in total expression levels of various KAR subunit mutants (Fig. 4 ), immunodetection of bands were optimised for the quantitative comparison of biotinylated and non-biotinylated (intracellular) bands for each construct. b Bar diagrams compare the biotinylated (surface) fractions of subunits expressed as % of total. Mutants with impaired LBDs (GluK2-T659V, GluK2-A487L and GluK3-T661V; red bars) show reduced cell surface expression (biotinylation) than the corresponding WT subunits (white bars). In contrast, LBD conversion mutants of GluK2 and GluK3 that retain ligand binding (GluK2-S689N/N690S, GluK2-A487T, GluK3-N691S; blue bars) show no significant change in cell-surface biotinylation compare to WT. Data are mean ± SEM ( n = 3), * p

    Journal: Neurochemical Research

    Article Title: Assembly and Trafficking of Homomeric and Heteromeric Kainate Receptors with Impaired Ligand Binding Sites

    doi: 10.1007/s11064-018-2654-0

    Figure Lengend Snippet: Thr/Val and Ala/Leu mutations reduce cell surface expression of GluK2 and GluK3 subunit proteins. a Transiently transfected HEK293 cells expressing wild type (GluK2-WT, GluK3-WT) or mutant subunits with impaired ligand binding sites (GluK2-T659V, GluK2-A487L, GluK3-T661V; red) or conversion mutants with functional ligand binding sites (GluK2-S689N/N690S, GluK2-A487T, GluK3-N691S; blue) were surface biotinylated 24 h after transfection. Following the solubilisation of membranes (total; T), biotin-labelled (cell surface exposed; S) proteins were separated from non-biotinylated (intracellular; I) proteins using streptavidin coated beads. The different subunit populations were analysed using immunoblotting with a GluK2/3 specific antibody. The β-actin content of the samples was analysed to confirm that biotinylation only labelled cell surface exposed proteins in transfected HEK293 cells. Due to differences in total expression levels of various KAR subunit mutants (Fig. 4 ), immunodetection of bands were optimised for the quantitative comparison of biotinylated and non-biotinylated (intracellular) bands for each construct. b Bar diagrams compare the biotinylated (surface) fractions of subunits expressed as % of total. Mutants with impaired LBDs (GluK2-T659V, GluK2-A487L and GluK3-T661V; red bars) show reduced cell surface expression (biotinylation) than the corresponding WT subunits (white bars). In contrast, LBD conversion mutants of GluK2 and GluK3 that retain ligand binding (GluK2-S689N/N690S, GluK2-A487T, GluK3-N691S; blue bars) show no significant change in cell-surface biotinylation compare to WT. Data are mean ± SEM ( n = 3), * p

    Article Snippet: To separate biotinylated cell surface and non-biotinylated intracellular proteins, the remaining fraction of lysate was incubated with a 1:1 mixture of streptavidin agarose beads (Sigma-Aldrich Company Ltd., Gillingham, Dorset, UK) for 3 h at 4 °C with mixing by rotation.

    Techniques: Expressing, Transfection, Mutagenesis, Ligand Binding Assay, Functional Assay, Immunodetection, Construct

    GluK5 co-expression restores the cell surface trafficking of GluK2 with Thr/Val or Ala/Leu mutations. a Wild type (WT) and mutant GluK2 subunits (GluK2-T659V, GluK2-A487L, GluK2-S689N/N690S, GluK2-A487T) were co-expressed with the GluK5 WT subunit in transiently transfected HEK293 cells. Following surface biotinylation, labelled proteins (S) were isolated using streptavidin coated beads and analysed using immunoblotting using anti-GluK2/3 and anti-GluK5 antibodies. T is 10% of total protein. The β-actin immunostaining confirmed that biotinylation only labelled cell surface exposed proteins. b Comparison of surface (biotinylated) GluK2 and GluK5 subunits (% of total) indicates no significant differences between the various LBD mutant and WT subunits (Data are mean ± SEM, n = 3, * p

    Journal: Neurochemical Research

    Article Title: Assembly and Trafficking of Homomeric and Heteromeric Kainate Receptors with Impaired Ligand Binding Sites

    doi: 10.1007/s11064-018-2654-0

    Figure Lengend Snippet: GluK5 co-expression restores the cell surface trafficking of GluK2 with Thr/Val or Ala/Leu mutations. a Wild type (WT) and mutant GluK2 subunits (GluK2-T659V, GluK2-A487L, GluK2-S689N/N690S, GluK2-A487T) were co-expressed with the GluK5 WT subunit in transiently transfected HEK293 cells. Following surface biotinylation, labelled proteins (S) were isolated using streptavidin coated beads and analysed using immunoblotting using anti-GluK2/3 and anti-GluK5 antibodies. T is 10% of total protein. The β-actin immunostaining confirmed that biotinylation only labelled cell surface exposed proteins. b Comparison of surface (biotinylated) GluK2 and GluK5 subunits (% of total) indicates no significant differences between the various LBD mutant and WT subunits (Data are mean ± SEM, n = 3, * p

    Article Snippet: To separate biotinylated cell surface and non-biotinylated intracellular proteins, the remaining fraction of lysate was incubated with a 1:1 mixture of streptavidin agarose beads (Sigma-Aldrich Company Ltd., Gillingham, Dorset, UK) for 3 h at 4 °C with mixing by rotation.

    Techniques: Expressing, Mutagenesis, Transfection, Isolation, Immunostaining

    Recombinant EDAvidin tetramerizes and binds to biotinylated proteins. (a) SDS-PAGE of purified recombinant proteins stained with Coomassie blue (1: (MWM: molecular weight marker); 2: denatured EDAvidin; 3: nondenatured EDAvidin). (b) Surface plasmon resonance analysis of the capacity of EDAvidin and streptavidin to bind biotinylated proteins. Biotinylated ovalbumin was coated into the chip, and EDAvidin or streptavidin were injected at different concentrations. The surface of the chip was regenerated by the injection of an excess of 2 μ M biotin before the injection of streptavidin (RU: surface plasmon resonance response units). (c) ELISA-based binding assays of EDAvidin to biotinylated proteins. Biotinylated or nonbiotinylated ovalbumin (OVA) and bovine serum albumin (BSA) were coated into the wells of ELISA plates. EDAvidin or EDA alone was added to the wells and after extensive washes, the plates were developed using rabbit polyclonal anti-EDA antibodies. (d) Binding assay of EDAvidin to biotinylated proteins by SDS-PAGEas 1: EDAvidin in its tetrameric form; 2: biotinylated OVA; 3: EDAvidin plus biotinylated OVA; 5: (MWM).

    Journal: BioMed Research International

    Article Title: A Fusion Protein between Streptavidin and the Endogenous TLR4 Ligand EDA Targets Biotinylated Antigens to Dendritic Cells and Induces T Cell Responses In Vivo

    doi: 10.1155/2013/864720

    Figure Lengend Snippet: Recombinant EDAvidin tetramerizes and binds to biotinylated proteins. (a) SDS-PAGE of purified recombinant proteins stained with Coomassie blue (1: (MWM: molecular weight marker); 2: denatured EDAvidin; 3: nondenatured EDAvidin). (b) Surface plasmon resonance analysis of the capacity of EDAvidin and streptavidin to bind biotinylated proteins. Biotinylated ovalbumin was coated into the chip, and EDAvidin or streptavidin were injected at different concentrations. The surface of the chip was regenerated by the injection of an excess of 2 μ M biotin before the injection of streptavidin (RU: surface plasmon resonance response units). (c) ELISA-based binding assays of EDAvidin to biotinylated proteins. Biotinylated or nonbiotinylated ovalbumin (OVA) and bovine serum albumin (BSA) were coated into the wells of ELISA plates. EDAvidin or EDA alone was added to the wells and after extensive washes, the plates were developed using rabbit polyclonal anti-EDA antibodies. (d) Binding assay of EDAvidin to biotinylated proteins by SDS-PAGEas 1: EDAvidin in its tetrameric form; 2: biotinylated OVA; 3: EDAvidin plus biotinylated OVA; 5: (MWM).

    Article Snippet: ELISA-Based Binding Assays of EDAvidin to Biotinylated Proteins OVA protein (grade III), BSA (Sigma), or the nonstructural NS3 protein from hepatitis C virus [ ] were biotinylated using sulfo-NHS-SS-Biotin (Thermo Scientific) following manufacturer's instruction.

    Techniques: Recombinant, SDS Page, Purification, Staining, Molecular Weight, Marker, SPR Assay, Chromatin Immunoprecipitation, Injection, Enzyme-linked Immunosorbent Assay, Binding Assay

    Western blot analysis of biotinylated cell extracts. A : Control samples are incubated without UDP-GalNAz, or double mutant enzymes or pretreatment with glycosidases. Coupling was performed using BCN-biotin. B : Loading control using Coomasie Blue Stain.

    Journal: Bioconjugate chemistry

    Article Title: Use of novel mutant galactosyltransferase for the bioconjugation of terminal N-Acetylglucosamine (GlcNAc) residues on live cell surface

    doi: 10.1021/bc300542z

    Figure Lengend Snippet: Western blot analysis of biotinylated cell extracts. A : Control samples are incubated without UDP-GalNAz, or double mutant enzymes or pretreatment with glycosidases. Coupling was performed using BCN-biotin. B : Loading control using Coomasie Blue Stain.

    Article Snippet: To purify biotin-labeled proteins, SDS was removed from 10 µg of glycosidase pretreated and BCN-biotinylated HeLa lysate proteins using Calbiosorb resin (Calbiochem) for 15 min on ice.

    Techniques: Western Blot, Incubation, Mutagenesis, Staining

    Endocytic assays demonstrating that LMTK2 knockdown decreased CFTR endocytosis in CFBE41o- cells. CFBE41o- cells stably expressing WT-CFTR were transfected with 10 n m siLMTK2 or the siRNA control ( siCTRL ) and cultured on collagen-coated tissue culture plates for 96 h to form monolayers. We previously showed that siCTRL does not affect CFTR endocytosis under similar cell culture conditions ( 10 ). The plasma membrane proteins were first biotinylated at 4 °C using cell membrane impermeable and cleavable EZ-Link™ Sulfo-NHS-SS-Biotin (Biotin). Cells were rapidly warmed to 37 °C for different periods of time after biotinylation, and, subsequently, the disulfide bonds on Sulfo-NHS-SS-biotinylated proteins remaining at the plasma membrane were reduced by l -glutathione ( GSH ) at 4 °C. At this point in the protocol, biotinylated proteins reside within the endosomal compartment. The amount of biotinylated CFTR at 4 °C and without the 37 °C warming was considered 100%. The amount of biotinylated CFTR remaining after the GSH treatment at 4 °C without warming to 37 °C was considered background and was subtracted from the amount of biotinylated CFTR remaining after warming to 37 °C at each time point. CFTR endocytosis was calculated after subtraction of the background and was expressed as the percent of CFTR remaining biotinylated before and after warming to 37 °C. Immunoblots ( A ) and summary of experiments ( B ) demonstrating that siLMTK2 decreased CFTR endocytosis. Please, note the increased abundance of CFTR before endocytosis ( A , the first lane) in siLMTK2 cells compared with siCTRL consistent with the increased plasma membrane abundance of CFTR at steady-state demonstrates in Fig. 2 . Ezrin expression in WCL was used as a loading control (data not shown). *, p

    Journal: The Journal of Biological Chemistry

    Article Title: LMTK2-mediated Phosphorylation Regulates CFTR Endocytosis in Human Airway Epithelial Cells *

    doi: 10.1074/jbc.M114.563742

    Figure Lengend Snippet: Endocytic assays demonstrating that LMTK2 knockdown decreased CFTR endocytosis in CFBE41o- cells. CFBE41o- cells stably expressing WT-CFTR were transfected with 10 n m siLMTK2 or the siRNA control ( siCTRL ) and cultured on collagen-coated tissue culture plates for 96 h to form monolayers. We previously showed that siCTRL does not affect CFTR endocytosis under similar cell culture conditions ( 10 ). The plasma membrane proteins were first biotinylated at 4 °C using cell membrane impermeable and cleavable EZ-Link™ Sulfo-NHS-SS-Biotin (Biotin). Cells were rapidly warmed to 37 °C for different periods of time after biotinylation, and, subsequently, the disulfide bonds on Sulfo-NHS-SS-biotinylated proteins remaining at the plasma membrane were reduced by l -glutathione ( GSH ) at 4 °C. At this point in the protocol, biotinylated proteins reside within the endosomal compartment. The amount of biotinylated CFTR at 4 °C and without the 37 °C warming was considered 100%. The amount of biotinylated CFTR remaining after the GSH treatment at 4 °C without warming to 37 °C was considered background and was subtracted from the amount of biotinylated CFTR remaining after warming to 37 °C at each time point. CFTR endocytosis was calculated after subtraction of the background and was expressed as the percent of CFTR remaining biotinylated before and after warming to 37 °C. Immunoblots ( A ) and summary of experiments ( B ) demonstrating that siLMTK2 decreased CFTR endocytosis. Please, note the increased abundance of CFTR before endocytosis ( A , the first lane) in siLMTK2 cells compared with siCTRL consistent with the increased plasma membrane abundance of CFTR at steady-state demonstrates in Fig. 2 . Ezrin expression in WCL was used as a loading control (data not shown). *, p

    Article Snippet: Cells were rapidly warmed to 37 °C for different periods of time after biotinylation and, subsequently, the disulfide bonds on Sulfo-NHS-SS-biotinylated proteins remaining at the plasma membrane were reduced by l -glutathione (GSH; Sigma-Aldrich) at 4 °C.

    Techniques: Stable Transfection, Expressing, Transfection, Cell Culture, Western Blot

    Quantification of cytosolic and plasma membrane-bound Hsp70 in CX+/CX− and Colo+/Colo− carcinoma sublines. Western blot analysis of biotinylated whole cell lysates (cytoplasm, upper graph), and plasma membranes (lower graph) of CX+/CX− and Colo+/Colo− tumor sublines. A corresponding Western blot was stained with the Hsp70 specific mAb cmHsp70.1. The figure shows a representative blot from three independent experiments. The Hsp70 surface phenotypes of the tumor sublines, as determined by flow cytometric analysis, are summarized in Table 1 .

    Journal: PLoS ONE

    Article Title: Tumor-Specific Hsp70 Plasma Membrane Localization Is Enabled by the Glycosphingolipid Gb3

    doi: 10.1371/journal.pone.0001925

    Figure Lengend Snippet: Quantification of cytosolic and plasma membrane-bound Hsp70 in CX+/CX− and Colo+/Colo− carcinoma sublines. Western blot analysis of biotinylated whole cell lysates (cytoplasm, upper graph), and plasma membranes (lower graph) of CX+/CX− and Colo+/Colo− tumor sublines. A corresponding Western blot was stained with the Hsp70 specific mAb cmHsp70.1. The figure shows a representative blot from three independent experiments. The Hsp70 surface phenotypes of the tumor sublines, as determined by flow cytometric analysis, are summarized in Table 1 .

    Article Snippet: Biotinylated membrane proteins were eluted from the column with 5 mM biotin in PBS containing 1% v/v Triton-X, concentrated on Centricon YM-3 columns (Millipore) and subjected to SDS-PAGE.

    Techniques: Western Blot, Staining, Flow Cytometry

    Characterization of neural cells in mouse striatal cultures (A-G). (A) Phase contrast photomicrograph of striatal cells at 5-7 days  in vitro . A majority of the cells are neurons; scale bar = 20 μm. (B) Astrocytes were characterized by large nuclei with multiple nucleoli and dispersed heterochromatin (Hoechst 33342 blue immunofluorescence) and glial fibrillary acidic (GFAP) immunofluorescence (Cy3 red fluorochrome); scale bar = 25 μm. (C-E) Subpopulations of striatal neurons possessed μ (C), κ (E), and to a lesser extent δ (not shown), opioid receptor immunoreactivity. D    E show phase contrast and κ opioid receptor immunofluorescent images of the same cells; scale bar = 20 μm. (F-G) Triple-label-identification of neurons, astrocytes and microglia in striatal cultures. Neurons were identified using anti-PGP 9.5 indirect immunofluorescence (Alexa 488, green product), while astrocytes were labeled using anti-GFAP indirect immunofluorescence (Alexa 546, red product) and microglia were detected using IB 4  biotinylated isolectin-conjugated (via avidin) to Alexa 350 (blue fluorescent product) (arrow) (F). A majority of the cells were neurons; microglia comprised 0.7% of the total cells. (G) A phase-contrast photomicrograph of the same cells as in Fig. 1F, some of which were non-viable/degenerating (hatched arrows); PGP 9.5 immunoreactivity is retained by many degenerating neurons; scale bar = 20 μm.

    Journal: Neuroscience

    Article Title: Synergistic Neurotoxicity of Opioids and Human Immunodeficiency Virus-1 Tat Protein in Striatal Neurons In Vitro

    doi:

    Figure Lengend Snippet: Characterization of neural cells in mouse striatal cultures (A-G). (A) Phase contrast photomicrograph of striatal cells at 5-7 days in vitro . A majority of the cells are neurons; scale bar = 20 μm. (B) Astrocytes were characterized by large nuclei with multiple nucleoli and dispersed heterochromatin (Hoechst 33342 blue immunofluorescence) and glial fibrillary acidic (GFAP) immunofluorescence (Cy3 red fluorochrome); scale bar = 25 μm. (C-E) Subpopulations of striatal neurons possessed μ (C), κ (E), and to a lesser extent δ (not shown), opioid receptor immunoreactivity. D E show phase contrast and κ opioid receptor immunofluorescent images of the same cells; scale bar = 20 μm. (F-G) Triple-label-identification of neurons, astrocytes and microglia in striatal cultures. Neurons were identified using anti-PGP 9.5 indirect immunofluorescence (Alexa 488, green product), while astrocytes were labeled using anti-GFAP indirect immunofluorescence (Alexa 546, red product) and microglia were detected using IB 4 biotinylated isolectin-conjugated (via avidin) to Alexa 350 (blue fluorescent product) (arrow) (F). A majority of the cells were neurons; microglia comprised 0.7% of the total cells. (G) A phase-contrast photomicrograph of the same cells as in Fig. 1F, some of which were non-viable/degenerating (hatched arrows); PGP 9.5 immunoreactivity is retained by many degenerating neurons; scale bar = 20 μm.

    Article Snippet: For triple label studies, neurons were detected using rabbit anti-human protein gene product (PGP) 9.5 antisera (1:1200 dilution; Ultraclone, Cambridge, UK) followed by goat anti-rabbit antibodies conjugated to Alexa 488 (Molecular Probes).

    Techniques: In Vitro, Immunofluorescence, Labeling, Avidin-Biotin Assay

    Binding interactions of ephrin Fc fusion proteins with Eph receptors. Apparent dissociation constant (K D ) values were obtained from curves measuring the binding of biotinylated ephrin Fc proteins to Eph receptor Fc proteins immobilized on ELISA plates.

    Journal: Cell Adhesion & Migration

    Article Title: Profiling Eph receptor expression in cells and tissues

    doi: 10.4161/cam.19620

    Figure Lengend Snippet: Binding interactions of ephrin Fc fusion proteins with Eph receptors. Apparent dissociation constant (K D ) values were obtained from curves measuring the binding of biotinylated ephrin Fc proteins to Eph receptor Fc proteins immobilized on ELISA plates.

    Article Snippet: After washing, the wells were incubated for 2 h with different concentrations of biotinylated ephrin Fc proteins (R & D Systems) in TBST (150 mM NaCl, 50 mM TRIS-HCl, pH 7.5 with 0.01% Tween 20) at room temperature, washed, and then incubated for 30 min with 0.5 μg/ml streptavidin conjugated to horseradish peroxidase (HRP) (Thermo Fisher Scientific, #21127) in TBST.

    Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay