tvb 3664  (Thermo Fisher)


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    Name:
    EZ Link NHS LC LC Biotin
    Description:
    Thermo Scientific EZ Link NHS LC LC Biotin is a doubly long chain NHS ester activated biotinylation reagent for labeling primary amines e g protein lysines whose membrane permeability enables it to be used for intracellular labeling Features of EZ Link NHS LC LC Biotin • Protein labeling biotinylate antibodies or other proteins for detection or purification using streptavidin probes or resins • Membrane permeable can be used to label inside cells intracellular • Amine reactive reacts with primary amines NH2 such as the side chain of lysines K or the amino termini of polypeptides • Irreversible forms permanent amide bonds spacer arm cannot be cleaved • Solubility must be dissolved in DMSO or DMF before further dilution in aqueous buffers • Long reach spacer arm total length added to target is 30 5 angstroms it consists of the biotin valeric acid group extended by a 13 atom chain NHS LC LC Biotin is succinimidyl 6 biotinamido 6 hexanamido hexanoate It is the longest of three EZ Link NHS Biotin Reagents that enable simple and efficient biotinylation of antibodies proteins and any other primary amine containing biomolecules in solution Differing only in their spacer arm lengths the three NHS ester reagents offer researchers the possibility of optimizing labeling and detection experiments where steric hindrance of biotin binding is an important factor Because they are uncharged and contain simple alkyl chain spacer arms these biotin compounds are membrane permeable and useful for intracellular labeling We manufacture biotin reagents to ensure the highest possible overall product integrity consistency and performance for the intended research applications N Hydroxysulfosuccinimide NHS esters of biotin are the most popular type of biotinylation reagent NHS activated biotins react efficiently with primary amino groups NH2 in alkaline buffers to form stable amide bonds Proteins e g antibodies typically have several primary amines that are available as targets for labeling including the side chain of lysine K residues and the N terminus of each polypeptide Varieties of biotin NHS ester reagents differ in length solubility cell permeability and cleavability Non sulfonated NHS biotins are cell permeable but must be dissolved in organic solvent such as DMSO or DMF Sulfo NHS biotins and those with pegylated spacers are directly water soluble but not membrane permeable Varieties containing disulfide bonds can be cleaved using reducing agents enabling the biotin group to be disconnected from the labeled protein
    Catalog Number:
    21343
    Price:
    None
    Applications:
    Protein Biology|Protein Labeling|Protein Labeling & Crosslinking
    Category:
    Labeling Detection Products
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    Structured Review

    Thermo Fisher tvb 3664
    TVB-3166 and <t>TVB-3664</t> small molecule FASN inhibitors. (A) Chemical structure of TVB-3166 and TVB-3664. (B) Pharmacokinetic analysis of TVB-3664 in mice. Drug levels were determined from 3 different mice for each time point following administration by oral gavage or intravenous injection. (Bayside Biosciences, San Jose, CA). For oral dosing, TVB-3664 was formulated at 3-V Biosciences in 100% PEG-400 and diluted with water to a final PEG concentration of 30% immediately before dosing. For iv dosing TVB-3664 was formulated Bioanalytical analysis was performed at 3-V Biosciences by mass spectrometry.
    Thermo Scientific EZ Link NHS LC LC Biotin is a doubly long chain NHS ester activated biotinylation reagent for labeling primary amines e g protein lysines whose membrane permeability enables it to be used for intracellular labeling Features of EZ Link NHS LC LC Biotin • Protein labeling biotinylate antibodies or other proteins for detection or purification using streptavidin probes or resins • Membrane permeable can be used to label inside cells intracellular • Amine reactive reacts with primary amines NH2 such as the side chain of lysines K or the amino termini of polypeptides • Irreversible forms permanent amide bonds spacer arm cannot be cleaved • Solubility must be dissolved in DMSO or DMF before further dilution in aqueous buffers • Long reach spacer arm total length added to target is 30 5 angstroms it consists of the biotin valeric acid group extended by a 13 atom chain NHS LC LC Biotin is succinimidyl 6 biotinamido 6 hexanamido hexanoate It is the longest of three EZ Link NHS Biotin Reagents that enable simple and efficient biotinylation of antibodies proteins and any other primary amine containing biomolecules in solution Differing only in their spacer arm lengths the three NHS ester reagents offer researchers the possibility of optimizing labeling and detection experiments where steric hindrance of biotin binding is an important factor Because they are uncharged and contain simple alkyl chain spacer arms these biotin compounds are membrane permeable and useful for intracellular labeling We manufacture biotin reagents to ensure the highest possible overall product integrity consistency and performance for the intended research applications N Hydroxysulfosuccinimide NHS esters of biotin are the most popular type of biotinylation reagent NHS activated biotins react efficiently with primary amino groups NH2 in alkaline buffers to form stable amide bonds Proteins e g antibodies typically have several primary amines that are available as targets for labeling including the side chain of lysine K residues and the N terminus of each polypeptide Varieties of biotin NHS ester reagents differ in length solubility cell permeability and cleavability Non sulfonated NHS biotins are cell permeable but must be dissolved in organic solvent such as DMSO or DMF Sulfo NHS biotins and those with pegylated spacers are directly water soluble but not membrane permeable Varieties containing disulfide bonds can be cleaved using reducing agents enabling the biotin group to be disconnected from the labeled protein
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    Images

    1) Product Images from "FASN Inhibition and Taxane Treatment Combine to Enhance Anti-tumor Efficacy in Diverse Xenograft Tumor Models through Disruption of Tubulin Palmitoylation and Microtubule Organization and FASN Inhibition-Mediated Effects on Oncogenic Signaling and Gene Expression"

    Article Title: FASN Inhibition and Taxane Treatment Combine to Enhance Anti-tumor Efficacy in Diverse Xenograft Tumor Models through Disruption of Tubulin Palmitoylation and Microtubule Organization and FASN Inhibition-Mediated Effects on Oncogenic Signaling and Gene Expression

    Journal: EBioMedicine

    doi: 10.1016/j.ebiom.2016.12.012

    TVB-3166 and TVB-3664 small molecule FASN inhibitors. (A) Chemical structure of TVB-3166 and TVB-3664. (B) Pharmacokinetic analysis of TVB-3664 in mice. Drug levels were determined from 3 different mice for each time point following administration by oral gavage or intravenous injection. (Bayside Biosciences, San Jose, CA). For oral dosing, TVB-3664 was formulated at 3-V Biosciences in 100% PEG-400 and diluted with water to a final PEG concentration of 30% immediately before dosing. For iv dosing TVB-3664 was formulated Bioanalytical analysis was performed at 3-V Biosciences by mass spectrometry.
    Figure Legend Snippet: TVB-3166 and TVB-3664 small molecule FASN inhibitors. (A) Chemical structure of TVB-3166 and TVB-3664. (B) Pharmacokinetic analysis of TVB-3664 in mice. Drug levels were determined from 3 different mice for each time point following administration by oral gavage or intravenous injection. (Bayside Biosciences, San Jose, CA). For oral dosing, TVB-3664 was formulated at 3-V Biosciences in 100% PEG-400 and diluted with water to a final PEG concentration of 30% immediately before dosing. For iv dosing TVB-3664 was formulated Bioanalytical analysis was performed at 3-V Biosciences by mass spectrometry.

    Techniques Used: Mouse Assay, Injection, Concentration Assay, Mass Spectrometry

    Combined treatment of tumor cells with TVB-3166 and paclitaxel in vitro increases soft agar colony growth inhibition and induction of apoptosis. (A) 22Rv1 prostate tumor cells (top panel) or CALU-6 NSCLC cells (bottom panel) were treated with a dose–response matrix of TVB-3166 and paclitaxel. 22Rv1 and CALU-6 tumor cells treated with 0.1 μM TVB-3166 and 0.001 μM paclitaxel showed increased inhibition of colony growth compared to treatment with each agent alone. Colony growth was inhibited completely with 0.1 μM TVB-3166 and 0.003 μM paclitaxel. (B) Intracellular paclitaxel concentration in CALU-6 tumor cells is unaffected by FASN inhibition. CALU-6 tumor cells were treated with DMSO, TVB-3166, or TVB-3664 for 24 (A) or 48 (B) hours. Cell lysates and supernatants were collected and the paclitaxel concentration in each matrix was determined by precipitation, extraction, and LC-MS-MS.
    Figure Legend Snippet: Combined treatment of tumor cells with TVB-3166 and paclitaxel in vitro increases soft agar colony growth inhibition and induction of apoptosis. (A) 22Rv1 prostate tumor cells (top panel) or CALU-6 NSCLC cells (bottom panel) were treated with a dose–response matrix of TVB-3166 and paclitaxel. 22Rv1 and CALU-6 tumor cells treated with 0.1 μM TVB-3166 and 0.001 μM paclitaxel showed increased inhibition of colony growth compared to treatment with each agent alone. Colony growth was inhibited completely with 0.1 μM TVB-3166 and 0.003 μM paclitaxel. (B) Intracellular paclitaxel concentration in CALU-6 tumor cells is unaffected by FASN inhibition. CALU-6 tumor cells were treated with DMSO, TVB-3166, or TVB-3664 for 24 (A) or 48 (B) hours. Cell lysates and supernatants were collected and the paclitaxel concentration in each matrix was determined by precipitation, extraction, and LC-MS-MS.

    Techniques Used: In Vitro, Inhibition, Concentration Assay, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    FASN inhibition disrupts tubulin palmitoylation, expression and microtubule organization. (A) TVB-3664 inhibits tubulin palmitoylation in NSCLC cell lines. Cells were treated with DMSO, TVB-3664 (48 h), or 2-bromopalimitate (18 h) as a positive control as indicated. Cell lysates were harvested and analyzed for palmitoylation of alpha-or beta-tubulin using acyl-biotin exchange chemistry followed by Western blot analysis. (B) TVB-3166 inhibits b-tubulin mRNA expression. 22Rv1 prostate tumor cells were treated with DMSO, TVB-3166, paclitaxel, or the combination of TVB-3166 and paclitaxel for 48 h in vitro. Three individual biological replicates were treated and used for analysis by RNA sequencing to quantitate mRNA levels. TVB-3166-treated cells showed a significant decrease in b-tubulin mRNA ( p
    Figure Legend Snippet: FASN inhibition disrupts tubulin palmitoylation, expression and microtubule organization. (A) TVB-3664 inhibits tubulin palmitoylation in NSCLC cell lines. Cells were treated with DMSO, TVB-3664 (48 h), or 2-bromopalimitate (18 h) as a positive control as indicated. Cell lysates were harvested and analyzed for palmitoylation of alpha-or beta-tubulin using acyl-biotin exchange chemistry followed by Western blot analysis. (B) TVB-3166 inhibits b-tubulin mRNA expression. 22Rv1 prostate tumor cells were treated with DMSO, TVB-3166, paclitaxel, or the combination of TVB-3166 and paclitaxel for 48 h in vitro. Three individual biological replicates were treated and used for analysis by RNA sequencing to quantitate mRNA levels. TVB-3166-treated cells showed a significant decrease in b-tubulin mRNA ( p

    Techniques Used: Inhibition, Expressing, Positive Control, Western Blot, In Vitro, RNA Sequencing Assay

    Immuno-fluorescent staining of β-tubulin in 22Rv1 cells with TVB-3664 and docetaxel and MRC5 cells with TVB-3664 and paclitaxel.
    Figure Legend Snippet: Immuno-fluorescent staining of β-tubulin in 22Rv1 cells with TVB-3664 and docetaxel and MRC5 cells with TVB-3664 and paclitaxel.

    Techniques Used: Staining

    2) 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

    3) Product Images from "Fluorophore Labeling, Nanodisc Reconstitution and Single-molecule Observation of a G Protein-coupled Receptor"

    Article Title: Fluorophore Labeling, Nanodisc Reconstitution and Single-molecule Observation of a G Protein-coupled Receptor

    Journal: Bio-protocol

    doi: 10.21769/BioProtoc.2332

    ) A. Typical TIRF image showing a 5 x 5 μm region (approximate dimensions) of immobilized Cy3-labeled β 2 AR reconstituted in nanodiscs. Each spot is due to the fluorescence emitted from an individual receptor-nanodisc complex. The very bright spots are due to a small fraction of receptor-nanodisc aggregates, which are readily identified and excluded from the data analysis. B. Corresponding TIRF image for a control surface lacking neutravidin. The number of fluorescent spots is greatly reduced relative to part A, indicating a negligible level of non-specific adsorption of receptor-nanodisc complexes. C. Representative fluorescence intensity versus time trace from a single nanodisc-bound receptor, showing repeated two-state intensity jumps prior to an irreversible photobleaching event. The red line is the best fit from Hidden Markov modeling. The single-step photobleaching transition confirms that a single receptor molecule is contained within the nanodisc.
    Figure Legend Snippet: ) A. Typical TIRF image showing a 5 x 5 μm region (approximate dimensions) of immobilized Cy3-labeled β 2 AR reconstituted in nanodiscs. Each spot is due to the fluorescence emitted from an individual receptor-nanodisc complex. The very bright spots are due to a small fraction of receptor-nanodisc aggregates, which are readily identified and excluded from the data analysis. B. Corresponding TIRF image for a control surface lacking neutravidin. The number of fluorescent spots is greatly reduced relative to part A, indicating a negligible level of non-specific adsorption of receptor-nanodisc complexes. C. Representative fluorescence intensity versus time trace from a single nanodisc-bound receptor, showing repeated two-state intensity jumps prior to an irreversible photobleaching event. The red line is the best fit from Hidden Markov modeling. The single-step photobleaching transition confirms that a single receptor molecule is contained within the nanodisc.

    Techniques Used: Labeling, Fluorescence, Adsorption

    4) Product Images from "A novel mechanism of myocyte degeneration involving the Ca2+-permeable growth factor-regulated channel"

    Article Title: A novel mechanism of myocyte degeneration involving the Ca2+-permeable growth factor-regulated channel

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200301101

    Effect of GRC antisense expression on BIO14.6 myotubes. (a) Immunoblot assay for GRC and β-dystroglycan (β-DG; top) and GRC immunohistochemistry (middle) of BIO14.6 myotubes infected with Ad.β-gal or Ad-antisense–GRC cDNA (Ad.asGRC). Cell surface GRC levels were estimated by labeling antisense-treated or nontreated myotubes with NHS-biotin and by further analyzing streptavidin agarose-bound (B) and -unbound (U) fractions by immunoblot assay with anti-GRC (bottom). (b and c) External Ca 2+ -induced changes in fluo-4 fluorescence and cyclic stretch-induced CK efflux from antisense-treated or nontreated myotubes. Other conditions were the same as those in Fig. 3 (b and c). Error bars show means ± SD and asterisks show P
    Figure Legend Snippet: Effect of GRC antisense expression on BIO14.6 myotubes. (a) Immunoblot assay for GRC and β-dystroglycan (β-DG; top) and GRC immunohistochemistry (middle) of BIO14.6 myotubes infected with Ad.β-gal or Ad-antisense–GRC cDNA (Ad.asGRC). Cell surface GRC levels were estimated by labeling antisense-treated or nontreated myotubes with NHS-biotin and by further analyzing streptavidin agarose-bound (B) and -unbound (U) fractions by immunoblot assay with anti-GRC (bottom). (b and c) External Ca 2+ -induced changes in fluo-4 fluorescence and cyclic stretch-induced CK efflux from antisense-treated or nontreated myotubes. Other conditions were the same as those in Fig. 3 (b and c). Error bars show means ± SD and asterisks show P

    Techniques Used: Expressing, Immunohistochemistry, Infection, Labeling, Fluorescence

    5) Product Images from "Aggregation Properties of the Small Nuclear Ribonucleoprotein U1-70K in Alzheimer Disease *"

    Article Title: Aggregation Properties of the Small Nuclear Ribonucleoprotein U1-70K in Alzheimer Disease *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.562959

    SDA-labeled rU1-70K fragments harboring the LC1 domain directly interact with aggregated U1-70K in AD brain. A, GST-tagged N- or C-terminal rU1-70K fragments (residues 1–99 and 182–310) were expressed in HEK cells, affinity-purified with glutathione-agarose, and assessed for purity by SDS-PAGE and Coomassie Blue staining ( left panel ). Purified fragments were derivatized with the amine-reactive photo-activatable cross-linker NHS-diazirine (SDA) ( right panel ). B, co-aggregation assays using Sarkosyl-insoluble fractions from AD brain were performed with unlabeled or SDA-labeled purified rU1-70K fragments (residues 1–99 or 182–310). Following UV treatment and fractionation, the cross-linked products were analyzed by Western blotting for both rU1-70K ( green ) and native U1-70K ( red ) ( top panel ). Higher molecular weight cross-linked species were only observed in co-aggregation experiments with SDA-labeled 182–310-residue rU1-70K fragment. Notably, lower molecular weight C-terminal fragments in the 182–310-residue rU1-70K co-aggregation assays were also observed to form cross-linked products. TDP-43 was used as a loading control ( bottom panel ). C, approximately 60% of native monomeric U1-70K from AD brain was cross-linked in co-aggregation assays using SDA-labeled 182–310-residue rU1-70K fragments compared with the control unlabeled fragments (no SDA). In contrast, ∼14% of native TDP-43 was cross-linked in these same experiments, which were performed in technical quadruplicate ( n = 4). Student's t test was performed for significance ( p
    Figure Legend Snippet: SDA-labeled rU1-70K fragments harboring the LC1 domain directly interact with aggregated U1-70K in AD brain. A, GST-tagged N- or C-terminal rU1-70K fragments (residues 1–99 and 182–310) were expressed in HEK cells, affinity-purified with glutathione-agarose, and assessed for purity by SDS-PAGE and Coomassie Blue staining ( left panel ). Purified fragments were derivatized with the amine-reactive photo-activatable cross-linker NHS-diazirine (SDA) ( right panel ). B, co-aggregation assays using Sarkosyl-insoluble fractions from AD brain were performed with unlabeled or SDA-labeled purified rU1-70K fragments (residues 1–99 or 182–310). Following UV treatment and fractionation, the cross-linked products were analyzed by Western blotting for both rU1-70K ( green ) and native U1-70K ( red ) ( top panel ). Higher molecular weight cross-linked species were only observed in co-aggregation experiments with SDA-labeled 182–310-residue rU1-70K fragment. Notably, lower molecular weight C-terminal fragments in the 182–310-residue rU1-70K co-aggregation assays were also observed to form cross-linked products. TDP-43 was used as a loading control ( bottom panel ). C, approximately 60% of native monomeric U1-70K from AD brain was cross-linked in co-aggregation assays using SDA-labeled 182–310-residue rU1-70K fragments compared with the control unlabeled fragments (no SDA). In contrast, ∼14% of native TDP-43 was cross-linked in these same experiments, which were performed in technical quadruplicate ( n = 4). Student's t test was performed for significance ( p

    Techniques Used: Labeling, Affinity Purification, SDS Page, Staining, Purification, Fractionation, Western Blot, Molecular Weight

    6) Product Images from "Mechanisms of osteopontin and CD44 as metastatic principles in prostate cancer cells"

    Article Title: Mechanisms of osteopontin and CD44 as metastatic principles in prostate cancer cells

    Journal: Molecular Cancer

    doi: 10.1186/1476-4598-6-18

    Analysis of CD44 surface expression and migration in different PC3 cell lines . A-C . Analyses shown in Figures A-C were performed in the following PC3 cell lines: PC3 (lane 1), PC3/OPN (lane 2), PC3/OPN (RGA) (lane 3), and PC3/SiRNA (lane 4). Cells were surface- labeled with NHS-Biotin and lysates were immunoprecipitated with an antibody to vCD44 (V3-10) (A) or MMP-9 (C). Also, as an internal control, a monoclonal antibody to actin was added to vCD44 immunoprecipitation. Actin immunoprecipitation was used as an internal control for normalization. Expression of variant forms of CD44 was observed in PC3 cell lines. Immunoprecipitation with a species-specific non-immune serum did not show any protein bands in the immunoblotting analysis (data not shown). Shorter exposure blot for PC3 (lane 5) and PC3/OPN (lane 6) is shown. The immunoblot shown in A was stripped and blotted with an actin antibody (B). Detection of surface expression of MMP-9 by immunoblotting with streptavidin-HRP is shown in C. No changes in the surface levels of MMP-9 indicate that biotinylation reaction was equally efficient in the indicated PC3 cell lines. The results represent one of three experiments performed. D and E: Wound healing assay. Phase-contrast micrographs of PC3, PC3/OPN, PC3/OPN (RGA) and PC3/SiRNA cells at 0 h and 48 h are shown. Results represent one of three experiments performed. Statistical analysis is provided as a graph at 0 h and 48 h in panel E. A significant increase in the migration of PC3/OPN, PC3, and PC3/OPN (RGA) cells was observed as compared with PC3/SiRNA cells. *** p
    Figure Legend Snippet: Analysis of CD44 surface expression and migration in different PC3 cell lines . A-C . Analyses shown in Figures A-C were performed in the following PC3 cell lines: PC3 (lane 1), PC3/OPN (lane 2), PC3/OPN (RGA) (lane 3), and PC3/SiRNA (lane 4). Cells were surface- labeled with NHS-Biotin and lysates were immunoprecipitated with an antibody to vCD44 (V3-10) (A) or MMP-9 (C). Also, as an internal control, a monoclonal antibody to actin was added to vCD44 immunoprecipitation. Actin immunoprecipitation was used as an internal control for normalization. Expression of variant forms of CD44 was observed in PC3 cell lines. Immunoprecipitation with a species-specific non-immune serum did not show any protein bands in the immunoblotting analysis (data not shown). Shorter exposure blot for PC3 (lane 5) and PC3/OPN (lane 6) is shown. The immunoblot shown in A was stripped and blotted with an actin antibody (B). Detection of surface expression of MMP-9 by immunoblotting with streptavidin-HRP is shown in C. No changes in the surface levels of MMP-9 indicate that biotinylation reaction was equally efficient in the indicated PC3 cell lines. The results represent one of three experiments performed. D and E: Wound healing assay. Phase-contrast micrographs of PC3, PC3/OPN, PC3/OPN (RGA) and PC3/SiRNA cells at 0 h and 48 h are shown. Results represent one of three experiments performed. Statistical analysis is provided as a graph at 0 h and 48 h in panel E. A significant increase in the migration of PC3/OPN, PC3, and PC3/OPN (RGA) cells was observed as compared with PC3/SiRNA cells. *** p

    Techniques Used: Expressing, Migration, Labeling, Immunoprecipitation, Variant Assay, Wound Healing Assay

    7) Product Images from "I-PLA2 Activation during Apoptosis Promotes the Exposure of Membrane Lysophosphatidylcholine Leading to Binding by Natural Immunoglobulin M Antibodies and Complement Activation"

    Article Title: I-PLA2 Activation during Apoptosis Promotes the Exposure of Membrane Lysophosphatidylcholine Leading to Binding by Natural Immunoglobulin M Antibodies and Complement Activation

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20020542

    IgM recruits C1q to apoptotic cells leading to C3 activation. (A) Live or apoptotic PBT cells were incubated with medium alone (−), NHS, HGS, or HGS reconstituted with IgM (1 mg/ml) as indicated for 30 min at 37°C. The cells were washed and C1q binding detected by Western blot analysis as described in Materials and Methods. For Western blot analysis, protein loading was compared by probing the same membrane with anti-ribosomal P antiserum (38 kD). Representative of three experiments. (B) Apoptotic PBT cells were incubated with purified human IgM (1 mg/ml) as above, washed and then incubated with purified human C1q (10 μg/ml) for 20 min at 37°C. C1q binding on the surface of apoptotic cells was detected by flow cytometry using a monoclonal anti–human C1q antibody. The results are expressed as the mean ± SD of three experiments. (C) Apoptotic PBT cells were incubated as in A. with either NHS, HGS, or HGS to which IgM was added to the final concentrations (μg/ml) indicated. These concentrations correspond to serial 10-fold dilutions of the normal serum concentration (1 mg/ml). Parallel experiments were performed with C1q depleted serum (C1q-D) to which serial 10-fold dilutions of C1q were added (normal serum concentration is 50 μg/ ml). C3b/bi binding was detected by flow cytometry and expressed as the percentage of cells positive for staining as in Fig. 1 E (mean ± SD of 3 experiments).
    Figure Legend Snippet: IgM recruits C1q to apoptotic cells leading to C3 activation. (A) Live or apoptotic PBT cells were incubated with medium alone (−), NHS, HGS, or HGS reconstituted with IgM (1 mg/ml) as indicated for 30 min at 37°C. The cells were washed and C1q binding detected by Western blot analysis as described in Materials and Methods. For Western blot analysis, protein loading was compared by probing the same membrane with anti-ribosomal P antiserum (38 kD). Representative of three experiments. (B) Apoptotic PBT cells were incubated with purified human IgM (1 mg/ml) as above, washed and then incubated with purified human C1q (10 μg/ml) for 20 min at 37°C. C1q binding on the surface of apoptotic cells was detected by flow cytometry using a monoclonal anti–human C1q antibody. The results are expressed as the mean ± SD of three experiments. (C) Apoptotic PBT cells were incubated as in A. with either NHS, HGS, or HGS to which IgM was added to the final concentrations (μg/ml) indicated. These concentrations correspond to serial 10-fold dilutions of the normal serum concentration (1 mg/ml). Parallel experiments were performed with C1q depleted serum (C1q-D) to which serial 10-fold dilutions of C1q were added (normal serum concentration is 50 μg/ ml). C3b/bi binding was detected by flow cytometry and expressed as the percentage of cells positive for staining as in Fig. 1 E (mean ± SD of 3 experiments).

    Techniques Used: Activation Assay, Incubation, Binding Assay, Western Blot, Purification, Flow Cytometry, Cytometry, Concentration Assay, Staining

    Proposed role of IgM in the binding and clearance of apoptotic cells. Apoptotic cells activate iPLA 2 , which results in exposure of lysophospholipids, including lyso-PtC, on the cell membrane. Under normal circumstances, lysoPtC is recognized by IgM which activates the classical complement pathway (A). As shown, not all complement activation is IgM dependent. Macrophages or dendritic cells phagocytose complement-coated cells, and produce immunosuppressive cytokines such as TGF-β (see references 50, 6, and 54 ; A). In contrast, when little IgM is available (B), either the cells undergo post-apoptotic necrosis and/or are seen by IgG antibodies. In either case, phagocytosis of this cargo leads to proinflammatory cytokine production (see references 50, and 6) and, possibly, maturation of dendritic cells (reference 55 ).
    Figure Legend Snippet: Proposed role of IgM in the binding and clearance of apoptotic cells. Apoptotic cells activate iPLA 2 , which results in exposure of lysophospholipids, including lyso-PtC, on the cell membrane. Under normal circumstances, lysoPtC is recognized by IgM which activates the classical complement pathway (A). As shown, not all complement activation is IgM dependent. Macrophages or dendritic cells phagocytose complement-coated cells, and produce immunosuppressive cytokines such as TGF-β (see references 50, 6, and 54 ; A). In contrast, when little IgM is available (B), either the cells undergo post-apoptotic necrosis and/or are seen by IgG antibodies. In either case, phagocytosis of this cargo leads to proinflammatory cytokine production (see references 50, and 6) and, possibly, maturation of dendritic cells (reference 55 ).

    Techniques Used: Binding Assay, Activation Assay

    IgM antibodies bind to components of lysophospholipids on the cell surface membrane. (A) Apoptotic cells were incubated in medium alone or medium containing 1 U of PLs (PLA 2 or PLD) for 30 min at 37°C or on ice (0°C). The cells were washed and then incubated with purified IgM or the TEPC15 anti-PC mAb for 20 min at 37°C. IgM binding to live cells is shown for comparison. Antibody binding was detected by flow cytometry and is expressed as the mean ± SD of 4 experiments. (B) Thymocytes were rendered apoptotic as in Fig. 1 and then exposed to 1U of type I or type III sPLA 2 or 1U PLD for 30 min at 37°C. They were then washed and incubated with wild-type (WT) or IgM-deficient (sIgM −/− ) serum followed by flow cytometry analysis for C3 binding. The results are expressed as the percentage of cells staining for C3 and are representative of two experiments. Cells incubated with heat-inactivated serum (HIS) or anti-Thy1 were used as negative and positive controls, respectively. (C) NHS was incubated with solid phase adsorbed lysophospholipids and then tested for binding to apoptotic cells. The results are expressed as inhibition of IgM binding, calculated as in Fig. 4 and are the mean ± SD of 3 experiments. (D) NHS was preincubated with varying concentration of PC, PS, or PE as shown for 30 min at 37°C. Inhibition of IgM binding to apoptotic cells was calculated as above. The results are expressed as the mean ± SD of 3 experiments. (E) Purified IgM or NHS was incubated with PC (PC-Cl) and the percentage inhibition of IgM binding to apoptotic cells determined by flow cytometry as in C. TEPC15, and an anti-β2 microglobulin mAb, were used as positive and negative controls, respectively. The results are expressed as the mean ± SD of 4 experiments.
    Figure Legend Snippet: IgM antibodies bind to components of lysophospholipids on the cell surface membrane. (A) Apoptotic cells were incubated in medium alone or medium containing 1 U of PLs (PLA 2 or PLD) for 30 min at 37°C or on ice (0°C). The cells were washed and then incubated with purified IgM or the TEPC15 anti-PC mAb for 20 min at 37°C. IgM binding to live cells is shown for comparison. Antibody binding was detected by flow cytometry and is expressed as the mean ± SD of 4 experiments. (B) Thymocytes were rendered apoptotic as in Fig. 1 and then exposed to 1U of type I or type III sPLA 2 or 1U PLD for 30 min at 37°C. They were then washed and incubated with wild-type (WT) or IgM-deficient (sIgM −/− ) serum followed by flow cytometry analysis for C3 binding. The results are expressed as the percentage of cells staining for C3 and are representative of two experiments. Cells incubated with heat-inactivated serum (HIS) or anti-Thy1 were used as negative and positive controls, respectively. (C) NHS was incubated with solid phase adsorbed lysophospholipids and then tested for binding to apoptotic cells. The results are expressed as inhibition of IgM binding, calculated as in Fig. 4 and are the mean ± SD of 3 experiments. (D) NHS was preincubated with varying concentration of PC, PS, or PE as shown for 30 min at 37°C. Inhibition of IgM binding to apoptotic cells was calculated as above. The results are expressed as the mean ± SD of 3 experiments. (E) Purified IgM or NHS was incubated with PC (PC-Cl) and the percentage inhibition of IgM binding to apoptotic cells determined by flow cytometry as in C. TEPC15, and an anti-β2 microglobulin mAb, were used as positive and negative controls, respectively. The results are expressed as the mean ± SD of 4 experiments.

    Techniques Used: Incubation, Proximity Ligation Assay, Purification, Binding Assay, Flow Cytometry, Cytometry, Staining, Inhibition, Concentration Assay

    IgM binds to apoptotic cells by its Fab domain. Fc and Fab fragments of IgM were prepared by tryptic digestion and were isolated by FPLC as described in Materials and Methods. (A) Purity of the fragments (20 μg per lane) was assessed by SDS -10% PAGE under reducing conditions and proteins detected by Commassie-Blue staining. Lane 1, molecular mass standards; lane 2, IgM before digestion; lane 3, Fcμ; lane 4, Fab′. (B) Isolated IgM, IgM heated to 65°C for 10 min, IgM Fcμ or Fab′ fragments (all tested at a concentration of 1 pM) were examined for binding to apoptotic PBT as in Fig. 1 except that IgM Fc or Fab′ binding were detected with polyclonal antibodies specific for the fragment. The results are expressed as the mean ± SD of four experiments.
    Figure Legend Snippet: IgM binds to apoptotic cells by its Fab domain. Fc and Fab fragments of IgM were prepared by tryptic digestion and were isolated by FPLC as described in Materials and Methods. (A) Purity of the fragments (20 μg per lane) was assessed by SDS -10% PAGE under reducing conditions and proteins detected by Commassie-Blue staining. Lane 1, molecular mass standards; lane 2, IgM before digestion; lane 3, Fcμ; lane 4, Fab′. (B) Isolated IgM, IgM heated to 65°C for 10 min, IgM Fcμ or Fab′ fragments (all tested at a concentration of 1 pM) were examined for binding to apoptotic PBT as in Fig. 1 except that IgM Fc or Fab′ binding were detected with polyclonal antibodies specific for the fragment. The results are expressed as the mean ± SD of four experiments.

    Techniques Used: Isolation, Fast Protein Liquid Chromatography, Polyacrylamide Gel Electrophoresis, Staining, Concentration Assay, Binding Assay

    Kinetics and specificity of IgM binding to apoptotic cells. (A) Apoptosis of PBT cells was induced as in Fig. 1 and at time 0, 2, 4 and 6 h, the cells were incubated in medium containing 20% NHS or HGS. Cells were analyzed by flow cytometry for Annexin V or IgM binding as well as for permeability to PI and trypan blue as indicated in the Figure. The results are expressed as the percentage of cells positive. The results are expressed as the mean ± SD of three experiments. (B) Purified IgM was incubated with liposomes containing either 50 or 500 ug/ml PtS or PtC for 30 min at 37°C. Samples were centrifuged and the supernates tested for binding to apoptotic cells. Annexin V and SUV (an anti-PtC specific mAb), were used as positive controls for binding to PtS and PtC, respectively. The results are expressed as percentage of inhibition of binding, calculated from (binding in medium − binding after preadsorbtion with liposome/binding in medium) × 100. The results are expressed as the mean ± SD of three experiments.
    Figure Legend Snippet: Kinetics and specificity of IgM binding to apoptotic cells. (A) Apoptosis of PBT cells was induced as in Fig. 1 and at time 0, 2, 4 and 6 h, the cells were incubated in medium containing 20% NHS or HGS. Cells were analyzed by flow cytometry for Annexin V or IgM binding as well as for permeability to PI and trypan blue as indicated in the Figure. The results are expressed as the percentage of cells positive. The results are expressed as the mean ± SD of three experiments. (B) Purified IgM was incubated with liposomes containing either 50 or 500 ug/ml PtS or PtC for 30 min at 37°C. Samples were centrifuged and the supernates tested for binding to apoptotic cells. Annexin V and SUV (an anti-PtC specific mAb), were used as positive controls for binding to PtS and PtC, respectively. The results are expressed as percentage of inhibition of binding, calculated from (binding in medium − binding after preadsorbtion with liposome/binding in medium) × 100. The results are expressed as the mean ± SD of three experiments.

    Techniques Used: Binding Assay, Incubation, Flow Cytometry, Cytometry, Permeability, Purification, Inhibition

    IgM activates complement on apoptotic cells. (A) Normal peripheral blood–derived T cells (PBT) were either incubated in medium or induced to undergo apoptosis by staurosporine (STS) as described in Materials and Methods. The cells were then incubated with either purified human IgG (10 mg/ml) or IgM (1 mg/ml) for 20 min at 37°C and examined for Ig binding by flow cytometry (thin line, live cells; thick line, apoptotic cells; dotted line, apoptotic cells incubated with second antibody alone). The change in mean channel fluorescence (Δ) between the cells incubated with second antibody alone versus IgG or IgM followed by second antibody is shown. Representative of five experiments. (B) Apoptotic PBT cells were incubated with either HGS or autologous NHS for 30 min followed by two-color flow cytometric analysis of IgM and C3b/bi binding. Representative of three experiments. (C) Apoptotic PBT cells were incubated with either 20% autologous NHS or HGS no. 2 and 3 for 30 min at 37°C. Deposition of cell surface C3b/bi and the MAC were quantified by flow cytometry as described in Materials and Methods. As a control for complement activity in the HGS sera, C3b/bi, and MAC deposition on T cells were quantified after incubation with an IgM monoclonal anti-β2 microglobulin antibody followed by either NHS or HGS as a source of complement. The results are expressed as complement component binding in HGS/complement component binding in NHS ×100 (mean ± SD of 4 experiments). (D) C57BL/6 (B6) thymocytes were incubated with 1 μM dexamethasone for 6 h to induce apoptosis. Apoptotic thymocytes were incubated with 20% of autologous wild-type serum or serum obtained from sIgM −/− deficient mice on a B6 or 129 background for 30 min at 37°C. C3 binding to the cells was quantified by flow cytometry. As a control, C3 deposition was also quantified following preincubation of the apoptotic cells with IgM anti-Thy.1 antibodies. The results are expressed as percentage of complement activation. Two experiments gave virtually identical results. (E) Apoptotic PBT cells prepared as in C, were incubated with NHS, HGS, or HGS reconstituted with purified human 10 mg/ml IgG or 1 mg/ml IgM. C3b/bi deposition was detected by flow cytometry and are expressed as the percentage of cells positive for staining (mean ± SD of 3 experiments).
    Figure Legend Snippet: IgM activates complement on apoptotic cells. (A) Normal peripheral blood–derived T cells (PBT) were either incubated in medium or induced to undergo apoptosis by staurosporine (STS) as described in Materials and Methods. The cells were then incubated with either purified human IgG (10 mg/ml) or IgM (1 mg/ml) for 20 min at 37°C and examined for Ig binding by flow cytometry (thin line, live cells; thick line, apoptotic cells; dotted line, apoptotic cells incubated with second antibody alone). The change in mean channel fluorescence (Δ) between the cells incubated with second antibody alone versus IgG or IgM followed by second antibody is shown. Representative of five experiments. (B) Apoptotic PBT cells were incubated with either HGS or autologous NHS for 30 min followed by two-color flow cytometric analysis of IgM and C3b/bi binding. Representative of three experiments. (C) Apoptotic PBT cells were incubated with either 20% autologous NHS or HGS no. 2 and 3 for 30 min at 37°C. Deposition of cell surface C3b/bi and the MAC were quantified by flow cytometry as described in Materials and Methods. As a control for complement activity in the HGS sera, C3b/bi, and MAC deposition on T cells were quantified after incubation with an IgM monoclonal anti-β2 microglobulin antibody followed by either NHS or HGS as a source of complement. The results are expressed as complement component binding in HGS/complement component binding in NHS ×100 (mean ± SD of 4 experiments). (D) C57BL/6 (B6) thymocytes were incubated with 1 μM dexamethasone for 6 h to induce apoptosis. Apoptotic thymocytes were incubated with 20% of autologous wild-type serum or serum obtained from sIgM −/− deficient mice on a B6 or 129 background for 30 min at 37°C. C3 binding to the cells was quantified by flow cytometry. As a control, C3 deposition was also quantified following preincubation of the apoptotic cells with IgM anti-Thy.1 antibodies. The results are expressed as percentage of complement activation. Two experiments gave virtually identical results. (E) Apoptotic PBT cells prepared as in C, were incubated with NHS, HGS, or HGS reconstituted with purified human 10 mg/ml IgG or 1 mg/ml IgM. C3b/bi deposition was detected by flow cytometry and are expressed as the percentage of cells positive for staining (mean ± SD of 3 experiments).

    Techniques Used: Derivative Assay, Incubation, Purification, Binding Assay, Flow Cytometry, Cytometry, Fluorescence, Activity Assay, Mouse Assay, Activation Assay, Staining

    BEL, an inhibitor of endogenous iPLA 2 attenuates IgM binding to apoptotic cells. (A) PBMT were incubated with staurosporine together with medium alone or medium containing the PLA 2 inhibitors, 10 μM BEL or 5 μM Shionogi-1 for 6 h (A and C) or the phosphatidate phosphohydrolase inhibitor, propranolol (12.5 μM) (B). The cells were then stained with Annexin V-FITC (A and B) and/or purified human IgM followed by FITC-anti–human IgM (B). To determine the effects of BEL on intracellular apoptotic events, cells were examined for caspase-3 activity by flow cytometry (C), PARP cleavage by Western blot analysis (D), or nuclear condensation by Hoechst staining and immunofluorescence (E) in the presence or absence of 10 μM BEL as described in Materials and Methods. For Western blot analysis (D), protein loading was compared by probing the same membrane with antiribosomal P antiserum (38 kD). A–C are mean ± SD of 3 experiments; D and E are representative of two experiments with identical results.
    Figure Legend Snippet: BEL, an inhibitor of endogenous iPLA 2 attenuates IgM binding to apoptotic cells. (A) PBMT were incubated with staurosporine together with medium alone or medium containing the PLA 2 inhibitors, 10 μM BEL or 5 μM Shionogi-1 for 6 h (A and C) or the phosphatidate phosphohydrolase inhibitor, propranolol (12.5 μM) (B). The cells were then stained with Annexin V-FITC (A and B) and/or purified human IgM followed by FITC-anti–human IgM (B). To determine the effects of BEL on intracellular apoptotic events, cells were examined for caspase-3 activity by flow cytometry (C), PARP cleavage by Western blot analysis (D), or nuclear condensation by Hoechst staining and immunofluorescence (E) in the presence or absence of 10 μM BEL as described in Materials and Methods. For Western blot analysis (D), protein loading was compared by probing the same membrane with antiribosomal P antiserum (38 kD). A–C are mean ± SD of 3 experiments; D and E are representative of two experiments with identical results.

    Techniques Used: Binding Assay, Incubation, Proximity Ligation Assay, Staining, Purification, Activity Assay, Flow Cytometry, Cytometry, Western Blot, Immunofluorescence

    8) Product Images from "Novel Evolved Immunoglobulin (Ig)-Binding Molecules Enhance the Detection of IgM against Hepatitis C Virus"

    Article Title: Novel Evolved Immunoglobulin (Ig)-Binding Molecules Enhance the Detection of IgM against Hepatitis C Virus

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0018477

    The enzyme-labeled LD5 exhibits enhanced binding activities for IgM and IgA. The binding activities of horseradish peroxidase (HRP)-labeled LD5 (HRP-LD5) or HRP-conjugated goat anti-human polyclonal antibodies (HRP-goat anti-human PcAb) to coated hIgM (A), hIgG (B) or hIgA (C) were examined by ELISA. The coating buffer was used as solvent control.
    Figure Legend Snippet: The enzyme-labeled LD5 exhibits enhanced binding activities for IgM and IgA. The binding activities of horseradish peroxidase (HRP)-labeled LD5 (HRP-LD5) or HRP-conjugated goat anti-human polyclonal antibodies (HRP-goat anti-human PcAb) to coated hIgM (A), hIgG (B) or hIgA (C) were examined by ELISA. The coating buffer was used as solvent control.

    Techniques Used: Labeling, Binding Assay, Enzyme-linked Immunosorbent Assay

    9) Product Images from "Novel Evolved Immunoglobulin (Ig)-Binding Molecules Enhance the Detection of IgM against Hepatitis C Virus"

    Article Title: Novel Evolved Immunoglobulin (Ig)-Binding Molecules Enhance the Detection of IgM against Hepatitis C Virus

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0018477

    The enzyme-labeled LD5 exhibits enhanced binding activities for IgM and IgA. The binding activities of horseradish peroxidase (HRP)-labeled LD5 (HRP-LD5) or HRP-conjugated goat anti-human polyclonal antibodies (HRP-goat anti-human PcAb) to coated hIgM (A), hIgG (B) or hIgA (C) were examined by ELISA. The coating buffer was used as solvent control.
    Figure Legend Snippet: The enzyme-labeled LD5 exhibits enhanced binding activities for IgM and IgA. The binding activities of horseradish peroxidase (HRP)-labeled LD5 (HRP-LD5) or HRP-conjugated goat anti-human polyclonal antibodies (HRP-goat anti-human PcAb) to coated hIgM (A), hIgG (B) or hIgA (C) were examined by ELISA. The coating buffer was used as solvent control.

    Techniques Used: Labeling, Binding Assay, Enzyme-linked Immunosorbent Assay

    10) Product Images from "Novel Evolved Immunoglobulin (Ig)-Binding Molecules Enhance the Detection of IgM against Hepatitis C Virus"

    Article Title: Novel Evolved Immunoglobulin (Ig)-Binding Molecules Enhance the Detection of IgM against Hepatitis C Virus

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0018477

    The enzyme-labeled LD5 exhibits enhanced binding activities for IgM and IgA. The binding activities of horseradish peroxidase (HRP)-labeled LD5 (HRP-LD5) or HRP-conjugated goat anti-human polyclonal antibodies (HRP-goat anti-human PcAb) to coated hIgM (A), hIgG (B) or hIgA (C) were examined by ELISA. The coating buffer was used as solvent control.
    Figure Legend Snippet: The enzyme-labeled LD5 exhibits enhanced binding activities for IgM and IgA. The binding activities of horseradish peroxidase (HRP)-labeled LD5 (HRP-LD5) or HRP-conjugated goat anti-human polyclonal antibodies (HRP-goat anti-human PcAb) to coated hIgM (A), hIgG (B) or hIgA (C) were examined by ELISA. The coating buffer was used as solvent control.

    Techniques Used: Labeling, Binding Assay, Enzyme-linked Immunosorbent Assay

    11) Product Images from "Beta-catenin signaling regulates barrier-specific gene expression in circumventricular organ and ocular vasculatures"

    Article Title: Beta-catenin signaling regulates barrier-specific gene expression in circumventricular organ and ocular vasculatures

    Journal: eLife

    doi: 10.7554/eLife.43257

    Stabilizing beta-catenin signaling suppresses EC fenestrae and reduces vascular permeability. ( A–F ) Transmission electron micrographs from control mice (left) and from age-matched mice with EC-specific deletion of Ctnnb1 exon 3 (right) showing representative sections of choriocapillaris ( A,B ), posterior pituitary ( C,D ), and choroid plexus ( E,F ). RPE, retinal pigment epithelium. Vertical arrows mark fenestrae. Neurohormone secretory granules are seen in the posterior pituitary parenchyma. Scale bar, 500 nm. ( G ) Quantification of the density of capillary EC fenestrae in age-matched and 4HT-treated control ( Ctnnb1 flex3/+ ) vs. Ctnnb1 exon 3 stabilized ( Ctnnb1 flex3/+ ;Pdgfb-CreER ) mice for the choriocapillaris, posterior pituitary, choroid plexus, and ciliary body. Each data-point represents all of the vascular wall length within a single 10 μm x 10 μm TEM image. Bars show mean ± S.D. Each cluster of data points represents a different mouse. For each location and genotype, 3–5 mice were analyzed. For each of the four anatomic locations, the p-values are calculated for all of the Ctnnb1 flex3/+ vs. all of the Ctnnb1 flex3/+ ;Pdgfb-CreER data-points. ( H,I ) Sagittal brain sections show vascular markers GLUT1 and PLVAP and perivascular accumulation of Sulfo-NHS-biotin (following IP injection) in the SFO and adjacent choroid plexus ( H ) and in the area postrema and adjacent choroid plexus ( I ) from ~P30 WT vs. Ctnnb1 flex3/+ ;Pdgfb-CreER mice. AP, area postrema; CP, choroid plexus. A, anterior; P, posterior. Scale bars in ( H ) and ( I ), 200 μm.
    Figure Legend Snippet: Stabilizing beta-catenin signaling suppresses EC fenestrae and reduces vascular permeability. ( A–F ) Transmission electron micrographs from control mice (left) and from age-matched mice with EC-specific deletion of Ctnnb1 exon 3 (right) showing representative sections of choriocapillaris ( A,B ), posterior pituitary ( C,D ), and choroid plexus ( E,F ). RPE, retinal pigment epithelium. Vertical arrows mark fenestrae. Neurohormone secretory granules are seen in the posterior pituitary parenchyma. Scale bar, 500 nm. ( G ) Quantification of the density of capillary EC fenestrae in age-matched and 4HT-treated control ( Ctnnb1 flex3/+ ) vs. Ctnnb1 exon 3 stabilized ( Ctnnb1 flex3/+ ;Pdgfb-CreER ) mice for the choriocapillaris, posterior pituitary, choroid plexus, and ciliary body. Each data-point represents all of the vascular wall length within a single 10 μm x 10 μm TEM image. Bars show mean ± S.D. Each cluster of data points represents a different mouse. For each location and genotype, 3–5 mice were analyzed. For each of the four anatomic locations, the p-values are calculated for all of the Ctnnb1 flex3/+ vs. all of the Ctnnb1 flex3/+ ;Pdgfb-CreER data-points. ( H,I ) Sagittal brain sections show vascular markers GLUT1 and PLVAP and perivascular accumulation of Sulfo-NHS-biotin (following IP injection) in the SFO and adjacent choroid plexus ( H ) and in the area postrema and adjacent choroid plexus ( I ) from ~P30 WT vs. Ctnnb1 flex3/+ ;Pdgfb-CreER mice. AP, area postrema; CP, choroid plexus. A, anterior; P, posterior. Scale bars in ( H ) and ( I ), 200 μm.

    Techniques Used: Permeability, Transmission Assay, Mouse Assay, Transmission Electron Microscopy, Injection

    12) Product Images from "Structure and vascular function of MEKK3–cerebral cavernous malformations 2 complex"

    Article Title: Structure and vascular function of MEKK3–cerebral cavernous malformations 2 complex

    Journal: Nature Communications

    doi: 10.1038/ncomms8937

    MEKK3 critically regulates the neonatal vascular permeability to small size molecule via suppressing the Rho signals. ( a – d ) In vivo leakage experiments show only small-molecular-weight tracers leaked from brain microvessels in Mekk3 iKO and iEC −/− neonatal mice. ( a ) Sulfo-NHS-biotin (556.6 dalton) was injected into the hearts of the tamoxifen-treated Mekk3 NCL or Mekk3 iKO neonatal pups. Brain sections were analysed for Sulfo-NHS-biotin leakage by staining. Objective lens power: 2.5 × . ( b – d ) Fluorescent-labelled tracers with different molecular weights were injected into tamoxifen-treated Mekk3 NCL or Mekk3 iEC −/− P7 neonatal pups' hearts. After euthanization, brains were fixed and sectioned and tracer leakage to the brain was determined. ( b ) Hoechst 33342 (616 dalton; blue) plus Dextran–Rhodamine (2 M Dalton; red), N =7, ( c ) Dextran–FITC (MW: 4K Dalton; green) plus Dextran–Rhodamine (2 M Dalton)(red), N=4, ( d ) Dextran–FITC (40 K Dalton; green) plus Dextran–Rhodamine (2 M Dalton; red). Bar graphs on the left of each panel show quantification of the relative leakage of the tracers normalized to the intensity of Dextran–Rhodamine, and shows no leakage in either NCL nor Mekk3 iEC −/− neonatal pups. Error bars indicate s.d. N =4. Objective lens power: 10 × . ( e ) ROCK inhibitor Y27632 partially rescues survival of Mekk3 iEC −/− neonatal pups. NCL or Mekk3 iEC −/− pups were treated with tamoxifen at P1, and continued daily in the absence (open circle and black diamond, respectively) or presence of Y27632 (cross and black triangle, respectively). Survival of pups was monitored daily until P20. ( f ) Hoechst 33342 (616 Dalton; blue) plus Dextran K–Rhodamine (2 M Dalton; red) were injected into tamoxifen-treated Mekk3 iEC −/− P7 neonatal pups' hearts fed either with water or Y27632. After euthanization, brains were fixed and sectioned at 30-μm thickness and leakage determined as described above. N =5. Objective lens power: 10 × . ( g ) Wild-type P7 neonatal pups were treated with cell-permeable wild-type Mekk3 -peptide (MEKK3 N-peptide ) or A6D/L7D mutated Mekk3 -peptide (MEKK3 mutant-N-peptide ). Brain leakage determined using Hoechst 33342 (blue) plus Dextran K–Rhodamine (red). N =5. Objective lens power: 10 × . ( h ) Proposed signalling pathway. The interactions of CCM2 and MEKK3 are critical for maintenance of vasculature integrity and permeability by control of Rho/ROCK signalling.
    Figure Legend Snippet: MEKK3 critically regulates the neonatal vascular permeability to small size molecule via suppressing the Rho signals. ( a – d ) In vivo leakage experiments show only small-molecular-weight tracers leaked from brain microvessels in Mekk3 iKO and iEC −/− neonatal mice. ( a ) Sulfo-NHS-biotin (556.6 dalton) was injected into the hearts of the tamoxifen-treated Mekk3 NCL or Mekk3 iKO neonatal pups. Brain sections were analysed for Sulfo-NHS-biotin leakage by staining. Objective lens power: 2.5 × . ( b – d ) Fluorescent-labelled tracers with different molecular weights were injected into tamoxifen-treated Mekk3 NCL or Mekk3 iEC −/− P7 neonatal pups' hearts. After euthanization, brains were fixed and sectioned and tracer leakage to the brain was determined. ( b ) Hoechst 33342 (616 dalton; blue) plus Dextran–Rhodamine (2 M Dalton; red), N =7, ( c ) Dextran–FITC (MW: 4K Dalton; green) plus Dextran–Rhodamine (2 M Dalton)(red), N=4, ( d ) Dextran–FITC (40 K Dalton; green) plus Dextran–Rhodamine (2 M Dalton; red). Bar graphs on the left of each panel show quantification of the relative leakage of the tracers normalized to the intensity of Dextran–Rhodamine, and shows no leakage in either NCL nor Mekk3 iEC −/− neonatal pups. Error bars indicate s.d. N =4. Objective lens power: 10 × . ( e ) ROCK inhibitor Y27632 partially rescues survival of Mekk3 iEC −/− neonatal pups. NCL or Mekk3 iEC −/− pups were treated with tamoxifen at P1, and continued daily in the absence (open circle and black diamond, respectively) or presence of Y27632 (cross and black triangle, respectively). Survival of pups was monitored daily until P20. ( f ) Hoechst 33342 (616 Dalton; blue) plus Dextran K–Rhodamine (2 M Dalton; red) were injected into tamoxifen-treated Mekk3 iEC −/− P7 neonatal pups' hearts fed either with water or Y27632. After euthanization, brains were fixed and sectioned at 30-μm thickness and leakage determined as described above. N =5. Objective lens power: 10 × . ( g ) Wild-type P7 neonatal pups were treated with cell-permeable wild-type Mekk3 -peptide (MEKK3 N-peptide ) or A6D/L7D mutated Mekk3 -peptide (MEKK3 mutant-N-peptide ). Brain leakage determined using Hoechst 33342 (blue) plus Dextran K–Rhodamine (red). N =5. Objective lens power: 10 × . ( h ) Proposed signalling pathway. The interactions of CCM2 and MEKK3 are critical for maintenance of vasculature integrity and permeability by control of Rho/ROCK signalling.

    Techniques Used: Permeability, In Vivo, Molecular Weight, Mouse Assay, Injection, Staining, Mutagenesis

    13) Product Images from "Crystal Structure of the Cul2-Rbx1-EloBC-VHL Ubiquitin Ligase Complex"

    Article Title: Crystal Structure of the Cul2-Rbx1-EloBC-VHL Ubiquitin Ligase Complex

    Journal: Structure(London, England:1993)

    doi: 10.1016/j.str.2017.04.009

    Biophysical Characterization of the Interaction between VBC and Cul2 (A) ITC data whereby VBC (200 μM) was titrated into Rbx1-Cul2 (20 μM) at 303 K. Under these conditions, the binding affinity of the interaction ( K D ) is 42 nM. (B) Temperature dependency of the thermodynamic parameters Δ H , Δ G and −TΔ S . The change in heating capacity, Δ C p , was derived from the change in enthalpy with the temperature. (C) Comparison of Δ C p values for similar CRL systems.
    Figure Legend Snippet: Biophysical Characterization of the Interaction between VBC and Cul2 (A) ITC data whereby VBC (200 μM) was titrated into Rbx1-Cul2 (20 μM) at 303 K. Under these conditions, the binding affinity of the interaction ( K D ) is 42 nM. (B) Temperature dependency of the thermodynamic parameters Δ H , Δ G and −TΔ S . The change in heating capacity, Δ C p , was derived from the change in enthalpy with the temperature. (C) Comparison of Δ C p values for similar CRL systems.

    Techniques Used: Binding Assay, Derivative Assay

    Swapping Residues and Selectivity for Cul2 or Cul5 (A) Structures of Cul5-SBC and Cul2-VBC aligned by the EloC subunit show residues involved in the electrostatic network created between substrate receptor and Cullin, in both cases. (B) AlphaLISA data show loss of binding affinity of V QRY BC toward Cul2 and rescue of binding of S KSD BC toward Cul2. The experiments were performed in quadruplicate and the results are an averaged value. The error bars represent the SD of each point. The fitting was performed with GraphPad Prism 7 software. (C and D) ITC data. Titrant solution (200 μM) was diluted into 20 μM titrate over 19 injections of 2 μL at 303 K. (C) Titration of SBC and S KSD BC into Rbx1-Cul2. (D) Titration of Cul5 NTD into V QRY BC and VBC. (E) Summary of the results obtained in the biophysical experiments.
    Figure Legend Snippet: Swapping Residues and Selectivity for Cul2 or Cul5 (A) Structures of Cul5-SBC and Cul2-VBC aligned by the EloC subunit show residues involved in the electrostatic network created between substrate receptor and Cullin, in both cases. (B) AlphaLISA data show loss of binding affinity of V QRY BC toward Cul2 and rescue of binding of S KSD BC toward Cul2. The experiments were performed in quadruplicate and the results are an averaged value. The error bars represent the SD of each point. The fitting was performed with GraphPad Prism 7 software. (C and D) ITC data. Titrant solution (200 μM) was diluted into 20 μM titrate over 19 injections of 2 μL at 303 K. (C) Titration of SBC and S KSD BC into Rbx1-Cul2. (D) Titration of Cul5 NTD into V QRY BC and VBC. (E) Summary of the results obtained in the biophysical experiments.

    Techniques Used: Binding Assay, Software, Titration

    Rbx1 Presents a New Orientation Superposition of the CTDs of six Cullins—Cul1 (PDB: 1U6G ), Cul2 (PDB: 5N4W ), Cul4 (PDB: 4A0C ), Cul5 (PDB: 3DPL ), Cul5∼NEDD8 (PDB: 3DQV ), and Glomulin-Rbx1-Cul1 (PDB: 4F52 )—complexed with Rbx1. The new structure of Rbx1 in complex with Cul2 unveils a novel orientation of its RING domain, resembling an en route conformation between Rbx1-Cul5 and Rbx1-Cul5∼NEDD8.
    Figure Legend Snippet: Rbx1 Presents a New Orientation Superposition of the CTDs of six Cullins—Cul1 (PDB: 1U6G ), Cul2 (PDB: 5N4W ), Cul4 (PDB: 4A0C ), Cul5 (PDB: 3DPL ), Cul5∼NEDD8 (PDB: 3DQV ), and Glomulin-Rbx1-Cul1 (PDB: 4F52 )—complexed with Rbx1. The new structure of Rbx1 in complex with Cul2 unveils a novel orientation of its RING domain, resembling an en route conformation between Rbx1-Cul5 and Rbx1-Cul5∼NEDD8.

    Techniques Used:

    14) Product Images from "Label-Free Plasmonic Detection of Biomolecular Binding by a Single Gold Nanorod"

    Article Title: Label-Free Plasmonic Detection of Biomolecular Binding by a Single Gold Nanorod

    Journal:

    doi: 10.1021/ac7017348

    (a and b) Scattering spectra of a single gold nanorod after sequential incubation in EG 3 SH/MHA (blue), biotin (red), and 10 nM streptavidin (black). (c and d) Scattering spectra of a single gold nanorod in EG 3 SH/MHA (blue), biotin (red), and 100 nM streptavidin
    Figure Legend Snippet: (a and b) Scattering spectra of a single gold nanorod after sequential incubation in EG 3 SH/MHA (blue), biotin (red), and 10 nM streptavidin (black). (c and d) Scattering spectra of a single gold nanorod in EG 3 SH/MHA (blue), biotin (red), and 100 nM streptavidin

    Techniques Used: Incubation

    Real-time measurement of the LSPR scattering peak centroid shifts of single biotin-conjugated gold nanorods incubated in 130 (black), 10 (blue), and 1 nM (red) streptavidin in PBS.
    Figure Legend Snippet: Real-time measurement of the LSPR scattering peak centroid shifts of single biotin-conjugated gold nanorods incubated in 130 (black), 10 (blue), and 1 nM (red) streptavidin in PBS.

    Techniques Used: Incubation

    15) Product Images from "Dynamic Dystroglycan Complexes Mediate Cell Entry of Lassa Virus"

    Article Title: Dynamic Dystroglycan Complexes Mediate Cell Entry of Lassa Virus

    Journal: mBio

    doi: 10.1128/mBio.02869-18

    Validation of candidate DG-binding proteins. (A) Coimmunoprecipitation (co-IP) of DG-associated scaffold proteins. Monolayers of A549 and SAEC were lysed in cold Triton X-100 containing buffer, and cleared lysates were subjected to IP with MAb VIA4 matrix (α-DG) using MAb anti-HA matrix (HA) as a control. Immunocomplexes were probed in Western blots for functional α-DG (MAb IIH6), β-DG (MAb 8D9), utrophin (MAb 20C5), α1-syntrophin (goat pAb 5941), β1-syntrophin (rabbit pAb 98977), β2-syntrophin (MAb 1351), and β-dystrobrevin (rabbit pAb 152133). Primary rabbit and goat antibodies as well as MAb IIH6 (mouse IgM) were detected as described in the legend to Fig. 1B and C . For detection of mouse MAb IgG, HRP-conjugated mouse TrueBlot ULTRA secondary antibody was used as detailed in Materials and Methods. For a positive control (+), total lysates of differentiated human myotubes were included. One representative example out of three independent experiments is shown. (B) Detection of sarcoglycans at the surfaces of A549 cells. Intact monolayers of A549 cells were subjected to cell surface biotinylation using the membrane-impermeable reagent sulfo-NHS-X-biotin (+) or reaction buffer only (−). After reaction quenching, cells were lysed, and biotinylated proteins were precipitated with streptavidin agarose beads. Proteins were eluted and probed in Western blots for β-DG (MAb 8D9), α-sarcoglycan (SG) (rabbit pAb R98), β-sarcoglycan (MAb 5B1), γ-sarcoglycan (MAb 21B5), δ-sarcoglycan (rabbit pAb R214), ε-sarcoglycan (rabbit pAb 155651), and sarcospan (SSN) (rabbit MAb 186730), followed by detection as described above for panel A. The positive control (+) was differentiated human myotube lysate. One representative example of two independent experiments is shown. (C) Flow chart for the live cell surface cross-linking approach. For details, please see text. (D) Chemical cross-linking of DG with sarcoglycans at the cell surface. Live intact A549 monolayers were treated with the membrane-impermeable thiol-cleavable cross-linking reagent DTSSP (+) or reaction buffer only (−). After quenching, cells were lysed, and cleared lysates were subjected to IP with MAb VIA4 matrix (α-DG) or anti-HA matrix (HA) as described above for panel A. Eluted proteins were treated with DTT and analyzed in Western blot probing for functional α-DG, β-DG, the indicated sacroglycans, and sarcospan as described above for panel B. Please note that the blots for α- and β-DG (top) correspond each to 1% of the material, and the blots for the sacroglycans and sarcospan each correspond to 15% of the sample. One representative example of two independent experiments is shown. (E) Schema of a working model of the DG complex in A549 cells (this study) compared to the DG complex in skeletal muscle (based on published data [ 40 , 41 ]). The α-DG-linked matriglycan sugar polymers and β-DG are indicated, as well as α-, β-, γ-, δ-, and ε-sarcoglycans (SG), sarcospan (SPN), α- and β-dystrobrevin (DTN), α1, β1, and β2-syntrophin (SNT), and nitric oxide synthase (nNOS).
    Figure Legend Snippet: Validation of candidate DG-binding proteins. (A) Coimmunoprecipitation (co-IP) of DG-associated scaffold proteins. Monolayers of A549 and SAEC were lysed in cold Triton X-100 containing buffer, and cleared lysates were subjected to IP with MAb VIA4 matrix (α-DG) using MAb anti-HA matrix (HA) as a control. Immunocomplexes were probed in Western blots for functional α-DG (MAb IIH6), β-DG (MAb 8D9), utrophin (MAb 20C5), α1-syntrophin (goat pAb 5941), β1-syntrophin (rabbit pAb 98977), β2-syntrophin (MAb 1351), and β-dystrobrevin (rabbit pAb 152133). Primary rabbit and goat antibodies as well as MAb IIH6 (mouse IgM) were detected as described in the legend to Fig. 1B and C . For detection of mouse MAb IgG, HRP-conjugated mouse TrueBlot ULTRA secondary antibody was used as detailed in Materials and Methods. For a positive control (+), total lysates of differentiated human myotubes were included. One representative example out of three independent experiments is shown. (B) Detection of sarcoglycans at the surfaces of A549 cells. Intact monolayers of A549 cells were subjected to cell surface biotinylation using the membrane-impermeable reagent sulfo-NHS-X-biotin (+) or reaction buffer only (−). After reaction quenching, cells were lysed, and biotinylated proteins were precipitated with streptavidin agarose beads. Proteins were eluted and probed in Western blots for β-DG (MAb 8D9), α-sarcoglycan (SG) (rabbit pAb R98), β-sarcoglycan (MAb 5B1), γ-sarcoglycan (MAb 21B5), δ-sarcoglycan (rabbit pAb R214), ε-sarcoglycan (rabbit pAb 155651), and sarcospan (SSN) (rabbit MAb 186730), followed by detection as described above for panel A. The positive control (+) was differentiated human myotube lysate. One representative example of two independent experiments is shown. (C) Flow chart for the live cell surface cross-linking approach. For details, please see text. (D) Chemical cross-linking of DG with sarcoglycans at the cell surface. Live intact A549 monolayers were treated with the membrane-impermeable thiol-cleavable cross-linking reagent DTSSP (+) or reaction buffer only (−). After quenching, cells were lysed, and cleared lysates were subjected to IP with MAb VIA4 matrix (α-DG) or anti-HA matrix (HA) as described above for panel A. Eluted proteins were treated with DTT and analyzed in Western blot probing for functional α-DG, β-DG, the indicated sacroglycans, and sarcospan as described above for panel B. Please note that the blots for α- and β-DG (top) correspond each to 1% of the material, and the blots for the sacroglycans and sarcospan each correspond to 15% of the sample. One representative example of two independent experiments is shown. (E) Schema of a working model of the DG complex in A549 cells (this study) compared to the DG complex in skeletal muscle (based on published data [ 40 , 41 ]). The α-DG-linked matriglycan sugar polymers and β-DG are indicated, as well as α-, β-, γ-, δ-, and ε-sarcoglycans (SG), sarcospan (SPN), α- and β-dystrobrevin (DTN), α1, β1, and β2-syntrophin (SNT), and nitric oxide synthase (nNOS).

    Techniques Used: Binding Assay, Co-Immunoprecipitation Assay, Western Blot, Functional Assay, Positive Control, Flow Cytometry

    Engagement of rLCMV-LASVGP affects receptor internalization. (A) Schematic of the virus-induced receptor internalization assay. For details, please see text. (B) Virus engagement affects receptor internalization. Monolayers of A549 cells were subjected to cell surface biotinylation with the thiol-cleavable membrane-impermeable reagent sulfo-NHS-SS-biotin in the cold. Labeled cells were then incubated with biotin-SS-labeled rLCMV-LASVGP (50 particles/cell) or a mock virus preparation for 1 h in the cold as described in the legend to Fig. 5D , followed by removal of unbound virus. Cells were rapidly shifted to 37°C at the indicated time points, chilled on ice, and treated with TCEP (+TCEP) or reaction buffer only (−TCEP). Residual TCEP was carefully quenched, and cells were lysed. Cleared lysates were subjected to IP with MAb VIA4 matrix (α-DG) or MAb 83.6 to GP2 as indicated. Biotinylated α-DG and LASV GP2 were detected with streptavidin-HRP in Western blots under nonreducing conditions using ECL. The anti-GP2 blots in the presence and absence of TCEP were exposed for 2 min and 10 s, respectively. The anti-α-DG blot with TCEP was exposed for 5 min, and the anti-α-DG blot without TCEP was exposed for 30 s. One representative example of three independent experiments is shown.
    Figure Legend Snippet: Engagement of rLCMV-LASVGP affects receptor internalization. (A) Schematic of the virus-induced receptor internalization assay. For details, please see text. (B) Virus engagement affects receptor internalization. Monolayers of A549 cells were subjected to cell surface biotinylation with the thiol-cleavable membrane-impermeable reagent sulfo-NHS-SS-biotin in the cold. Labeled cells were then incubated with biotin-SS-labeled rLCMV-LASVGP (50 particles/cell) or a mock virus preparation for 1 h in the cold as described in the legend to Fig. 5D , followed by removal of unbound virus. Cells were rapidly shifted to 37°C at the indicated time points, chilled on ice, and treated with TCEP (+TCEP) or reaction buffer only (−TCEP). Residual TCEP was carefully quenched, and cells were lysed. Cleared lysates were subjected to IP with MAb VIA4 matrix (α-DG) or MAb 83.6 to GP2 as indicated. Biotinylated α-DG and LASV GP2 were detected with streptavidin-HRP in Western blots under nonreducing conditions using ECL. The anti-GP2 blots in the presence and absence of TCEP were exposed for 2 min and 10 s, respectively. The anti-α-DG blot with TCEP was exposed for 5 min, and the anti-α-DG blot without TCEP was exposed for 30 s. One representative example of three independent experiments is shown.

    Techniques Used: Labeling, Incubation, Western Blot

    The molecular composition of the cellular DG complex can affect virus uptake. (A) Schematic representation of a working model of the DG-associated sarcoglycan complex in A549 cells and skeletal muscle. (B) Reconstitution of a muscle-type sarcoglycan complex in A549 cells. A549 cells were infected with recombinant AdV5 vectors expressing α-, β-, γ-, and δ-sarcoglycan (SG) and sarcospan (SSN) (A549m) or equal amounts of AdV5 expressing GFP (A549). After 48 h, cells were subjected to cell surface biotinylation with sulfo-NHS-X-biotin (biotin) or reaction buffer only (control) as described in the legend to Fig. 3B , and expression of the indicated proteins was assessed by Western blotting as described in the legend to Fig. 3 . The mildly enhanced signal for functional α-DG and the weaker band for ε-sarcoglycan were consistently observed. One representative example of three independent experiments is shown. (C) Schematic of the virus internalization assay (for details, please see text). (D) Expression of a muscle-type sarcoglycan complement in A549m cells affects viral uptake. A549m and A549 cells were chilled on ice and incubated with biotin-S-S-labeled rLCMV-LASVGP (50 particles/cell) for 1 h in the cold. Unbound virus was removed, and cells were shifted to 37°C. After the indicated time points, cells were chilled on ice and treated with TCEP (+TCEP) or reaction buffer only (−TCEP). After quenching of residual TCEP, cells were lysed, viral GP was isolated by IP with MAb 83.6 to GP2. Biotinylated GP2 was detected with streptavidin-HRP in Western blots under nonreducing conditions using enhanced chemiluminescence (ECL). The top blot (+TCEP) was exposed for 5 min, and the bottom blot (−TCEP) was exposed for 30 s. One representative example of three independent experiments is shown. (E) Quantification of the three independent experiments (D). The intensity of GP2 signals in the presence or absence of TCEP was assessed by densitometry, followed by calculation of the signal ratios. For normalization, the value for the 15-min time point for A549 control cells was set at 1. Data are means ± SD ( n = 3). (F) Infection of A549m and A549 cells. A549m and A549 cells in panel B were incubated with 300 PFU/well rLCMV-LASVGP (LASV) and rLCMV-VSVG (VSV), and infection was detected as described in the legend to Fig. 1D . Data are means ± SD ( n = 3). One representative example of three independent experiments is shown.
    Figure Legend Snippet: The molecular composition of the cellular DG complex can affect virus uptake. (A) Schematic representation of a working model of the DG-associated sarcoglycan complex in A549 cells and skeletal muscle. (B) Reconstitution of a muscle-type sarcoglycan complex in A549 cells. A549 cells were infected with recombinant AdV5 vectors expressing α-, β-, γ-, and δ-sarcoglycan (SG) and sarcospan (SSN) (A549m) or equal amounts of AdV5 expressing GFP (A549). After 48 h, cells were subjected to cell surface biotinylation with sulfo-NHS-X-biotin (biotin) or reaction buffer only (control) as described in the legend to Fig. 3B , and expression of the indicated proteins was assessed by Western blotting as described in the legend to Fig. 3 . The mildly enhanced signal for functional α-DG and the weaker band for ε-sarcoglycan were consistently observed. One representative example of three independent experiments is shown. (C) Schematic of the virus internalization assay (for details, please see text). (D) Expression of a muscle-type sarcoglycan complement in A549m cells affects viral uptake. A549m and A549 cells were chilled on ice and incubated with biotin-S-S-labeled rLCMV-LASVGP (50 particles/cell) for 1 h in the cold. Unbound virus was removed, and cells were shifted to 37°C. After the indicated time points, cells were chilled on ice and treated with TCEP (+TCEP) or reaction buffer only (−TCEP). After quenching of residual TCEP, cells were lysed, viral GP was isolated by IP with MAb 83.6 to GP2. Biotinylated GP2 was detected with streptavidin-HRP in Western blots under nonreducing conditions using enhanced chemiluminescence (ECL). The top blot (+TCEP) was exposed for 5 min, and the bottom blot (−TCEP) was exposed for 30 s. One representative example of three independent experiments is shown. (E) Quantification of the three independent experiments (D). The intensity of GP2 signals in the presence or absence of TCEP was assessed by densitometry, followed by calculation of the signal ratios. For normalization, the value for the 15-min time point for A549 control cells was set at 1. Data are means ± SD ( n = 3). (F) Infection of A549m and A549 cells. A549m and A549 cells in panel B were incubated with 300 PFU/well rLCMV-LASVGP (LASV) and rLCMV-VSVG (VSV), and infection was detected as described in the legend to Fig. 1D . Data are means ± SD ( n = 3). One representative example of three independent experiments is shown.

    Techniques Used: Infection, Recombinant, Expressing, Western Blot, Functional Assay, Incubation, Labeling, Isolation

    Differential signaling is required for steady-state DG turnover and DG-mediated viral entry. (A) Inhibition of rLCMV-LASVGP infection with selected inhibitors. A549 cells were pretreated with the Dyngo 4a, EIPA, jasplakinoline, IPA3, and EMD 1214063 at the indicated concentrations for 30 min, followed by infection with the rLCMV-LASVGP (LASV), rLCMV-VSVG (VSV), or AdV5 expressing GFP (AdV5) at 200 PFU/well in the presence of drug. After 1 h, cells were washed three times with medium containing 20 mM ammonium chloride, followed by 16 h of incubation in the presence of the lysosomotropic agent. Infection of rLCMV-LASVGP and rLCMV-VSVG was detected by IFA and AdV5-GFP by direct fluorescence as described in the legend to Fig. 1D . Data are means ± SD ( n = 3). (B) Pulse-chase assay of cell surface DG in the presence of inhibitors. Intact monolayers of A549 cells were pretreated with the indicated drugs (Dyngo 4A, EIPA, EMD 1214063, and IPA3 [40 µM each] and jasplakinoline [2 µM]), chilled on ice, and subjected to cell surface biotinylation with NHS-X-biotin as described in the legend to Fig. 6A . Biotinylated proteins were precipitated from 90% of lysates with agarose beads, whereas 10% of lysates underwent total protein extraction. Beta-DG was detected in Western blots as described in the legend to Fig. 1B . One representative example of three independent experiments is shown.
    Figure Legend Snippet: Differential signaling is required for steady-state DG turnover and DG-mediated viral entry. (A) Inhibition of rLCMV-LASVGP infection with selected inhibitors. A549 cells were pretreated with the Dyngo 4a, EIPA, jasplakinoline, IPA3, and EMD 1214063 at the indicated concentrations for 30 min, followed by infection with the rLCMV-LASVGP (LASV), rLCMV-VSVG (VSV), or AdV5 expressing GFP (AdV5) at 200 PFU/well in the presence of drug. After 1 h, cells were washed three times with medium containing 20 mM ammonium chloride, followed by 16 h of incubation in the presence of the lysosomotropic agent. Infection of rLCMV-LASVGP and rLCMV-VSVG was detected by IFA and AdV5-GFP by direct fluorescence as described in the legend to Fig. 1D . Data are means ± SD ( n = 3). (B) Pulse-chase assay of cell surface DG in the presence of inhibitors. Intact monolayers of A549 cells were pretreated with the indicated drugs (Dyngo 4A, EIPA, EMD 1214063, and IPA3 [40 µM each] and jasplakinoline [2 µM]), chilled on ice, and subjected to cell surface biotinylation with NHS-X-biotin as described in the legend to Fig. 6A . Biotinylated proteins were precipitated from 90% of lysates with agarose beads, whereas 10% of lysates underwent total protein extraction. Beta-DG was detected in Western blots as described in the legend to Fig. 1B . One representative example of three independent experiments is shown.

    Techniques Used: Inhibition, Infection, Expressing, Incubation, Immunofluorescence, Fluorescence, Pulse Chase, Protein Extraction, Western Blot

    Steady-state DG uptake and viral entry kinetics. (A) Pulse-chase assay to assess steady-state DG turnover in uninfected cells. Intact monolayers of A549 and MDCKII cells were chilled on ice and subjected to cell surface biotinylation with sulfo-NHS-biotin, followed by reaction quenching in the cold. Cells were washed, prewarmed complete medium was added, and the temperature was shifted to 37°C to restore membrane fluidity. At the indicated time points, cells were chilled on ice, and lysis was performed with cold detergent buffer. Biotinylated proteins were precipitated from 80% of lysates with agarose beads, whereas 20% of lysates underwent WGA purification. As a specificity control for cell surface biotinylation, blots were probed for tubulin in the biotinylated fraction, including 1% of total lysate as a positive control (+). Please note the negligible signal for tubulin in the biotinylated protein fraction, validating the specificity of cell surface labeling. Functional α-DG and β-DG were detected in Western blots as described in the legend to Fig. 1B . One representative example of three independent experiments is shown. (B) Quantification of one out of three representative experiments by densitometry, followed by calculation of the signal ratios of biotinylated α-DG/total α-DG (α-DG biotin /α-DG total ). (C) Generation of LAMP-1 null A549 cells. LAMP-1 was deleted from A549 cells by CRISPR/Cas9 as detailed in Materials and Methods and efficiency of depletion was assessed by Western blotting in seven individual clonal LAMP-1 null A549 cell lines using α-tubulin (Tub) as a loading control. (D) DG-dependent rVSVΔG-LASVGP infection of A549 cells depends on LAMP-1. The LAMP-1 null and control A549 cells (C) were infected with rVSV-LASVGP and rVSV-LCMVGP at 300 PFU/well for 1 h. Infection was assessed by direct fluorescence detection of the GFP reporter as described in the legend to Fig. 1D . Data are means plus SD ( n = 3). (E) Kinetics of the endosomal escape of virus. rLCMV-LASVGP and rVSV-VSVG at 300 PFU/well were attached to monolayers of the indicated cells in the cold for 2 h in complete medium. Unbound virus was removed, and the cells were rapidly shifted to 37°C. At the indicated time points, 20 mM ammonium chloride was added and left throughout the experiment. After 16 h, infection was assessed by IFA as described in the legend to Fig. 1D . The means ± SD ( n = 3) are given. Please note that the half-time of endosomal escape for rLCMV-LASVGP is
    Figure Legend Snippet: Steady-state DG uptake and viral entry kinetics. (A) Pulse-chase assay to assess steady-state DG turnover in uninfected cells. Intact monolayers of A549 and MDCKII cells were chilled on ice and subjected to cell surface biotinylation with sulfo-NHS-biotin, followed by reaction quenching in the cold. Cells were washed, prewarmed complete medium was added, and the temperature was shifted to 37°C to restore membrane fluidity. At the indicated time points, cells were chilled on ice, and lysis was performed with cold detergent buffer. Biotinylated proteins were precipitated from 80% of lysates with agarose beads, whereas 20% of lysates underwent WGA purification. As a specificity control for cell surface biotinylation, blots were probed for tubulin in the biotinylated fraction, including 1% of total lysate as a positive control (+). Please note the negligible signal for tubulin in the biotinylated protein fraction, validating the specificity of cell surface labeling. Functional α-DG and β-DG were detected in Western blots as described in the legend to Fig. 1B . One representative example of three independent experiments is shown. (B) Quantification of one out of three representative experiments by densitometry, followed by calculation of the signal ratios of biotinylated α-DG/total α-DG (α-DG biotin /α-DG total ). (C) Generation of LAMP-1 null A549 cells. LAMP-1 was deleted from A549 cells by CRISPR/Cas9 as detailed in Materials and Methods and efficiency of depletion was assessed by Western blotting in seven individual clonal LAMP-1 null A549 cell lines using α-tubulin (Tub) as a loading control. (D) DG-dependent rVSVΔG-LASVGP infection of A549 cells depends on LAMP-1. The LAMP-1 null and control A549 cells (C) were infected with rVSV-LASVGP and rVSV-LCMVGP at 300 PFU/well for 1 h. Infection was assessed by direct fluorescence detection of the GFP reporter as described in the legend to Fig. 1D . Data are means plus SD ( n = 3). (E) Kinetics of the endosomal escape of virus. rLCMV-LASVGP and rVSV-VSVG at 300 PFU/well were attached to monolayers of the indicated cells in the cold for 2 h in complete medium. Unbound virus was removed, and the cells were rapidly shifted to 37°C. At the indicated time points, 20 mM ammonium chloride was added and left throughout the experiment. After 16 h, infection was assessed by IFA as described in the legend to Fig. 1D . The means ± SD ( n = 3) are given. Please note that the half-time of endosomal escape for rLCMV-LASVGP is

    Techniques Used: Pulse Chase, Lysis, Whole Genome Amplification, Purification, Positive Control, Labeling, Functional Assay, Western Blot, CRISPR, Infection, Fluorescence, Immunofluorescence

    16) Product Images from "Release probability of hippocampal glutamatergic terminals scales with the size of the active zone"

    Article Title: Release probability of hippocampal glutamatergic terminals scales with the size of the active zone

    Journal: Nature neuroscience

    doi: 10.1038/nn.3137

    Measurement of volume averaged [Ca 2+ ] transients in CA3 pyramidal cell local axon terminals. ( a ) Two-photon image of a CA3 pyramidal cell filled with Alexa594 (red) and Fluo5F. ( b ) Higher magnification view of two boutons. White lines indicate the position of the line scans. ( c ) Neurolucida reconstruction of the cell shown in panel a . The majority of the axon (red) is truncated for clarity. Boxed area, enlarged in panel f, showing the scanned axon collateral segment. ( d ) Individual [Ca 2+ ] transients in 25 axon terminals (shown in panel f ) of the pyramidal cell. ( e ) Distribution of the peak amplitudes measured in 4 cells. ( f ) The two-photon image (upper panel) is superimposed (middle panel) on the transmitted light microscopic image (lower panel) following aldehyde fixation and visualization of the intracellular biocytin with diaminobenzidine. ( g ) [Ca 2+ ] transients from two boutons are shown (b3, b7). ( h-i ) Electron microscopic images (left) and 3D reconstructions (right) of two boutons (b3: blue, b7: green) that established excitatory synapses on pyramidal cell spines (yellow, arrowheads demarcate the PSDs). ( j ) Peak amplitude of the [Ca 2+ ] transients does not correlate with the bouton volume (p = 0.3, n = 27 boutons, n = 4 cells, n = 4 animals). ( k ) The peak [Ca 2+ ] shows a significant (p
    Figure Legend Snippet: Measurement of volume averaged [Ca 2+ ] transients in CA3 pyramidal cell local axon terminals. ( a ) Two-photon image of a CA3 pyramidal cell filled with Alexa594 (red) and Fluo5F. ( b ) Higher magnification view of two boutons. White lines indicate the position of the line scans. ( c ) Neurolucida reconstruction of the cell shown in panel a . The majority of the axon (red) is truncated for clarity. Boxed area, enlarged in panel f, showing the scanned axon collateral segment. ( d ) Individual [Ca 2+ ] transients in 25 axon terminals (shown in panel f ) of the pyramidal cell. ( e ) Distribution of the peak amplitudes measured in 4 cells. ( f ) The two-photon image (upper panel) is superimposed (middle panel) on the transmitted light microscopic image (lower panel) following aldehyde fixation and visualization of the intracellular biocytin with diaminobenzidine. ( g ) [Ca 2+ ] transients from two boutons are shown (b3, b7). ( h-i ) Electron microscopic images (left) and 3D reconstructions (right) of two boutons (b3: blue, b7: green) that established excitatory synapses on pyramidal cell spines (yellow, arrowheads demarcate the PSDs). ( j ) Peak amplitude of the [Ca 2+ ] transients does not correlate with the bouton volume (p = 0.3, n = 27 boutons, n = 4 cells, n = 4 animals). ( k ) The peak [Ca 2+ ] shows a significant (p

    Techniques Used:

    17) Product Images from "Activation of Group I Metabotropic Glutamate Receptors on Main Olfactory Bulb Granule Cells and Periglomerular Cells Enhances Synaptic Inhibition of Mitral Cells"

    Article Title: Activation of Group I Metabotropic Glutamate Receptors on Main Olfactory Bulb Granule Cells and Periglomerular Cells Enhances Synaptic Inhibition of Mitral Cells

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    doi: 10.1523/JNEUROSCI.5495-06.2007

    DHPG failed to increase mIPSC frequency in sub-GL slices. A1 , A2 , Examples of biocytin-filled mitral cells recorded in intact ( A1 ) and sub-GL ( A2 ) slices; arrows mark the apical dendrites. Note that in A2 , the GL and ONL have been excised and the mitral cell apical dendrite is truncated within the EPL. B , Voltage-clamp recordings in normal ACSF show that the frequency of sIPSCs in mitral cells is higher in intact slices (top trace) than in sub-GL slices (bottom trace). C , DHPG readily increased sIPSCs in a mitral cell recorded in a sub-GL slice. D , Cumulative inter-sIPSC interval distributions of the mitral cell in C showed that DHPG significantly increased the frequency of sIPSCs ( p
    Figure Legend Snippet: DHPG failed to increase mIPSC frequency in sub-GL slices. A1 , A2 , Examples of biocytin-filled mitral cells recorded in intact ( A1 ) and sub-GL ( A2 ) slices; arrows mark the apical dendrites. Note that in A2 , the GL and ONL have been excised and the mitral cell apical dendrite is truncated within the EPL. B , Voltage-clamp recordings in normal ACSF show that the frequency of sIPSCs in mitral cells is higher in intact slices (top trace) than in sub-GL slices (bottom trace). C , DHPG readily increased sIPSCs in a mitral cell recorded in a sub-GL slice. D , Cumulative inter-sIPSC interval distributions of the mitral cell in C showed that DHPG significantly increased the frequency of sIPSCs ( p

    Techniques Used:

    18) Product Images from "Inactivation of Tm6sf2, a Gene Defective in Fatty Liver Disease, Impairs Lipidation but Not Secretion of Very Low Density Lipoproteins *"

    Article Title: Inactivation of Tm6sf2, a Gene Defective in Fatty Liver Disease, Impairs Lipidation but Not Secretion of Very Low Density Lipoproteins *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.719955

    Plasma levels of lipids, lipoproteins, PCSK9, and apoB in Tm6sf2 −/− mice. A ( left ), plasma TG and cholesterol levels were measured in chow-fed male WT, Tm6sf2 +/− , and Tm6sf2 −/− mice ( n = 5 mice/group, 13 weeks old) using enzymatic assays. A ( right ), fast protein liquid chromatography (FPLC) profiles of plasma samples pooled from WT and Tm6sf2 −/− mice (4 male mice/group, 10 weeks old). Cholesterol ( top ) and TG ( bottom ) were measured in each fraction. Experiments were repeated twice with similar results. B , levels of plasma PCSK9 in 11-week-old male chow-fed male mice. Mice ( n = 5) were metabolically synchronized for 3 days by fasting from 8:00 a.m. to 8:00 p.m. and refed overnight. Blood was collected after the last refeeding period (at 8:00 a.m.), and the plasma levels of PCSK9 were detected using an ELISA as described under “Experimental Procedures.” C , plasma (0.2 μl) was size-fractionated on a 4–12% gradient SDS-polyacrylamide gel, and levels of apoB-48 and apoB-100 were determined by immunoblotting analysis using a rabbit polyclonal antibody (Abcam, ab20737; 1:1,000) The signal was detected and quantified using a LI-COR Odyssey Fc imager. Fibronectin was used as a loading control. Values are means ± S.E. ( error bars ). D , VLDL particles from plasma of WT and KO female mice ( n = 3, 20 weeks old) were visualized by electron microscopy as described under “Experimental Procedures.” The size distribution of VLDL particles in 10 randomly selected images was analyzed and compared using ImageJ software as described under “Experimental Procedures.” *, p
    Figure Legend Snippet: Plasma levels of lipids, lipoproteins, PCSK9, and apoB in Tm6sf2 −/− mice. A ( left ), plasma TG and cholesterol levels were measured in chow-fed male WT, Tm6sf2 +/− , and Tm6sf2 −/− mice ( n = 5 mice/group, 13 weeks old) using enzymatic assays. A ( right ), fast protein liquid chromatography (FPLC) profiles of plasma samples pooled from WT and Tm6sf2 −/− mice (4 male mice/group, 10 weeks old). Cholesterol ( top ) and TG ( bottom ) were measured in each fraction. Experiments were repeated twice with similar results. B , levels of plasma PCSK9 in 11-week-old male chow-fed male mice. Mice ( n = 5) were metabolically synchronized for 3 days by fasting from 8:00 a.m. to 8:00 p.m. and refed overnight. Blood was collected after the last refeeding period (at 8:00 a.m.), and the plasma levels of PCSK9 were detected using an ELISA as described under “Experimental Procedures.” C , plasma (0.2 μl) was size-fractionated on a 4–12% gradient SDS-polyacrylamide gel, and levels of apoB-48 and apoB-100 were determined by immunoblotting analysis using a rabbit polyclonal antibody (Abcam, ab20737; 1:1,000) The signal was detected and quantified using a LI-COR Odyssey Fc imager. Fibronectin was used as a loading control. Values are means ± S.E. ( error bars ). D , VLDL particles from plasma of WT and KO female mice ( n = 3, 20 weeks old) were visualized by electron microscopy as described under “Experimental Procedures.” The size distribution of VLDL particles in 10 randomly selected images was analyzed and compared using ImageJ software as described under “Experimental Procedures.” *, p

    Techniques Used: Mouse Assay, Fast Protein Liquid Chromatography, Metabolic Labelling, Enzyme-linked Immunosorbent Assay, Electron Microscopy, Software

    19) Product Images from "Purification and Characterization of Mammalian Glucose Transporters Expressed in Pichia Pastoris"

    Article Title: Purification and Characterization of Mammalian Glucose Transporters Expressed in Pichia Pastoris

    Journal: Protein expression and purification

    doi: 10.1016/j.pep.2009.10.011

    Photolabeling of aglyco-GLUT1 and aglyco-GLUT4 with PEG-biotincap-ATB-BMPA Eluted fractions from the Ni-column were desalted by gel filtration and analyzed on 10 % SDS polyacrylamide gels and detected by staining with Coomassie G250 and by western blot analysis using specific antibodies. a : molecular weight markers; b and c : aglyco-GLUT1; f and g : aglyco-GLUT4. Fifteen micrograms of purified protein was incubated in the presence of 4 µM PEG-biotincap-ATB-BMPA without or with1 µM cytochalasin B and then subjected to irradiation for 1 min at 18 °C. The reaction mixes were centrifuged through G-25 Sepharose spin columns and the flow-throughs were loaded onto 10 % SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and probed with streptavidin-HRP.
    Figure Legend Snippet: Photolabeling of aglyco-GLUT1 and aglyco-GLUT4 with PEG-biotincap-ATB-BMPA Eluted fractions from the Ni-column were desalted by gel filtration and analyzed on 10 % SDS polyacrylamide gels and detected by staining with Coomassie G250 and by western blot analysis using specific antibodies. a : molecular weight markers; b and c : aglyco-GLUT1; f and g : aglyco-GLUT4. Fifteen micrograms of purified protein was incubated in the presence of 4 µM PEG-biotincap-ATB-BMPA without or with1 µM cytochalasin B and then subjected to irradiation for 1 min at 18 °C. The reaction mixes were centrifuged through G-25 Sepharose spin columns and the flow-throughs were loaded onto 10 % SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and probed with streptavidin-HRP.

    Techniques Used: Filtration, Staining, Western Blot, Molecular Weight, Purification, Incubation, Irradiation, Flow Cytometry

    20) Product Images from "Discovery of a microbial transglutaminase enabling highly site-specific labeling of proteins"

    Article Title: Discovery of a microbial transglutaminase enabling highly site-specific labeling of proteins

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M117.797811

    Enzymatic biotinylation by MTG and KalbTG and functional characterization of anti-TSH IgGs tagged C-terminally either with LLQGA or YRYRQ motifs. Shown are size-exclusion chromatography profiles of SA-FLUO–IgG-biotin complexes and unbound antibodies. Black chromatograms , protein absorbance (280 nm); red chromatograms , fluorescein absorbance (494 nm). A , SA-FLUO analytics of LLQGA-tagged antibody (TU1.20), labeled with MTG and a 100-fold excess of biotin-dPEG(23)-NH 2 . Peaks 2 , 3 , and 4 , SA-FLUO molecules complexed with two, one, or no biotinylated IgGs, respectively. Peak 1 , larger, undefined SA-FLUO–IgG complexes. B , SA-FLUO analytics of LLQGA-tagged antibody (TU1.20), incubated with KalbTG and a 100-fold excess of biotin-dPEG(23)-NH 2 . Peaks 1 and 2 , unbiotinylated IgG and unbound SA-FLUO, respectively. C , SA-FLUO analytics of YRYRQGGS-tagged antibody (TU1.20), labeled with KalbTG and a 25-fold excess of biotin-dPEG(23)-NH 2 . Peaks 1 , 2 , and 3 , SA-FLUO molecules complexed with two, one, or no biotinylated IgGs, respectively. D and E , enhanced selectivity of KalbTG for K-tag amino donors. SA-FLUO analytics of YRYRQGGS-tagged antibodies (TU1.20) conjugated by KalbTG with a 5-fold excess of either K-tag-biotin label ( D ) or biotin-dPEG(23)-NH 2 label ( E ). Peaks 1 , 2 , and 4 , SA-FLUO molecules complexed with two, one, or no biotinylated IgGs, respectively. Peak 3 , unbiotinylated IgG. F , performance of the purified IgG-biotin conjugates from A (LLQGA MTG NH 2 -biotin), C (YRYRQ KalbTG NH 2 -biotin), and D (YRYRQ KalbTG K-tag-biotin) in the TSH Elecsys immunoassay. The TSH sandwich assay principle is shown schematically.
    Figure Legend Snippet: Enzymatic biotinylation by MTG and KalbTG and functional characterization of anti-TSH IgGs tagged C-terminally either with LLQGA or YRYRQ motifs. Shown are size-exclusion chromatography profiles of SA-FLUO–IgG-biotin complexes and unbound antibodies. Black chromatograms , protein absorbance (280 nm); red chromatograms , fluorescein absorbance (494 nm). A , SA-FLUO analytics of LLQGA-tagged antibody (TU1.20), labeled with MTG and a 100-fold excess of biotin-dPEG(23)-NH 2 . Peaks 2 , 3 , and 4 , SA-FLUO molecules complexed with two, one, or no biotinylated IgGs, respectively. Peak 1 , larger, undefined SA-FLUO–IgG complexes. B , SA-FLUO analytics of LLQGA-tagged antibody (TU1.20), incubated with KalbTG and a 100-fold excess of biotin-dPEG(23)-NH 2 . Peaks 1 and 2 , unbiotinylated IgG and unbound SA-FLUO, respectively. C , SA-FLUO analytics of YRYRQGGS-tagged antibody (TU1.20), labeled with KalbTG and a 25-fold excess of biotin-dPEG(23)-NH 2 . Peaks 1 , 2 , and 3 , SA-FLUO molecules complexed with two, one, or no biotinylated IgGs, respectively. D and E , enhanced selectivity of KalbTG for K-tag amino donors. SA-FLUO analytics of YRYRQGGS-tagged antibodies (TU1.20) conjugated by KalbTG with a 5-fold excess of either K-tag-biotin label ( D ) or biotin-dPEG(23)-NH 2 label ( E ). Peaks 1 , 2 , and 4 , SA-FLUO molecules complexed with two, one, or no biotinylated IgGs, respectively. Peak 3 , unbiotinylated IgG. F , performance of the purified IgG-biotin conjugates from A (LLQGA MTG NH 2 -biotin), C (YRYRQ KalbTG NH 2 -biotin), and D (YRYRQ KalbTG K-tag-biotin) in the TSH Elecsys immunoassay. The TSH sandwich assay principle is shown schematically.

    Techniques Used: Functional Assay, Size-exclusion Chromatography, Labeling, Incubation, Purification

    21) Product Images from "Intracellular translocation and differential accumulation of cell-penetrating peptides in bovine spermatozoa: evaluation of efficient delivery vectors that do not compromise human sperm motility"

    Article Title: Intracellular translocation and differential accumulation of cell-penetrating peptides in bovine spermatozoa: evaluation of efficient delivery vectors that do not compromise human sperm motility

    Journal: Human Reproduction (Oxford, England)

    doi: 10.1093/humrep/det064

    CPP-mediated protein delivery into bovine spermatozoa. ( A ) Biotinyl-CPP constructs of tat and penetratin were synthesized and complexed with avidin Terxas Red (TXR) at a 3:1 molar ratio to assess the utility of CPP for the intracellular delivery of large
    Figure Legend Snippet: CPP-mediated protein delivery into bovine spermatozoa. ( A ) Biotinyl-CPP constructs of tat and penetratin were synthesized and complexed with avidin Terxas Red (TXR) at a 3:1 molar ratio to assess the utility of CPP for the intracellular delivery of large

    Techniques Used: Conditioned Place Preference, Construct, Synthesized, Avidin-Biotin Assay

    22) Product Images from "Pimozide suppresses cancer cell migration and tumor metastasis through binding to ARPC2, a subunit of the Arp2/3 complex, et al. Pimozide suppresses cancer cell migration and tumor metastasis through binding to ARPC2, a subunit of the Arp2/3 complex"

    Article Title: Pimozide suppresses cancer cell migration and tumor metastasis through binding to ARPC2, a subunit of the Arp2/3 complex, et al. Pimozide suppresses cancer cell migration and tumor metastasis through binding to ARPC2, a subunit of the Arp2/3 complex

    Journal: Cancer Science

    doi: 10.1111/cas.14205

    Direct binding of pimozide with ARPC2. A, DLD‐1 cell lysates were incubated in the presence or absence of pimozide (100 or 200 μmol/L) for 1 h at room temperature, followed by proteolysis with various pronases in a dose‐dependent way. GAPDH, which served as the loading control, is relatively resistant to proteolysis (n = 3). B, DLD‐1 cells were treated for 12 h with 10 μmol/L pimozide, and then CETSA was carried out to measure binding ability. Pimozide increased the thermal stability of ARPC2 compared with DMSO. Vinculin is a nontarget protein of pimozide (n = 3). Band intensity was quantified using the MultiGauge program. C, DLD‐1 cell lysates was incubated with 20 μmol/L biotinyl benproperine (biotinyl‐Benp) and competed with pimozide at the indicated concentration. Proteins were captured with NeutrAvidin‐Agarose resin (ThermoFisher Scientific Inc., Waltham, MA, USA) and eluted proteins were analyzed by western blotting (n = 2). D, Chemical structure of N‐methyl‐pimozide. E, Cell migration assay of DLD‐1 cells that were treated with DMSO, pimozide, or N‐methyl‐pimozide for 18 h and quantification of the migrated cells (n = 3). Scale bars, 200 μm. F, Pull‐down assay with biotinyl‐Benp was done in the absence or presence of compounds as competitor (100 μM). Bound proteins on the beads were separated by SDS‐PAGE, and western blot was carried out using anti‐ARPC2 and anti‐GAPDH antibodies (n = 2). Data represent the means ± SD; * P
    Figure Legend Snippet: Direct binding of pimozide with ARPC2. A, DLD‐1 cell lysates were incubated in the presence or absence of pimozide (100 or 200 μmol/L) for 1 h at room temperature, followed by proteolysis with various pronases in a dose‐dependent way. GAPDH, which served as the loading control, is relatively resistant to proteolysis (n = 3). B, DLD‐1 cells were treated for 12 h with 10 μmol/L pimozide, and then CETSA was carried out to measure binding ability. Pimozide increased the thermal stability of ARPC2 compared with DMSO. Vinculin is a nontarget protein of pimozide (n = 3). Band intensity was quantified using the MultiGauge program. C, DLD‐1 cell lysates was incubated with 20 μmol/L biotinyl benproperine (biotinyl‐Benp) and competed with pimozide at the indicated concentration. Proteins were captured with NeutrAvidin‐Agarose resin (ThermoFisher Scientific Inc., Waltham, MA, USA) and eluted proteins were analyzed by western blotting (n = 2). D, Chemical structure of N‐methyl‐pimozide. E, Cell migration assay of DLD‐1 cells that were treated with DMSO, pimozide, or N‐methyl‐pimozide for 18 h and quantification of the migrated cells (n = 3). Scale bars, 200 μm. F, Pull‐down assay with biotinyl‐Benp was done in the absence or presence of compounds as competitor (100 μM). Bound proteins on the beads were separated by SDS‐PAGE, and western blot was carried out using anti‐ARPC2 and anti‐GAPDH antibodies (n = 2). Data represent the means ± SD; * P

    Techniques Used: Binding Assay, Incubation, Concentration Assay, Western Blot, Cell Migration Assay, Pull Down Assay, SDS Page

    23) Product Images from "Norrin-induced Frizzled4 endocytosis and endo-lysosomal trafficking control retinal angiogenesis and barrier function"

    Article Title: Norrin-induced Frizzled4 endocytosis and endo-lysosomal trafficking control retinal angiogenesis and barrier function

    Journal: Nature Communications

    doi: 10.1038/ncomms16050

    Inhibition of endo-lysosomal trafficking in vascular endothelial cells in vivo . ( a ) Blood-CNS barrier defects are revealed in the cortex and hippocampus of Cre-activated Rosa 26 LSL-VPS4 EQ mice transcardially perfused with Sulfo-NHS-biotin by staining with Streptavidin coupled to Alexa 555. ( b ) Barrier defects in the molecular layer of the cerebellum. ( c ) Retinal sections show failure of intraretinal vascularization and strongly increased PLVAP expression in VPS4 EQ expressing mice. These defects recapitulate hallmark phenotypes of impaired Norrin/FZD4 signalling in the retinal vasculature. NFL, nerve fiber layer; IPL, inner plexiform layer; OPL, outer plexiform layer. ( d ) Confocal projections of the retinal vasculature in P10 whole mount retinas stained with IsolectinB4-Alexa488. Characteristic Norrin LOF phenotypes are observed. Progression towards the rim of the retina (indicated by white dashed line) is delayed (distance from the vascular front to the rim of the retina indicated by white bar) and the microscopic appearance of the vascular front is altered (arrowheads). ( e , f ) Optical sections of the boxed areas in d show separate representations of the vascular plexuses located at different depth levels of the retina. Normal vasculature is present in the nerve fiber layer (NFL) and outer plexiform layer (OPL) in control mice, whereas intraretinal capillaries in the OPL of VPS4 EQ expressing mice are absent. ( g ) Colour depth projections show the vascular beds of the boxed areas in d as single projection, in which distinct depth levels are colour coded. These projections reveal the characteristic glomeruloid vascular malformations (yellow structures, arrowheads) in the nerve fiber layer vasculature, similar to the defects described in Fzd4 , Ndp , Lrp5 and Tspan12 mutant mice.
    Figure Legend Snippet: Inhibition of endo-lysosomal trafficking in vascular endothelial cells in vivo . ( a ) Blood-CNS barrier defects are revealed in the cortex and hippocampus of Cre-activated Rosa 26 LSL-VPS4 EQ mice transcardially perfused with Sulfo-NHS-biotin by staining with Streptavidin coupled to Alexa 555. ( b ) Barrier defects in the molecular layer of the cerebellum. ( c ) Retinal sections show failure of intraretinal vascularization and strongly increased PLVAP expression in VPS4 EQ expressing mice. These defects recapitulate hallmark phenotypes of impaired Norrin/FZD4 signalling in the retinal vasculature. NFL, nerve fiber layer; IPL, inner plexiform layer; OPL, outer plexiform layer. ( d ) Confocal projections of the retinal vasculature in P10 whole mount retinas stained with IsolectinB4-Alexa488. Characteristic Norrin LOF phenotypes are observed. Progression towards the rim of the retina (indicated by white dashed line) is delayed (distance from the vascular front to the rim of the retina indicated by white bar) and the microscopic appearance of the vascular front is altered (arrowheads). ( e , f ) Optical sections of the boxed areas in d show separate representations of the vascular plexuses located at different depth levels of the retina. Normal vasculature is present in the nerve fiber layer (NFL) and outer plexiform layer (OPL) in control mice, whereas intraretinal capillaries in the OPL of VPS4 EQ expressing mice are absent. ( g ) Colour depth projections show the vascular beds of the boxed areas in d as single projection, in which distinct depth levels are colour coded. These projections reveal the characteristic glomeruloid vascular malformations (yellow structures, arrowheads) in the nerve fiber layer vasculature, similar to the defects described in Fzd4 , Ndp , Lrp5 and Tspan12 mutant mice.

    Techniques Used: Inhibition, In Vivo, Mouse Assay, Staining, Expressing, Mutagenesis

    24) Product Images from "The Endoplasmic Reticulum Membrane Is Permeable to Small Molecules"

    Article Title: The Endoplasmic Reticulum Membrane Is Permeable to Small Molecules

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E03-05-0325

    Modification of metabolically labeled ER proteins occurs only upon cell permeabilization. (A) HeLa cells expressing HA-tagged MHC class I heavy chains with lysine residues only in the luminal domain (HA-heavy chain; K-R) were labeled with [ 35 S]methionine and -cysteine. The cells were permeabilized with digitonin (Dig), as indicated, and treated with the amino group modifying reagent sulfo-NHS-biotin. The heavy chains were immunoprecipitated with HA antibodies. 20% of the immunoprecipitated material was analyzed directly by SDS-PAGE (lanes 1-4), and 80% was incubated with streptavidin beads to detect biotinylated material (lanes 5-8). Where indicated, one-half of each sample was treated with Endo H. All samples were subjected to SDS-PAGE followed by autoradiography. (B) Hybridoma B cells expressing IgG were analyzed as in A, except that the labeled IgG was collected with protein A/G-Sepharose.
    Figure Legend Snippet: Modification of metabolically labeled ER proteins occurs only upon cell permeabilization. (A) HeLa cells expressing HA-tagged MHC class I heavy chains with lysine residues only in the luminal domain (HA-heavy chain; K-R) were labeled with [ 35 S]methionine and -cysteine. The cells were permeabilized with digitonin (Dig), as indicated, and treated with the amino group modifying reagent sulfo-NHS-biotin. The heavy chains were immunoprecipitated with HA antibodies. 20% of the immunoprecipitated material was analyzed directly by SDS-PAGE (lanes 1-4), and 80% was incubated with streptavidin beads to detect biotinylated material (lanes 5-8). Where indicated, one-half of each sample was treated with Endo H. All samples were subjected to SDS-PAGE followed by autoradiography. (B) Hybridoma B cells expressing IgG were analyzed as in A, except that the labeled IgG was collected with protein A/G-Sepharose.

    Techniques Used: Modification, Metabolic Labelling, Labeling, Expressing, Immunoprecipitation, SDS Page, Incubation, Autoradiography

    Rough microsomes are permeable to a modification reagent. Radiolabeled full-length ppαF was translocated into rough microsomes. The membranes were isolated and incubated with different concentrations of sulfo-NHS-biotin, an amino group-modifying reagent, for 10 min on ice or at 30°C. Where indicated, 0.5% TX-100 was present during the modification reaction. One portion of the samples was analyzed directly (total), the other was incubated with streptavidin beads and the bound fraction was analyzed (beads). All samples were subjected to SDS-PAGE and autoradiography. ppαF, gppαF, pro-α-factor without or with three carbohydrate chains, respectively. Molecular masses (in kilodaltons) are indicated on the right side.
    Figure Legend Snippet: Rough microsomes are permeable to a modification reagent. Radiolabeled full-length ppαF was translocated into rough microsomes. The membranes were isolated and incubated with different concentrations of sulfo-NHS-biotin, an amino group-modifying reagent, for 10 min on ice or at 30°C. Where indicated, 0.5% TX-100 was present during the modification reaction. One portion of the samples was analyzed directly (total), the other was incubated with streptavidin beads and the bound fraction was analyzed (beads). All samples were subjected to SDS-PAGE and autoradiography. ppαF, gppαF, pro-α-factor without or with three carbohydrate chains, respectively. Molecular masses (in kilodaltons) are indicated on the right side.

    Techniques Used: Modification, Isolation, Incubation, SDS Page, Autoradiography

    The ER membrane does not constitute a significant barrier to small molecules. HeLa cells were permeabilized with 0.04% digitonin (A) or SLO (B) and incubated with 0.5 mM sulfo-NHS-LC-biotin for different periods of time. Biotinylated proteins were recovered after incubation with streptavidin beads and analyzed by SDS-PAGE and immunoblotting with antibodies to different proteins. The bands were quantitated using a Fujix PhosporIimager and the software Image Gauge 3.0.
    Figure Legend Snippet: The ER membrane does not constitute a significant barrier to small molecules. HeLa cells were permeabilized with 0.04% digitonin (A) or SLO (B) and incubated with 0.5 mM sulfo-NHS-LC-biotin for different periods of time. Biotinylated proteins were recovered after incubation with streptavidin beads and analyzed by SDS-PAGE and immunoblotting with antibodies to different proteins. The bands were quantitated using a Fujix PhosporIimager and the software Image Gauge 3.0.

    Techniques Used: Incubation, SDS Page, Software

    Time course of modification of ppαF. (A) Radiolabeled full-length ppαF with a cysteine at position 56 was translocated into rough microsomes. The samples were centrifuged resulting in P and S fractions. The membranes were incubated with 50 μM MB for different time periods on ice. One-half of the samples was analyzed after TCA precipitation; the other was incubated with streptavidin beads and the bound material was analyzed. ppαF, gppαF, pro-α-factor without or with three carbohydrate chains, respectively. (B) Quantification of the experiment in A.
    Figure Legend Snippet: Time course of modification of ppαF. (A) Radiolabeled full-length ppαF with a cysteine at position 56 was translocated into rough microsomes. The samples were centrifuged resulting in P and S fractions. The membranes were incubated with 50 μM MB for different time periods on ice. One-half of the samples was analyzed after TCA precipitation; the other was incubated with streptavidin beads and the bound material was analyzed. ppαF, gppαF, pro-α-factor without or with three carbohydrate chains, respectively. (B) Quantification of the experiment in A.

    Techniques Used: Modification, Incubation, TCA Precipitation

    An endogenous luminal ER protein is accessible to a modification reagent. (A) Ribosome-stripped microsomes (PK-RM) were incubated with 100 μM MB on ice for different time periods in the absence or presence of 0.5% TX-100. The samples were immunoprecipitated with antibodies to PDI, subjected to SDS-PAGE, transferred onto a nitrocellulose filter, and incubated with peroxidase-coupled streptavidin. (B) As in A, but immunoprecipitation with antibodies to Sec61α.
    Figure Legend Snippet: An endogenous luminal ER protein is accessible to a modification reagent. (A) Ribosome-stripped microsomes (PK-RM) were incubated with 100 μM MB on ice for different time periods in the absence or presence of 0.5% TX-100. The samples were immunoprecipitated with antibodies to PDI, subjected to SDS-PAGE, transferred onto a nitrocellulose filter, and incubated with peroxidase-coupled streptavidin. (B) As in A, but immunoprecipitation with antibodies to Sec61α.

    Techniques Used: Modification, Incubation, Immunoprecipitation, SDS Page

    Ribosome-stripped ER membranes are permeable to small molecules. (A) Ribosomes carrying a radiolabeled fragment of ppαF (86mer) with a single cysteine at position 56 were incubated with ribosome-stripped microsomes (PK-RM). After treatment with puromycin to release the nascent chains into the ER lumen, the samples were incubated with 0.1 mM MB or 0.075 mM MPB for 10 min on ice, either in the absence of detergent, or in the presence of 0.5% TX-100 or 0.1% digitonin, as indicated. One-half of the samples was analyzed after precipitation with TCA (total), the other was subjected to incubation with streptavidin beads, and the bound (beads) and unbound (supernatant) fractions were analyzed. All samples were analyzed by SDS-PAGE and autoradiography. The stars indicate molecules with one to three carbohydrate chains. (B) Full-length ppαF with a single cysteine at position 56 was imported into untreated RMs. Membranes were pelleted before the modification reaction was carried out. The samples were then treated for 10 min on ice with the indicated concentrations of MB in the absence or presence of 0.5% Triton X-100. One half of the sample was analyzed directly (total), the other was subjected to incubation with streptavidin beads and the bound material was analyzed (beads). All samples were subjected to SDS-PAGE and autoradiography. ppαF, gppαF, pro-α-factor without or with one to three carbohydrate chains, respectively. Molecular masses (in kilodaltons) are indicated on the right side.
    Figure Legend Snippet: Ribosome-stripped ER membranes are permeable to small molecules. (A) Ribosomes carrying a radiolabeled fragment of ppαF (86mer) with a single cysteine at position 56 were incubated with ribosome-stripped microsomes (PK-RM). After treatment with puromycin to release the nascent chains into the ER lumen, the samples were incubated with 0.1 mM MB or 0.075 mM MPB for 10 min on ice, either in the absence of detergent, or in the presence of 0.5% TX-100 or 0.1% digitonin, as indicated. One-half of the samples was analyzed after precipitation with TCA (total), the other was subjected to incubation with streptavidin beads, and the bound (beads) and unbound (supernatant) fractions were analyzed. All samples were analyzed by SDS-PAGE and autoradiography. The stars indicate molecules with one to three carbohydrate chains. (B) Full-length ppαF with a single cysteine at position 56 was imported into untreated RMs. Membranes were pelleted before the modification reaction was carried out. The samples were then treated for 10 min on ice with the indicated concentrations of MB in the absence or presence of 0.5% Triton X-100. One half of the sample was analyzed directly (total), the other was subjected to incubation with streptavidin beads and the bound material was analyzed (beads). All samples were subjected to SDS-PAGE and autoradiography. ppαF, gppαF, pro-α-factor without or with one to three carbohydrate chains, respectively. Molecular masses (in kilodaltons) are indicated on the right side.

    Techniques Used: Incubation, SDS Page, Autoradiography, Modification

    A small molecule passes into the ER but not into lysosomes or the trans -Golgi. HeLa cells (2 × 10 6 ) were permeabilized with SLO, 0.04% digitonin (Dig), or 0.1% digitonin as indicated. Then 0.5 mM sulfo-NHS-LC-biotin was added for 30 min on ice. After solubilization in detergent, cell lysates were incubated with streptavidin agarose beads, and the bound material was analyzed by SDS-PAGE and immunoblotting with antibodies to various proteins. Hex B, hexosaminidase B; Cath D, cathepsin D; βGT1; β-1,4-galactosyl transferase. For βGT1, twice as much sample was used. Cell lysate corresponding to 10% of the input material was loaded as a control (lane 1).
    Figure Legend Snippet: A small molecule passes into the ER but not into lysosomes or the trans -Golgi. HeLa cells (2 × 10 6 ) were permeabilized with SLO, 0.04% digitonin (Dig), or 0.1% digitonin as indicated. Then 0.5 mM sulfo-NHS-LC-biotin was added for 30 min on ice. After solubilization in detergent, cell lysates were incubated with streptavidin agarose beads, and the bound material was analyzed by SDS-PAGE and immunoblotting with antibodies to various proteins. Hex B, hexosaminidase B; Cath D, cathepsin D; βGT1; β-1,4-galactosyl transferase. For βGT1, twice as much sample was used. Cell lysate corresponding to 10% of the input material was loaded as a control (lane 1).

    Techniques Used: Incubation, SDS Page

    25) Product Images from "Surface-Associated Plasminogen Binding of Cryptococcus neoformans Promotes Extracellular Matrix Invasion"

    Article Title: Surface-Associated Plasminogen Binding of Cryptococcus neoformans Promotes Extracellular Matrix Invasion

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0005780

    Plasminogen binds selectively and specifically to the cell-surface of intact C. neoformans strains. (A–B) Conversion of plasminogen (Plg) into plasmin heavy chain (Pla H ) and light chain (Pla L ) on the surface of intact C. neoformans serotype D and A strains. (A) Serotype D strain JEC21 was incubated in the presence or absence of plasminogen, tissue plasminogen activator (tPA), and/or the plasmin inhibitor aprotinin in phosphate-buffered saline with BSA. Cell wall proteins were released by boiling labeled cells in SDS-extraction buffer and fractionated by SDS-PAGE, transferred to PVDF, and Western blotted with polyclonal anti-plasminogen antibody. Lane descriptions as follow: cells (JEC21) only (1), 100 µg plasminogen (2), plasminogen and 100 ng tPA (3), plasminogen, tPA, and 1 unit aprotinin (4). (B) Serotype A strains C23 and A1 38-2 were incubated in the presence or absence of plasminogen and/or tPA for 4 hrs at 37°C prior to Western blot analysis as described above. Lanes: cells (C23) only (1), C23 with 15 µg plasminogen (2), C23 with plasminogen and 100 ng tPA (3), cells (A1 38-2) only (4), A1 38-2 with 15 µg plasminogen (5), and A1 38-2 with plasminogen and tPA (6). (C) Plasminogen associates with the cell wall of intact cells. Cells (1×10 10 ) from log phase cultures (JEC21) were incubated 4 hr at 37°C in the presence (lane 1) or absence (lane 3) of 50 µg plasminogen and separated into cell wall and cytosol components, as described in Methods . Membranes (lane 2, 4) from cell walls were extracted and each fraction examined for the presence of plasminogen by Western blot analysis. Sample loading was uniform at 5 µg per well. (D) Sulfo-NHS-biotin and plasminogen compete for cell-surface binding sites. Log phase cells (JEC21) were initially labeled with sulfo-NHS-biotin in 0-, 1-, 10-, 100-fold molar equivalents of plasminogen then labeled 1 hr at 37°C with 50 µg plasminogen (lanes 1–4, respectively).
    Figure Legend Snippet: Plasminogen binds selectively and specifically to the cell-surface of intact C. neoformans strains. (A–B) Conversion of plasminogen (Plg) into plasmin heavy chain (Pla H ) and light chain (Pla L ) on the surface of intact C. neoformans serotype D and A strains. (A) Serotype D strain JEC21 was incubated in the presence or absence of plasminogen, tissue plasminogen activator (tPA), and/or the plasmin inhibitor aprotinin in phosphate-buffered saline with BSA. Cell wall proteins were released by boiling labeled cells in SDS-extraction buffer and fractionated by SDS-PAGE, transferred to PVDF, and Western blotted with polyclonal anti-plasminogen antibody. Lane descriptions as follow: cells (JEC21) only (1), 100 µg plasminogen (2), plasminogen and 100 ng tPA (3), plasminogen, tPA, and 1 unit aprotinin (4). (B) Serotype A strains C23 and A1 38-2 were incubated in the presence or absence of plasminogen and/or tPA for 4 hrs at 37°C prior to Western blot analysis as described above. Lanes: cells (C23) only (1), C23 with 15 µg plasminogen (2), C23 with plasminogen and 100 ng tPA (3), cells (A1 38-2) only (4), A1 38-2 with 15 µg plasminogen (5), and A1 38-2 with plasminogen and tPA (6). (C) Plasminogen associates with the cell wall of intact cells. Cells (1×10 10 ) from log phase cultures (JEC21) were incubated 4 hr at 37°C in the presence (lane 1) or absence (lane 3) of 50 µg plasminogen and separated into cell wall and cytosol components, as described in Methods . Membranes (lane 2, 4) from cell walls were extracted and each fraction examined for the presence of plasminogen by Western blot analysis. Sample loading was uniform at 5 µg per well. (D) Sulfo-NHS-biotin and plasminogen compete for cell-surface binding sites. Log phase cells (JEC21) were initially labeled with sulfo-NHS-biotin in 0-, 1-, 10-, 100-fold molar equivalents of plasminogen then labeled 1 hr at 37°C with 50 µg plasminogen (lanes 1–4, respectively).

    Techniques Used: Proximity Ligation Assay, Incubation, Labeling, SDS Page, Western Blot, Binding Assay

    26) Product Images from "?1-Adrenergic Receptor Recycles Via a Membranous Organelle, Recycling Endosome, by Binding with Sorting Nexin27"

    Article Title: ?1-Adrenergic Receptor Recycles Via a Membranous Organelle, Recycling Endosome, by Binding with Sorting Nexin27

    Journal: The Journal of Membrane Biology

    doi: 10.1007/s00232-013-9571-6

    Steady-state localization and internalization of β 1 -AR upon Iso stimulation in COS-1 cells. β 1 -AR cells were treated without ( a ) or with ( b ) cycloheximide ( CHX , 50 μg/ml) for 4 h and stained with anti-HA or anti-GM130 Abs. Images were captured using the LSM500. c Internalization of β 1 -AR from the cell surface. β 1 -AR cells were biotinylated using 0.1 mg/ml NHS-sulfo-biotin on ice for 30 min, quenched with 0.1 M glycine, washed with prewarmed DMEM thrice and then incubated with 2.5 × 10 −7 (+) or 10 −6 M (++) of Iso for 2 h. Biotinylated proteins concentrated with avidin-agarose were subjected to SDS-PAGE followed by Western blotting ( upper panel cell surface proteins probed with anti-β 1 -AR Ab; middle and lower panels total proteins probed with anti-β 1 -AR Ab or anti-β-actin Ab, respectively). Bar 20 μm
    Figure Legend Snippet: Steady-state localization and internalization of β 1 -AR upon Iso stimulation in COS-1 cells. β 1 -AR cells were treated without ( a ) or with ( b ) cycloheximide ( CHX , 50 μg/ml) for 4 h and stained with anti-HA or anti-GM130 Abs. Images were captured using the LSM500. c Internalization of β 1 -AR from the cell surface. β 1 -AR cells were biotinylated using 0.1 mg/ml NHS-sulfo-biotin on ice for 30 min, quenched with 0.1 M glycine, washed with prewarmed DMEM thrice and then incubated with 2.5 × 10 −7 (+) or 10 −6 M (++) of Iso for 2 h. Biotinylated proteins concentrated with avidin-agarose were subjected to SDS-PAGE followed by Western blotting ( upper panel cell surface proteins probed with anti-β 1 -AR Ab; middle and lower panels total proteins probed with anti-β 1 -AR Ab or anti-β-actin Ab, respectively). Bar 20 μm

    Techniques Used: Staining, Incubation, Avidin-Biotin Assay, SDS Page, Western Blot

    27) Product Images from "Protein disulfide isomerase plasma levels in healthy humans reveal proteomic signatures involved in contrasting endothelial phenotypes"

    Article Title: Protein disulfide isomerase plasma levels in healthy humans reveal proteomic signatures involved in contrasting endothelial phenotypes

    Journal: Redox Biology

    doi: 10.1016/j.redox.2019.101142

    Plasma PDI levels in healthy individuals. A. Distribution of plasma PDI levels are represented by box plot, indicating the median plus lower and upper quartiles ( n = 35). B. Frequency distribution of plasma PDI levels shown in (A). C. Graphic representation of plasma PDI from selected individuals assessed over time. Each box plot represents one individual and their different plasma samples collected over time ( n = 10–15 samples for each individual), as detailed in the text. The dashed line designates the median value (330 pg/mL) of the whole population shown in (A). Values ≤ 330 pg/mL were designated as PDI-poor plasma and > 330 pg/mL as PDI-rich plasma. Data represent median plus lower and upper quartiles. D. Detection of PDI redox state in human plasma. Plasma samples labelled with either EZ-Link Sulfo-NHS-Biotin or MPB were acetone precipitated (to remove unbounded biotinylated probes) and protein pellets were re-suspended in PBS-tween. Samples were added to a plate coated with anti-PDI antibody and after this incubation probed with a monoclonal antibody to biotin (peroxidase conjugate). Bar graphics represent the percentage of reduced or non-reduced PDI comparing MPB vs. EZ-Link Sulpho-NHS-Biotin labelling. Numbers represent different individuals. Data represent mean ± SEM from 3 independent experiments.
    Figure Legend Snippet: Plasma PDI levels in healthy individuals. A. Distribution of plasma PDI levels are represented by box plot, indicating the median plus lower and upper quartiles ( n = 35). B. Frequency distribution of plasma PDI levels shown in (A). C. Graphic representation of plasma PDI from selected individuals assessed over time. Each box plot represents one individual and their different plasma samples collected over time ( n = 10–15 samples for each individual), as detailed in the text. The dashed line designates the median value (330 pg/mL) of the whole population shown in (A). Values ≤ 330 pg/mL were designated as PDI-poor plasma and > 330 pg/mL as PDI-rich plasma. Data represent median plus lower and upper quartiles. D. Detection of PDI redox state in human plasma. Plasma samples labelled with either EZ-Link Sulfo-NHS-Biotin or MPB were acetone precipitated (to remove unbounded biotinylated probes) and protein pellets were re-suspended in PBS-tween. Samples were added to a plate coated with anti-PDI antibody and after this incubation probed with a monoclonal antibody to biotin (peroxidase conjugate). Bar graphics represent the percentage of reduced or non-reduced PDI comparing MPB vs. EZ-Link Sulpho-NHS-Biotin labelling. Numbers represent different individuals. Data represent mean ± SEM from 3 independent experiments.

    Techniques Used: Incubation

    28) 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

    29) Product Images from "Establishment and Comparison of Two Different Diagnostic Platforms for Detection of DENV1 NS1 Protein"

    Article Title: Establishment and Comparison of Two Different Diagnostic Platforms for Detection of DENV1 NS1 Protein

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms161126069

    Development of an ELISA-based diagnostic platform for DENV1. ( A ) Direct ELISA was used to compare a panel of mAbs against serial dilutions of immunoaffinity-purified DENV1 NS1 protein. The ELISA plate was coated with a three-fold dilution of purified DENV1 NS1 protein. After washing, the coated NS1 protein was detected with the indicated mAb at a concentration of 1 μg/mL. Normal mouse IgG (NM-IgG) was used as the negative control; ( B ) Schematic describing the diagnostic platform; ( C ) Standard curve of DENV1 NS1 in different buffer systems. DENV1 NS1-specific mAb, DA15-3, was used to coat an ELISA plate at a concentration of 50 μg/mL. Immunoaffinity-purified DENV1 NS1 protein was diluted three-fold in different buffers and incubated with the capture mAb. NS1 protein was detected through the scheme shown in panel ( B ). Data points represent the mean ± standard deviation for three replicates. The dashed line represents the cut off value. NHS: normal human serum.
    Figure Legend Snippet: Development of an ELISA-based diagnostic platform for DENV1. ( A ) Direct ELISA was used to compare a panel of mAbs against serial dilutions of immunoaffinity-purified DENV1 NS1 protein. The ELISA plate was coated with a three-fold dilution of purified DENV1 NS1 protein. After washing, the coated NS1 protein was detected with the indicated mAb at a concentration of 1 μg/mL. Normal mouse IgG (NM-IgG) was used as the negative control; ( B ) Schematic describing the diagnostic platform; ( C ) Standard curve of DENV1 NS1 in different buffer systems. DENV1 NS1-specific mAb, DA15-3, was used to coat an ELISA plate at a concentration of 50 μg/mL. Immunoaffinity-purified DENV1 NS1 protein was diluted three-fold in different buffers and incubated with the capture mAb. NS1 protein was detected through the scheme shown in panel ( B ). Data points represent the mean ± standard deviation for three replicates. The dashed line represents the cut off value. NHS: normal human serum.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Diagnostic Assay, Direct ELISA, Purification, Concentration Assay, Negative Control, Incubation, Standard Deviation

    30) Product Images from "Influence of Porcine Intervertebral Disc Matrix on Stem Cell Differentiation"

    Article Title: Influence of Porcine Intervertebral Disc Matrix on Stem Cell Differentiation

    Journal: Journal of Functional Biomaterials

    doi: 10.3390/jfb2030155

    Biotinylation of porcine nucleus pulposus proteins by transglutaminase mediated incorporation of glutamine and lysine probes. Extract of nucleus pulposus in 100 mM HEPES buffer (pH 7) and 2 mM monobiotinylcadaverine (probe for accessible glutamine residues, lines Q) or 0.13 mM of a biotinylated glutamine dipeptide (probe for accessible lysine residues, lines K) were incubated with 1 U/mL TGase at 37 °C for 1.5 h (lines NP). For comparison, extract of nucleus pulposus without TGase (control) and purified bovine type II collagen (B-CII) were used. Staining was performed using streptavidin alkaline phosphatase conjugates and BCIP/NBT as described [ 13 ]. Line M displays a prestained molecular marker mixture.
    Figure Legend Snippet: Biotinylation of porcine nucleus pulposus proteins by transglutaminase mediated incorporation of glutamine and lysine probes. Extract of nucleus pulposus in 100 mM HEPES buffer (pH 7) and 2 mM monobiotinylcadaverine (probe for accessible glutamine residues, lines Q) or 0.13 mM of a biotinylated glutamine dipeptide (probe for accessible lysine residues, lines K) were incubated with 1 U/mL TGase at 37 °C for 1.5 h (lines NP). For comparison, extract of nucleus pulposus without TGase (control) and purified bovine type II collagen (B-CII) were used. Staining was performed using streptavidin alkaline phosphatase conjugates and BCIP/NBT as described [ 13 ]. Line M displays a prestained molecular marker mixture.

    Techniques Used: Incubation, Purification, Staining, Marker

    31) Product Images from "Role of the Scavenger Receptor MARCO in Alveolar Macrophage Binding of Unopsonized Environmental Particles "

    Article Title: Role of the Scavenger Receptor MARCO in Alveolar Macrophage Binding of Unopsonized Environmental Particles

    Journal: The Journal of Experimental Medicine

    doi:

    Immunoprecipitation of the AM surface protein recognized by mAb PAL-1. Hamster AMs were surface-labeled with sulfo-NHS-biotin, extracted with Triton X-100, precleared with anti–mouse IgM magnetic beads, and immunoprecipitated with mAb PAL-1 or a control IgM bound to anti–mouse magnetic beads. Immunoprecipitates were analyzed using reducing (R) or nonreducing (NR) SDS-PAGE as described in Materials and Methods. Relative molecular mass is indicated (kD).
    Figure Legend Snippet: Immunoprecipitation of the AM surface protein recognized by mAb PAL-1. Hamster AMs were surface-labeled with sulfo-NHS-biotin, extracted with Triton X-100, precleared with anti–mouse IgM magnetic beads, and immunoprecipitated with mAb PAL-1 or a control IgM bound to anti–mouse magnetic beads. Immunoprecipitates were analyzed using reducing (R) or nonreducing (NR) SDS-PAGE as described in Materials and Methods. Relative molecular mass is indicated (kD).

    Techniques Used: Immunoprecipitation, Affinity Magnetic Separation, Labeling, Magnetic Beads, SDS Page

    32) Product Images from "Hepatitis C Virus (HCV) Envelope Glycoproteins E1 and E2 Contain Reduced Cysteine Residues Essential for Virus Entry *"

    Article Title: Hepatitis C Virus (HCV) Envelope Glycoproteins E1 and E2 Contain Reduced Cysteine Residues Essential for Virus Entry *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.269605

    Free sulfhydryl groups are present on both E1 and E2 in HCVpp. A , HCVpp or no envelope control particles (empty) were labeled with maleimide-biotin or NHS-biotin. Particles were lysed and reduced by addition of DTT prior to capture of biotinylated proteins by streptavadin-agarose. For M135-treated samples, HCVpp were incubated with 1 m m M135 followed by labeling with maleimide-biotin or NHS-biotin. Samples were run on reducing SDS-PAGE and Western blotted with anti-E2 and anti-E1 MAbs H52 and A4, respectively. Molecular weight markers are shown to the left. Input E1, E2, and HIV-1 capsid protein p24 were determined by Western blot (probed with mAbs A4, H52, and 183, respectively) for each sample prior to streptavadin-agarose pulldown. B and C , HCVpp were incubated with 5–500 n m M135 prior to labeling as described above. The resulting Western blots of E2 ( B ) and E1 ( C ) labeled by maleimide-biotin (×) or NHS-biotin (●) were quantified by densitometry analyses (representative assay).
    Figure Legend Snippet: Free sulfhydryl groups are present on both E1 and E2 in HCVpp. A , HCVpp or no envelope control particles (empty) were labeled with maleimide-biotin or NHS-biotin. Particles were lysed and reduced by addition of DTT prior to capture of biotinylated proteins by streptavadin-agarose. For M135-treated samples, HCVpp were incubated with 1 m m M135 followed by labeling with maleimide-biotin or NHS-biotin. Samples were run on reducing SDS-PAGE and Western blotted with anti-E2 and anti-E1 MAbs H52 and A4, respectively. Molecular weight markers are shown to the left. Input E1, E2, and HIV-1 capsid protein p24 were determined by Western blot (probed with mAbs A4, H52, and 183, respectively) for each sample prior to streptavadin-agarose pulldown. B and C , HCVpp were incubated with 5–500 n m M135 prior to labeling as described above. The resulting Western blots of E2 ( B ) and E1 ( C ) labeled by maleimide-biotin (×) or NHS-biotin (●) were quantified by densitometry analyses (representative assay).

    Techniques Used: Labeling, Incubation, SDS Page, Western Blot, Molecular Weight

    CD81 or heparin binding in isolation does not affect the oxidation state of E1 or E2. HCVpp were incubated with heparin sodium salt, dimeric MBP-LEL(113–201), or mutant MBP-LEL(113–201)F186S prior to purification and labeling with maleimide- or NHS-biotin. Particles were lysed and reduced by addition of DTT then applied to streptavidin agarose for capture of biotinylated proteins. Samples were run on reducing SDS-PAGE and Western blotted with anti-E2 and anti-E1 mAbs (H52 and A4, respectively). The ratio of maleimide-biotin and NHS-biotin labeling was calculated for E1 and E2 ± heparin, MBP-LEL(113–201), and MBP-LEL(113–201)F186S by densitometry analyses of Western blots produced from three independent experiments (means ± S.E.).
    Figure Legend Snippet: CD81 or heparin binding in isolation does not affect the oxidation state of E1 or E2. HCVpp were incubated with heparin sodium salt, dimeric MBP-LEL(113–201), or mutant MBP-LEL(113–201)F186S prior to purification and labeling with maleimide- or NHS-biotin. Particles were lysed and reduced by addition of DTT then applied to streptavidin agarose for capture of biotinylated proteins. Samples were run on reducing SDS-PAGE and Western blotted with anti-E2 and anti-E1 mAbs (H52 and A4, respectively). The ratio of maleimide-biotin and NHS-biotin labeling was calculated for E1 and E2 ± heparin, MBP-LEL(113–201), and MBP-LEL(113–201)F186S by densitometry analyses of Western blots produced from three independent experiments (means ± S.E.).

    Techniques Used: Binding Assay, Isolation, Incubation, Mutagenesis, Purification, Labeling, SDS Page, Western Blot, Produced

    33) Product Images from "Aggregation of Sodium Channels Induced by a Postnatally Upregulated Isoform of Agrin"

    Article Title: Aggregation of Sodium Channels Induced by a Postnatally Upregulated Isoform of Agrin

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.16-21-06775.1996

    NaCh and agrin labeling of Agrin8-transfected CHO cells after 1 d of culture on Matrigel-coated coverslips. A , CHO cell labeling with secondary antibodies alone. Fluorescence when anti-NaCh ( A1 ) and anti-agrin ( A2 ) antibodies were omitted. A3 , Nomarski image. B1 , CHO cell labeling with “blocked” anti-NaCh antibody. Notice that there is some non-NaCh labeling of the CHO cells. B2 , Weak, diffuse labeling when primary antibody to agrin (same protocol as A2 ) was omitted. B3 , Nomarski image. C1 , NaCh labeling of CHO cells. This signal is attributable both to a low level of endogenous NaCh expression in some CHO cells and to nonspecific labeling by the anti-NaCh antibody (see B1 ). C2 , Agrin labeling of CHO cells. Notice that expression of agrin is heterogeneous between and within cells. C3 , Nomarski image. Scale bars: A , 20 μm; B , C , 10 μm.
    Figure Legend Snippet: NaCh and agrin labeling of Agrin8-transfected CHO cells after 1 d of culture on Matrigel-coated coverslips. A , CHO cell labeling with secondary antibodies alone. Fluorescence when anti-NaCh ( A1 ) and anti-agrin ( A2 ) antibodies were omitted. A3 , Nomarski image. B1 , CHO cell labeling with “blocked” anti-NaCh antibody. Notice that there is some non-NaCh labeling of the CHO cells. B2 , Weak, diffuse labeling when primary antibody to agrin (same protocol as A2 ) was omitted. B3 , Nomarski image. C1 , NaCh labeling of CHO cells. This signal is attributable both to a low level of endogenous NaCh expression in some CHO cells and to nonspecific labeling by the anti-NaCh antibody (see B1 ). C2 , Agrin labeling of CHO cells. Notice that expression of agrin is heterogeneous between and within cells. C3 , Nomarski image. Scale bars: A , 20 μm; B , C , 10 μm.

    Techniques Used: Labeling, Transfection, Fluorescence, Expressing

    34) Product Images from "The Endoplasmic Reticulum Membrane Is Permeable to Small Molecules"

    Article Title: The Endoplasmic Reticulum Membrane Is Permeable to Small Molecules

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E03-05-0325

    Modification of metabolically labeled ER proteins occurs only upon cell permeabilization. (A) HeLa cells expressing HA-tagged MHC class I heavy chains with lysine residues only in the luminal domain (HA-heavy chain; K-R) were labeled with [ 35 S]methionine and -cysteine. The cells were permeabilized with digitonin (Dig), as indicated, and treated with the amino group modifying reagent sulfo-NHS-biotin. The heavy chains were immunoprecipitated with HA antibodies. 20% of the immunoprecipitated material was analyzed directly by SDS-PAGE (lanes 1-4), and 80% was incubated with streptavidin beads to detect biotinylated material (lanes 5-8). Where indicated, one-half of each sample was treated with Endo H. All samples were subjected to SDS-PAGE followed by autoradiography. (B) Hybridoma B cells expressing IgG were analyzed as in A, except that the labeled IgG was collected with protein A/G-Sepharose.
    Figure Legend Snippet: Modification of metabolically labeled ER proteins occurs only upon cell permeabilization. (A) HeLa cells expressing HA-tagged MHC class I heavy chains with lysine residues only in the luminal domain (HA-heavy chain; K-R) were labeled with [ 35 S]methionine and -cysteine. The cells were permeabilized with digitonin (Dig), as indicated, and treated with the amino group modifying reagent sulfo-NHS-biotin. The heavy chains were immunoprecipitated with HA antibodies. 20% of the immunoprecipitated material was analyzed directly by SDS-PAGE (lanes 1-4), and 80% was incubated with streptavidin beads to detect biotinylated material (lanes 5-8). Where indicated, one-half of each sample was treated with Endo H. All samples were subjected to SDS-PAGE followed by autoradiography. (B) Hybridoma B cells expressing IgG were analyzed as in A, except that the labeled IgG was collected with protein A/G-Sepharose.

    Techniques Used: Modification, Metabolic Labelling, Labeling, Expressing, Immunoprecipitation, SDS Page, Incubation, Autoradiography

    Rough microsomes are permeable to a modification reagent. Radiolabeled full-length ppαF was translocated into rough microsomes. The membranes were isolated and incubated with different concentrations of sulfo-NHS-biotin, an amino group-modifying reagent, for 10 min on ice or at 30°C. Where indicated, 0.5% TX-100 was present during the modification reaction. One portion of the samples was analyzed directly (total), the other was incubated with streptavidin beads and the bound fraction was analyzed (beads). All samples were subjected to SDS-PAGE and autoradiography. ppαF, gppαF, pro-α-factor without or with three carbohydrate chains, respectively. Molecular masses (in kilodaltons) are indicated on the right side.
    Figure Legend Snippet: Rough microsomes are permeable to a modification reagent. Radiolabeled full-length ppαF was translocated into rough microsomes. The membranes were isolated and incubated with different concentrations of sulfo-NHS-biotin, an amino group-modifying reagent, for 10 min on ice or at 30°C. Where indicated, 0.5% TX-100 was present during the modification reaction. One portion of the samples was analyzed directly (total), the other was incubated with streptavidin beads and the bound fraction was analyzed (beads). All samples were subjected to SDS-PAGE and autoradiography. ppαF, gppαF, pro-α-factor without or with three carbohydrate chains, respectively. Molecular masses (in kilodaltons) are indicated on the right side.

    Techniques Used: Modification, Isolation, Incubation, SDS Page, Autoradiography

    The ER membrane does not constitute a significant barrier to small molecules. HeLa cells were permeabilized with 0.04% digitonin (A) or SLO (B) and incubated with 0.5 mM sulfo-NHS-LC-biotin for different periods of time. Biotinylated proteins were recovered after incubation with streptavidin beads and analyzed by SDS-PAGE and immunoblotting with antibodies to different proteins. The bands were quantitated using a Fujix PhosporIimager and the software Image Gauge 3.0.
    Figure Legend Snippet: The ER membrane does not constitute a significant barrier to small molecules. HeLa cells were permeabilized with 0.04% digitonin (A) or SLO (B) and incubated with 0.5 mM sulfo-NHS-LC-biotin for different periods of time. Biotinylated proteins were recovered after incubation with streptavidin beads and analyzed by SDS-PAGE and immunoblotting with antibodies to different proteins. The bands were quantitated using a Fujix PhosporIimager and the software Image Gauge 3.0.

    Techniques Used: Incubation, SDS Page, Software

    A small molecule passes into the ER but not into lysosomes or the trans -Golgi. HeLa cells (2 × 10 6 ) were permeabilized with SLO, 0.04% digitonin (Dig), or 0.1% digitonin as indicated. Then 0.5 mM sulfo-NHS-LC-biotin was added for 30 min on ice. After solubilization in detergent, cell lysates were incubated with streptavidin agarose beads, and the bound material was analyzed by SDS-PAGE and immunoblotting with antibodies to various proteins. Hex B, hexosaminidase B; Cath D, cathepsin D; βGT1; β-1,4-galactosyl transferase. For βGT1, twice as much sample was used. Cell lysate corresponding to 10% of the input material was loaded as a control (lane 1).
    Figure Legend Snippet: A small molecule passes into the ER but not into lysosomes or the trans -Golgi. HeLa cells (2 × 10 6 ) were permeabilized with SLO, 0.04% digitonin (Dig), or 0.1% digitonin as indicated. Then 0.5 mM sulfo-NHS-LC-biotin was added for 30 min on ice. After solubilization in detergent, cell lysates were incubated with streptavidin agarose beads, and the bound material was analyzed by SDS-PAGE and immunoblotting with antibodies to various proteins. Hex B, hexosaminidase B; Cath D, cathepsin D; βGT1; β-1,4-galactosyl transferase. For βGT1, twice as much sample was used. Cell lysate corresponding to 10% of the input material was loaded as a control (lane 1).

    Techniques Used: Incubation, SDS Page

    35) Product Images from "Neurokinin 3 Receptor Immunoreactivity in the Septal Region, Preoptic Area and Hypothalamus of the Female Sheep: Colocalization in Neurokinin B Cells of the Arcuate Nucleus but not in Gonadotrophin-Releasing Hormone Neurones"

    Article Title: Neurokinin 3 Receptor Immunoreactivity in the Septal Region, Preoptic Area and Hypothalamus of the Female Sheep: Colocalization in Neurokinin B Cells of the Arcuate Nucleus but not in Gonadotrophin-Releasing Hormone Neurones

    Journal: Journal of neuroendocrinology

    doi: 10.1111/j.1365-2826.2009.01930.x

    Confocal images of representative sections of the ARC (A, B) and POA (C, D). Images represent 1 µm optical sections of tissue processed for immunofluorescent detection of NK3R (red) and counterstained with fluorescent Nissl (green). Arrows indicate fluorescent particles dissociated from the cell membrane. Arrow-heads indicate Nissl-stained nucleolus. Scale bars, 10 µm.
    Figure Legend Snippet: Confocal images of representative sections of the ARC (A, B) and POA (C, D). Images represent 1 µm optical sections of tissue processed for immunofluorescent detection of NK3R (red) and counterstained with fluorescent Nissl (green). Arrows indicate fluorescent particles dissociated from the cell membrane. Arrow-heads indicate Nissl-stained nucleolus. Scale bars, 10 µm.

    Techniques Used: Staining

    36) Product Images from "FASN Inhibition and Taxane Treatment Combine to Enhance Anti-tumor Efficacy in Diverse Xenograft Tumor Models through Disruption of Tubulin Palmitoylation and Microtubule Organization and FASN Inhibition-Mediated Effects on Oncogenic Signaling and Gene Expression"

    Article Title: FASN Inhibition and Taxane Treatment Combine to Enhance Anti-tumor Efficacy in Diverse Xenograft Tumor Models through Disruption of Tubulin Palmitoylation and Microtubule Organization and FASN Inhibition-Mediated Effects on Oncogenic Signaling and Gene Expression

    Journal: EBioMedicine

    doi: 10.1016/j.ebiom.2016.12.012

    Combined treatment of NSCLC tumor xenografts with TVB-3166 and a taxane (paclitaxel or docetaxel) inhibits tumor growth synergistically compared to single agent activity. A minimum of 10 mice per treatment group was used in all studies. (A) CALU6 NSCLC adenocarcinoma cell line. (B) A549 NSCLC adenocarcinoma cell line. (C) CTG-0165_P + 6 NSCLC adenocarcinoma patient-derived tumor. TVB-3166 was dosed once daily by oral gavage at 60 mg/kg. Paclitaxel was dosed once every 4 days by intravenous administration at 10 mg/kg. Docetaxel was dosed once every 7 days by intravenous administration at 8 or 6 mg/kg. In groups dosed with both TVB-3166 (60 mg/kg) and paclitaxel (10 mg/kg) or docetaxel (8 or 6 mg/kg), TVB-3166 was administered 2 h before taxane administration. Animals were randomized according to tumor size and drug treatment was started when the mean tumor size was 150–200 mm 3 . Tumors and blood samples were harvested 2 h after the last dose. TVB-3166 and paclitaxel plasma and tumor drug concentrations were determined by mass spectrometry. The in-life phase for the CALU-6 (A) and A549 (B) studies was performed at Crown Biosciences (Santa Clara, CA; Beijing, China). The in-life phase for the CTG-0165_P + 6 study (C) was performed at Champions Oncology (Baltimore, MD).
    Figure Legend Snippet: Combined treatment of NSCLC tumor xenografts with TVB-3166 and a taxane (paclitaxel or docetaxel) inhibits tumor growth synergistically compared to single agent activity. A minimum of 10 mice per treatment group was used in all studies. (A) CALU6 NSCLC adenocarcinoma cell line. (B) A549 NSCLC adenocarcinoma cell line. (C) CTG-0165_P + 6 NSCLC adenocarcinoma patient-derived tumor. TVB-3166 was dosed once daily by oral gavage at 60 mg/kg. Paclitaxel was dosed once every 4 days by intravenous administration at 10 mg/kg. Docetaxel was dosed once every 7 days by intravenous administration at 8 or 6 mg/kg. In groups dosed with both TVB-3166 (60 mg/kg) and paclitaxel (10 mg/kg) or docetaxel (8 or 6 mg/kg), TVB-3166 was administered 2 h before taxane administration. Animals were randomized according to tumor size and drug treatment was started when the mean tumor size was 150–200 mm 3 . Tumors and blood samples were harvested 2 h after the last dose. TVB-3166 and paclitaxel plasma and tumor drug concentrations were determined by mass spectrometry. The in-life phase for the CALU-6 (A) and A549 (B) studies was performed at Crown Biosciences (Santa Clara, CA; Beijing, China). The in-life phase for the CTG-0165_P + 6 study (C) was performed at Champions Oncology (Baltimore, MD).

    Techniques Used: Activity Assay, Mouse Assay, CTG Assay, Derivative Assay, Mass Spectrometry

    37) Product Images from "Pimozide suppresses cancer cell migration and tumor metastasis through binding to ARPC2, a subunit of the Arp2/3 complex, et al. Pimozide suppresses cancer cell migration and tumor metastasis through binding to ARPC2, a subunit of the Arp2/3 complex"

    Article Title: Pimozide suppresses cancer cell migration and tumor metastasis through binding to ARPC2, a subunit of the Arp2/3 complex, et al. Pimozide suppresses cancer cell migration and tumor metastasis through binding to ARPC2, a subunit of the Arp2/3 complex

    Journal: Cancer Science

    doi: 10.1111/cas.14205

    Direct binding of pimozide with ARPC2. A, DLD‐1 cell lysates were incubated in the presence or absence of pimozide (100 or 200 μmol/L) for 1 h at room temperature, followed by proteolysis with various pronases in a dose‐dependent way. GAPDH, which served as the loading control, is relatively resistant to proteolysis (n = 3). B, DLD‐1 cells were treated for 12 h with 10 μmol/L pimozide, and then CETSA was carried out to measure binding ability. Pimozide increased the thermal stability of ARPC2 compared with DMSO. Vinculin is a nontarget protein of pimozide (n = 3). Band intensity was quantified using the MultiGauge program. C, DLD‐1 cell lysates was incubated with 20 μmol/L biotinyl benproperine (biotinyl‐Benp) and competed with pimozide at the indicated concentration. Proteins were captured with NeutrAvidin‐Agarose resin (ThermoFisher Scientific Inc., Waltham, MA, USA) and eluted proteins were analyzed by western blotting (n = 2). D, Chemical structure of N‐methyl‐pimozide. E, Cell migration assay of DLD‐1 cells that were treated with DMSO, pimozide, or N‐methyl‐pimozide for 18 h and quantification of the migrated cells (n = 3). Scale bars, 200 μm. F, Pull‐down assay with biotinyl‐Benp was done in the absence or presence of compounds as competitor (100 μM). Bound proteins on the beads were separated by SDS‐PAGE, and western blot was carried out using anti‐ARPC2 and anti‐GAPDH antibodies (n = 2). Data represent the means ± SD; * P
    Figure Legend Snippet: Direct binding of pimozide with ARPC2. A, DLD‐1 cell lysates were incubated in the presence or absence of pimozide (100 or 200 μmol/L) for 1 h at room temperature, followed by proteolysis with various pronases in a dose‐dependent way. GAPDH, which served as the loading control, is relatively resistant to proteolysis (n = 3). B, DLD‐1 cells were treated for 12 h with 10 μmol/L pimozide, and then CETSA was carried out to measure binding ability. Pimozide increased the thermal stability of ARPC2 compared with DMSO. Vinculin is a nontarget protein of pimozide (n = 3). Band intensity was quantified using the MultiGauge program. C, DLD‐1 cell lysates was incubated with 20 μmol/L biotinyl benproperine (biotinyl‐Benp) and competed with pimozide at the indicated concentration. Proteins were captured with NeutrAvidin‐Agarose resin (ThermoFisher Scientific Inc., Waltham, MA, USA) and eluted proteins were analyzed by western blotting (n = 2). D, Chemical structure of N‐methyl‐pimozide. E, Cell migration assay of DLD‐1 cells that were treated with DMSO, pimozide, or N‐methyl‐pimozide for 18 h and quantification of the migrated cells (n = 3). Scale bars, 200 μm. F, Pull‐down assay with biotinyl‐Benp was done in the absence or presence of compounds as competitor (100 μM). Bound proteins on the beads were separated by SDS‐PAGE, and western blot was carried out using anti‐ARPC2 and anti‐GAPDH antibodies (n = 2). Data represent the means ± SD; * P

    Techniques Used: Binding Assay, Incubation, Concentration Assay, Western Blot, Cell Migration Assay, Pull Down Assay, SDS Page

    38) 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

    39) Product Images from "Plasmodium falciparum liver stage antigen-1 is cross-linked by tissue transglutaminase"

    Article Title: Plasmodium falciparum liver stage antigen-1 is cross-linked by tissue transglutaminase

    Journal: Malaria Journal

    doi: 10.1186/1475-2875-10-14

    Assessment of LSA-NRC cross-linking by TG2 . A. SDS PAGE analysis of LSA-NRC samples after various times of incubation with 100 μg/ml of either gpTG2 (i) or hTG2 (ii). * indicates the band representing TG2 (MW - 76.6 kDa). B. SDS PAGE analysis of LSA-NRC samples after various times of incubation with 100 μg/ml of gpTG2 in the absence of CaCl 2 indicating dependence of cross-linking on Ca + . C. Western analysis of LSA-NRC samples after incubation with lysates of human cell line SK-N-BE(2) (i) or its stably transfected derivative, TGA, that over-expresses hTG2 (ii). Blots were probed with anti-LSA-NRC polyclonal antibodies. D. Plate based colorimetric analysis of LSA-NRC TG2 mediated cross-linking. Change in absorbance at 405 nm is shown as a function of TG2 concentration. Open circles - hTG2; Open triangles - gpTG2; Open squares - gpTG2 in the absence of CaCl 2 ; closed triangles - in the absence of TG2. Error bars show variation of 3 experiments.
    Figure Legend Snippet: Assessment of LSA-NRC cross-linking by TG2 . A. SDS PAGE analysis of LSA-NRC samples after various times of incubation with 100 μg/ml of either gpTG2 (i) or hTG2 (ii). * indicates the band representing TG2 (MW - 76.6 kDa). B. SDS PAGE analysis of LSA-NRC samples after various times of incubation with 100 μg/ml of gpTG2 in the absence of CaCl 2 indicating dependence of cross-linking on Ca + . C. Western analysis of LSA-NRC samples after incubation with lysates of human cell line SK-N-BE(2) (i) or its stably transfected derivative, TGA, that over-expresses hTG2 (ii). Blots were probed with anti-LSA-NRC polyclonal antibodies. D. Plate based colorimetric analysis of LSA-NRC TG2 mediated cross-linking. Change in absorbance at 405 nm is shown as a function of TG2 concentration. Open circles - hTG2; Open triangles - gpTG2; Open squares - gpTG2 in the absence of CaCl 2 ; closed triangles - in the absence of TG2. Error bars show variation of 3 experiments.

    Techniques Used: SDS Page, Incubation, Western Blot, Stable Transfection, Transfection, Concentration Assay

    P. falciparum LSA-1 in human liver hepatocytes . P. falciparum sporozoites were injected intravenously into transgenic, chimeric mice possessing functioning human livers. Liver nodules were collected 5 or 6 days after injection, fixed and sectioned. Sections containing developing parasites were probed with antibody and detected by immunofluorescence. (A) A 5-day infected liver section probed with mouse polyclonal sera against LSA-NRC. (B) A 6-day infected liver treated as in (A). (C) A 6-day infected liver probed with mAb 71A3F1 that recognizes the TG2 formed isopeptide bond between glutamine and lysine. (D) As in (C) but using another mAb, 81D1C2, that also recognizes the TG2 isopeptide bond [ 52 ]
    Figure Legend Snippet: P. falciparum LSA-1 in human liver hepatocytes . P. falciparum sporozoites were injected intravenously into transgenic, chimeric mice possessing functioning human livers. Liver nodules were collected 5 or 6 days after injection, fixed and sectioned. Sections containing developing parasites were probed with antibody and detected by immunofluorescence. (A) A 5-day infected liver section probed with mouse polyclonal sera against LSA-NRC. (B) A 6-day infected liver treated as in (A). (C) A 6-day infected liver probed with mAb 71A3F1 that recognizes the TG2 formed isopeptide bond between glutamine and lysine. (D) As in (C) but using another mAb, 81D1C2, that also recognizes the TG2 isopeptide bond [ 52 ]

    Techniques Used: Injection, Transgenic Assay, Mouse Assay, Immunofluorescence, Infection

    Analysis of cross-linking site . A. PAGE analysis of LSA-NRC TG2-cross-linking in the absence (i) or presence (ii) of peptide corresponding to the major repeat sequence of LSA-1. B. RP-HPLC analysis of a peptide corresponding to the major repeat sequence of LSA-1 before (i) and after (ii) gpTG2 treatment for 2 h at 100 μg/ml gpTG2. Position of monomers [retention time 23.3 min] (1), dimers [retention time 24.5 min] (2) and trimers [retention time 25.6 min] (3) are indicated. (ii). C. Tertiary structure of a single LSA-1 major repeat as predicted by Robetta modeling. Arrows indicate glutamines and lysines predicted to be involved in TG2 mediated cross-linking. D. PAGE analysis of gpTG2 cross-linking of LSA-NRC-C (i) and LSA-NRC-N (ii). * indicates band formed by the gpTG2 enzyme (MW - 76.6 kDa).
    Figure Legend Snippet: Analysis of cross-linking site . A. PAGE analysis of LSA-NRC TG2-cross-linking in the absence (i) or presence (ii) of peptide corresponding to the major repeat sequence of LSA-1. B. RP-HPLC analysis of a peptide corresponding to the major repeat sequence of LSA-1 before (i) and after (ii) gpTG2 treatment for 2 h at 100 μg/ml gpTG2. Position of monomers [retention time 23.3 min] (1), dimers [retention time 24.5 min] (2) and trimers [retention time 25.6 min] (3) are indicated. (ii). C. Tertiary structure of a single LSA-1 major repeat as predicted by Robetta modeling. Arrows indicate glutamines and lysines predicted to be involved in TG2 mediated cross-linking. D. PAGE analysis of gpTG2 cross-linking of LSA-NRC-C (i) and LSA-NRC-N (ii). * indicates band formed by the gpTG2 enzyme (MW - 76.6 kDa).

    Techniques Used: Polyacrylamide Gel Electrophoresis, Sequencing, High Performance Liquid Chromatography

    PfCK2α regulation of TG2 LSA-1 cross-linking . (A) PAGE analysis of LSA-NRC incubated with PfCK2 α. Coomassie stained samples (i). Autoradiograph of gel in (i) (ii). Lane 1 - LSA-NRC incubated with PfCK2α; lane 2 - LSA-NRC incubated with inactivated PfCK2α; lane 3 - LSA-NRC; lane 4 - PfCK2α. (B) PAGE analysis of samples taken at various time points from non-phosphorylated (i) and phosphorylated (ii) LSA-NRC incubated with gpTG2.
    Figure Legend Snippet: PfCK2α regulation of TG2 LSA-1 cross-linking . (A) PAGE analysis of LSA-NRC incubated with PfCK2 α. Coomassie stained samples (i). Autoradiograph of gel in (i) (ii). Lane 1 - LSA-NRC incubated with PfCK2α; lane 2 - LSA-NRC incubated with inactivated PfCK2α; lane 3 - LSA-NRC; lane 4 - PfCK2α. (B) PAGE analysis of samples taken at various time points from non-phosphorylated (i) and phosphorylated (ii) LSA-NRC incubated with gpTG2.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Incubation, Staining, Autoradiography

    40) Product Images from "Terpestacin Inhibits Tumor Angiogenesis by Targeting UQCRB of Mitochondrial Complex III and Suppressing Hypoxia-induced Reactive Oxygen Species Production and Cellular Oxygen Sensing *"

    Article Title: Terpestacin Inhibits Tumor Angiogenesis by Targeting UQCRB of Mitochondrial Complex III and Suppressing Hypoxia-induced Reactive Oxygen Species Production and Cellular Oxygen Sensing *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.087809

    Identification of terpestacin-binding protein using phage display affinity selection. A , the structures of terpestacin and the molecular probes, including coumarin-conjugated terpestacin ( ter-coumarin ) and the biotinylated terpestacin derivatives ( BT1
    Figure Legend Snippet: Identification of terpestacin-binding protein using phage display affinity selection. A , the structures of terpestacin and the molecular probes, including coumarin-conjugated terpestacin ( ter-coumarin ) and the biotinylated terpestacin derivatives ( BT1

    Techniques Used: Binding Assay, Selection

    Effects of terpestacin on mitochondrial membrane potential (ΔΨ m ) and mitochondrial respiration. A , HT1080 cells were incubated for 4 h with terpestacin ( Ter , 20 or 50 μ m ) or radicicol ( Rad , 10 μ m ) and then stained with
    Figure Legend Snippet: Effects of terpestacin on mitochondrial membrane potential (ΔΨ m ) and mitochondrial respiration. A , HT1080 cells were incubated for 4 h with terpestacin ( Ter , 20 or 50 μ m ) or radicicol ( Rad , 10 μ m ) and then stained with

    Techniques Used: Incubation, Staining

    Effects of terpestacin on mitochondrial ROS generation and HIF-1α stability. A , intracellular ROS levels were determined by the 2′,7′-dichlorofluorescein ( DCF ) fluorescence. HT1080 cells were pretreated with terpestacin ( Ter , 30
    Figure Legend Snippet: Effects of terpestacin on mitochondrial ROS generation and HIF-1α stability. A , intracellular ROS levels were determined by the 2′,7′-dichlorofluorescein ( DCF ) fluorescence. HT1080 cells were pretreated with terpestacin ( Ter , 30

    Techniques Used: Fluorescence

    Molecular interactions and subcellular localization of terpestacin. A , effects of various competitors on the binding between UQCRB phages and immobilized terpestacin. All of the competitors were used at a concentration of 100 μ m . Ter , terpestacin;
    Figure Legend Snippet: Molecular interactions and subcellular localization of terpestacin. A , effects of various competitors on the binding between UQCRB phages and immobilized terpestacin. All of the competitors were used at a concentration of 100 μ m . Ter , terpestacin;

    Techniques Used: Binding Assay, Concentration Assay

    Effect of terpestacin on tumor angiogenesis in vivo . Mice bearing FM3A breast tumors were treated with saline ( control ) or terpestacin ( Ter , one-third of LD 50 , LD 50 = 7.5 μ m ). A , time course of dynamic contrast-enhanced magnetic resonance signal
    Figure Legend Snippet: Effect of terpestacin on tumor angiogenesis in vivo . Mice bearing FM3A breast tumors were treated with saline ( control ) or terpestacin ( Ter , one-third of LD 50 , LD 50 = 7.5 μ m ). A , time course of dynamic contrast-enhanced magnetic resonance signal

    Techniques Used: In Vivo, Mouse Assay

    Docking model of terpestacin-UQCRB complex. A , electrostatic surface representation of the hydrophobic pocket of UQCRB bound with terpestacin. The red color denotes electronegative charge potential, and the blue color denotes electropositive charge potential.
    Figure Legend Snippet: Docking model of terpestacin-UQCRB complex. A , electrostatic surface representation of the hydrophobic pocket of UQCRB bound with terpestacin. The red color denotes electronegative charge potential, and the blue color denotes electropositive charge potential.

    Techniques Used:

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    Article Title: The ARP 2/3 complex mediates endothelial barrier function and recovery
    Article Snippet: .. Next, EZ-Link NHS-LC-LC-Biotin (Thermo Scientific, Rockford, IL, USA) dissolved in DMSO (5.7 mg/mL) was added to the gelatin solution to a final concentration of 0.57 mg/mL and allowed to conjugate for 1 h at RT while stirring. ..

    Incubation:

    Article Title: Structural Based Screening of Antiandrogen Targeting Activation Function-2 Binding Site
    Article Snippet: .. The purified AR was biotinylated (EZ-Link® NHS-Biotin Reagents, Cat. # 21343, Thermo) by 3:1, and then incubated for 1 h at room temperature. ..

    Binding Assay:

    Article Title: EsGLUT4 and CHHBP are involved in the regulation of glucose homeostasis in the crustacean Eriocheir sinensis
    Article Snippet: .. First, the rEsGLUT4 intracellular and extracellular regions were labeled with biotin using EZ-Link™ NHS-LC-LC-Biotin (Thermo Scientific, USA) to enable binding to the streptavidin biosensor. .. In detail, the appropriate volume of biotin was dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 10 mM.

    Labeling:

    Article Title: Ensembles of Bidirectional Kinesin Cin8 Produce Additive Forces in Both Directions of Movement
    Article Snippet: .. To generate biotinylated tubulin, Alexa647-tubulin, or NEM-tubulin, tubulin was covalently labeled with either EZ-Link NHS-LC-LC-biotin (21343, Life Technologies, Carlsbad, CA), Alexa Fluor 647-NHS (A-20006, Life Technologies), or N-(ethylmaleimide) (NEM) (Sigma-Aldrich, St. Louis, MO), respectively, essentially as described previously ( ). ..

    Article Title: EsGLUT4 and CHHBP are involved in the regulation of glucose homeostasis in the crustacean Eriocheir sinensis
    Article Snippet: .. First, the rEsGLUT4 intracellular and extracellular regions were labeled with biotin using EZ-Link™ NHS-LC-LC-Biotin (Thermo Scientific, USA) to enable binding to the streptavidin biosensor. .. In detail, the appropriate volume of biotin was dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 10 mM.

    Purification:

    Article Title: Structural Based Screening of Antiandrogen Targeting Activation Function-2 Binding Site
    Article Snippet: .. The purified AR was biotinylated (EZ-Link® NHS-Biotin Reagents, Cat. # 21343, Thermo) by 3:1, and then incubated for 1 h at room temperature. ..

    Article Title: Nanobody-Displaying Flagellar Nanotubes
    Article Snippet: .. Purified sfGFP was biotinylated using EZ-link Sulpho-NHS-LC-LC Biotin (Thermo Scientific) following the manufacturer’s instructions. .. Purification of the FliC-aGFP_ENH fusion protein was performed as follows: the bacterial pellet from 1 L overnight culture (at 37 °C in 3% yeast extract) of the selected cell line was collected by centrifugation at 4000 g for 30 min and suspended in 5 ml PBS.

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    Thermo Fisher sulfo nhs ss biotin
    miR-17 and 221 promote endocytic recycling of integrin β1 in macrophages A–C. THP1-differentiated macrophages were transfected with miR-17 or 221 mimics. Surface proteins of the macrophages were labeled with EZ-Link <t>Sulfo-NHS-SS-Biotin,</t> followed by surface integrin internalization ( A ), endocytic recycling ( B ), and degradation assay ( C ). <t>Biotinylated</t> proteins were precipitated with streptavindin, followed by Western blot analysis. Data represent mean ± SD (n = 3 per group).
    Sulfo Nhs Ss Biotin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 200 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher biotin x dhpe lipids
    Single <t>Biotin-X-DHPE</t> molecules tagged with Streptavidin-Alexa546 were tracked on the curved supported lipid bilayers (ROC = 28 nm) in space and time. ( a ) example trajectories (blue) show the heterogeneous dynamics observed on both flat (white) and curved (black) regions. Scale bar = 1 µm; ( b ) the average step a molecule takes over 0.228 s (5 frames) when starting at a region of curvature (white) or at a flat region (grey); ( c ) the distribution of steps observed at curved and flat regions (shown in Figure S3 ) was fitted to Equation 3 to obtain the diffusion coefficients and the percentage of steps moving at that rate. The average D is plotted for t = 0.091, 0.228 and 0.456. Note that there is no fast component for the tracks that start at regions of curvature.
    Biotin X Dhpe Lipids, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    miR-17 and 221 promote endocytic recycling of integrin β1 in macrophages A–C. THP1-differentiated macrophages were transfected with miR-17 or 221 mimics. Surface proteins of the macrophages were labeled with EZ-Link Sulfo-NHS-SS-Biotin, followed by surface integrin internalization ( A ), endocytic recycling ( B ), and degradation assay ( C ). Biotinylated proteins were precipitated with streptavindin, followed by Western blot analysis. Data represent mean ± SD (n = 3 per group).

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

    Article Title: Lung epithelial cell-derived microvesicles regulate macrophage migration via microRNA-17/221-induced integrin beta1 recycling

    doi: 10.4049/jimmunol.1700165

    Figure Lengend Snippet: miR-17 and 221 promote endocytic recycling of integrin β1 in macrophages A–C. THP1-differentiated macrophages were transfected with miR-17 or 221 mimics. Surface proteins of the macrophages were labeled with EZ-Link Sulfo-NHS-SS-Biotin, followed by surface integrin internalization ( A ), endocytic recycling ( B ), and degradation assay ( C ). Biotinylated proteins were precipitated with streptavindin, followed by Western blot analysis. Data represent mean ± SD (n = 3 per group).

    Article Snippet: For the internalization assay, macrophage-surface proteins were biotinylated with 0.5 mg/mL Sulfo-NHS-SS Biotin (Thermo Fisher Scientific) for 20 min at 4 °C, followed by washing with TBS.

    Techniques: Transfection, Labeling, Degradation Assay, Western Blot

    Knockdown of α-taxilin impedes the recycling of Tfn. (A) HeLaS3 cells transfected with control or α-taxilin siRNA (#3) were treated with sulfo-NHS-SS-biotin at 4°C, and then the cells were incubated at 37°C for the indicated periods of time. Cells were treated with MesNa to remove biotin remaining on the plasma membrane, and then the cell lysates were precipitated with neutravidin-agarose beads. The precipitates were probed with an anti-TfnR antibody (biotinylated TfnR). The cell lysates used for precipitation were probed with anti-TfnR, anti-α-taxilin and anti-clathrin heavy chain antibodies. The results shown are representative of three independent experiments. (B) The amount of internalized TfnR in (A) was quantified using Image J software. The results shown are means ± s.e.m. of the ratio of internalized TfnR at the indicated time periods to biotinylated TfnR at time zero without MesNa treatment from three independent experiments. P -values (control cells vs. α-taxilin knockdown cells at 2.5, 5, 10 min) determined by Student's t -test was not significant. (C) HeLaS3 cells transfected with control or α-taxilin siRNA (#3) were serum starved for 3 h, and then the cells were incubated with Tfn-488 at 37°C for 1 h. In the case of treatment with leupeptin, the cells were preincubated with leupeptin (200 μg/ml) 1 h prior to Tfn-488 labeling. After washing out unbound Tfn-488, the cells were incubated at 37°C for various time periods in the presence or absence of leupeptin (200 μg/ml). Scale bars, 10 μm. (D) The intensity of Tfn-488 signal of HeLaS3 cells untreated with leupeptin in (C) was expressed as signal intensity per unit area. At each time point, signal intensity of at least 20 cells was measured from three independent experiments. The results shown are means ± s.e.m. of the ratio of Tfn-488 at each time point to Tfn-488 at time zero. Values at time zero are set to 1.0. P -values (control cells vs. α-taxilin knockdown cells at 10, 20, 40 min) are determined by Student's t -test. *, P

    Journal: PLoS ONE

    Article Title: ?-Taxilin Interacts with Sorting Nexin 4 and Participates in the Recycling Pathway of Transferrin Receptor

    doi: 10.1371/journal.pone.0093509

    Figure Lengend Snippet: Knockdown of α-taxilin impedes the recycling of Tfn. (A) HeLaS3 cells transfected with control or α-taxilin siRNA (#3) were treated with sulfo-NHS-SS-biotin at 4°C, and then the cells were incubated at 37°C for the indicated periods of time. Cells were treated with MesNa to remove biotin remaining on the plasma membrane, and then the cell lysates were precipitated with neutravidin-agarose beads. The precipitates were probed with an anti-TfnR antibody (biotinylated TfnR). The cell lysates used for precipitation were probed with anti-TfnR, anti-α-taxilin and anti-clathrin heavy chain antibodies. The results shown are representative of three independent experiments. (B) The amount of internalized TfnR in (A) was quantified using Image J software. The results shown are means ± s.e.m. of the ratio of internalized TfnR at the indicated time periods to biotinylated TfnR at time zero without MesNa treatment from three independent experiments. P -values (control cells vs. α-taxilin knockdown cells at 2.5, 5, 10 min) determined by Student's t -test was not significant. (C) HeLaS3 cells transfected with control or α-taxilin siRNA (#3) were serum starved for 3 h, and then the cells were incubated with Tfn-488 at 37°C for 1 h. In the case of treatment with leupeptin, the cells were preincubated with leupeptin (200 μg/ml) 1 h prior to Tfn-488 labeling. After washing out unbound Tfn-488, the cells were incubated at 37°C for various time periods in the presence or absence of leupeptin (200 μg/ml). Scale bars, 10 μm. (D) The intensity of Tfn-488 signal of HeLaS3 cells untreated with leupeptin in (C) was expressed as signal intensity per unit area. At each time point, signal intensity of at least 20 cells was measured from three independent experiments. The results shown are means ± s.e.m. of the ratio of Tfn-488 at each time point to Tfn-488 at time zero. Values at time zero are set to 1.0. P -values (control cells vs. α-taxilin knockdown cells at 10, 20, 40 min) are determined by Student's t -test. *, P

    Article Snippet: Biotinylation recycling assay using cleavable sulfo-NHS-SS-biotin Cell surface proteins of HeLaS3 cells were biotinylated with 0.5 mg/ml sulfo-NHS-SS-biotin as described above.

    Techniques: Transfection, Incubation, Software, Labeling

    aPKC is required for the cell-surface localization of SD components in vivo . ( A ) aPKC cKO and control mice at P10 or P11 were transcardially perfused with 2 mg/ml sulfo-NHS-SS-biotin/PBSCM for 5 min. Then, the kidneys were lysed and biotinylated proteins isolated with streptavidin sepharose and detected by immunoblot. The white arrowhead represents the mature-glycosylated, cell-surface form, and the black arrowhead represents the N -glycosylated, ER-form of nephrin. ( B ) Quantification of the results in (A). The values were normalized to control mice and are the mean ± SD of three independent experiments. The P values were determined by two-tailed Student’s t -test. ( C ) The cell-surface biotinylated aPKC cKO and control kidney were immunostained with nephrin, neph1 or podocin and biotin ( Supplementary Fig. S5 ), and the colocalization coefficient was calculated with LAS-AF software provided by Leica. The P values were determined by two-tailed Student’s t -test. ( D ) The ultrathin cryosections of aPKC cKO and control kidney were labelled with anti-nephrin or anti-podocin antibodies followed by 10 nm gold particle-conjugated secondary antibody. Black arrowheads represent nephrin localized to the intracellular region, and white arrowheads represent nephrin localized to the rough ER. FP, foot process; GBM, glomerular basement membrane. ( E ) The distance of gold particle-labelled nephrin or podocin from the plasma membrane in aPKC cKO podocytes was compared with that of the control kidney. The P values were determined by two-tailed Mann–Whitney U -test.

    Journal: Journal of Biochemistry

    Article Title: aPKCλ maintains the integrity of the glomerular slit diaphragm through trafficking of nephrin to the cell surface

    doi: 10.1093/jb/mvu022

    Figure Lengend Snippet: aPKC is required for the cell-surface localization of SD components in vivo . ( A ) aPKC cKO and control mice at P10 or P11 were transcardially perfused with 2 mg/ml sulfo-NHS-SS-biotin/PBSCM for 5 min. Then, the kidneys were lysed and biotinylated proteins isolated with streptavidin sepharose and detected by immunoblot. The white arrowhead represents the mature-glycosylated, cell-surface form, and the black arrowhead represents the N -glycosylated, ER-form of nephrin. ( B ) Quantification of the results in (A). The values were normalized to control mice and are the mean ± SD of three independent experiments. The P values were determined by two-tailed Student’s t -test. ( C ) The cell-surface biotinylated aPKC cKO and control kidney were immunostained with nephrin, neph1 or podocin and biotin ( Supplementary Fig. S5 ), and the colocalization coefficient was calculated with LAS-AF software provided by Leica. The P values were determined by two-tailed Student’s t -test. ( D ) The ultrathin cryosections of aPKC cKO and control kidney were labelled with anti-nephrin or anti-podocin antibodies followed by 10 nm gold particle-conjugated secondary antibody. Black arrowheads represent nephrin localized to the intracellular region, and white arrowheads represent nephrin localized to the rough ER. FP, foot process; GBM, glomerular basement membrane. ( E ) The distance of gold particle-labelled nephrin or podocin from the plasma membrane in aPKC cKO podocytes was compared with that of the control kidney. The P values were determined by two-tailed Mann–Whitney U -test.

    Article Snippet: Briefly, the isolated glomeruli, HCT116-nephrin cells or HeLa Tet-On Advanced cells were washed with PBS containing 0.1 mM CaCl2 and 1 mM MgCl2 (PBSCM), and cell-surface proteins biotinylated with 0.5 mg/ml sulfo-NHS-SS-biotin (Thermo Scientific, Waltham, MA, USA) in PBSCM at 4°C for 30 min. Then, the glomeruli or cells were washed with 20 mM glycine in PBSCM at 4°C for 15 min to quench free sulfo-NSH-SS-biotin.

    Techniques: In Vivo, Mouse Assay, Isolation, Two Tailed Test, Software, MANN-WHITNEY

    Single Biotin-X-DHPE molecules tagged with Streptavidin-Alexa546 were tracked on the curved supported lipid bilayers (ROC = 28 nm) in space and time. ( a ) example trajectories (blue) show the heterogeneous dynamics observed on both flat (white) and curved (black) regions. Scale bar = 1 µm; ( b ) the average step a molecule takes over 0.228 s (5 frames) when starting at a region of curvature (white) or at a flat region (grey); ( c ) the distribution of steps observed at curved and flat regions (shown in Figure S3 ) was fitted to Equation 3 to obtain the diffusion coefficients and the percentage of steps moving at that rate. The average D is plotted for t = 0.091, 0.228 and 0.456. Note that there is no fast component for the tracks that start at regions of curvature.

    Journal: Membranes

    Article Title: Single Lipid Molecule Dynamics on Supported Lipid Bilayers with Membrane Curvature

    doi: 10.3390/membranes7010015

    Figure Lengend Snippet: Single Biotin-X-DHPE molecules tagged with Streptavidin-Alexa546 were tracked on the curved supported lipid bilayers (ROC = 28 nm) in space and time. ( a ) example trajectories (blue) show the heterogeneous dynamics observed on both flat (white) and curved (black) regions. Scale bar = 1 µm; ( b ) the average step a molecule takes over 0.228 s (5 frames) when starting at a region of curvature (white) or at a flat region (grey); ( c ) the distribution of steps observed at curved and flat regions (shown in Figure S3 ) was fitted to Equation 3 to obtain the diffusion coefficients and the percentage of steps moving at that rate. The average D is plotted for t = 0.091, 0.228 and 0.456. Note that there is no fast component for the tracks that start at regions of curvature.

    Article Snippet: The dynamics of Biotin-X-DHPE lipids were detected by in situ labeling with Alexa Fluor 546 (Thermo Fisher) conjugated to streptavidin (Strep-546).

    Techniques: Diffusion-based Assay