streptavidin -- horseradish Thermo Fisher Search Results


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  • 99
    Thermo Fisher streptavidin
    Interaction of HMGB1 and tail-truncated derivatives with chromatin. ( a ) Schematic of HMGB1. The sequence of the C-terminal intrinsically disordered acidic tail is shown, with the positions of the various truncations. ( b ) Label-transfer for HMGB1 and its truncated products modified with the biotin label-transfer reagent Sulfo-SBED, either alone or after incubation with linker-histone-depleted chromatin. After cross-linking, and cleavage of the reagent, proteins were resolved by SDS/PAGE and transferred to nitrocellulose membrane. The biotin label was detected by probing sequentially with <t>streptavidin</t> and anti-streptavidin (i), and the identity of the H3 band was confirmed by stripping and re-probing the same blot with anti-H3 (ii).
    Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8270 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher streptavidin horseradish conjugate
    Interaction of HMGB1 and tail-truncated derivatives with chromatin. ( a ) Schematic of HMGB1. The sequence of the C-terminal intrinsically disordered acidic tail is shown, with the positions of the various truncations. ( b ) Label-transfer for HMGB1 and its truncated products modified with the biotin label-transfer reagent Sulfo-SBED, either alone or after incubation with linker-histone-depleted chromatin. After cross-linking, and cleavage of the reagent, proteins were resolved by SDS/PAGE and transferred to nitrocellulose membrane. The biotin label was detected by probing sequentially with <t>streptavidin</t> and anti-streptavidin (i), and the identity of the H3 band was confirmed by stripping and re-probing the same blot with anti-H3 (ii).
    Streptavidin Horseradish Conjugate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher horseradish conjugated streptavidin
    Interaction of HMGB1 and tail-truncated derivatives with chromatin. ( a ) Schematic of HMGB1. The sequence of the C-terminal intrinsically disordered acidic tail is shown, with the positions of the various truncations. ( b ) Label-transfer for HMGB1 and its truncated products modified with the biotin label-transfer reagent Sulfo-SBED, either alone or after incubation with linker-histone-depleted chromatin. After cross-linking, and cleavage of the reagent, proteins were resolved by SDS/PAGE and transferred to nitrocellulose membrane. The biotin label was detected by probing sequentially with <t>streptavidin</t> and anti-streptavidin (i), and the identity of the H3 band was confirmed by stripping and re-probing the same blot with anti-H3 (ii).
    Horseradish Conjugated Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher streptavidin hrp
    CD9 regulates MHC-II internalization and recycling in mature MoDCs. (A and B) Immature (A) and LPS-matured (B) WT and CD9 KO MoDCs were incubated with biotinylated MHC-II antibodies for 1 h at 4°C, washed, and incubated for different times at 37°C, and then MHC-II surface expression was detected by flow cytometry after <t>streptavidin</t> labeling in CD11c + cells. Data represent mean fold changes ± SEM of results from three independent experiments performed in triplicate and were analyzed by two-way ANOVA with Bonferroni's post hoc multiple-comparison test. (C and D) Measurement of MHC-II internalization (C) and recycling (D) in mature WT and CD9 KO MoDCs by cell biotinylation assay. After incubation with biotin (blue in the schemes) at 4°C, cells were kept at 37°C (internalization), and the remaining surface biotins were removed by MESNA washing. (D) For recycling experiments, cells were further incubated for different times at 37°C (recycling), and surface biotins removed by MESNA washing. Cell lysates were immunoprecipitated with an anti-MHC-II antibody (M5/114), and biotinylated proteins were detected after membrane incubation with <t>streptavidin-HRP</t> (StrepHRP). Membranes were reprobed with MHC-II antibody for loading measurement. The blots shown are from representative experiments. (C) Only a fractional amount from cells incubated at 4°C in the absence of MESNA (0 min) was loaded. The graph shows the biotinylated MHC-II/total MHC-II signal ratio. The graph shows data determined as follows: [1 − (biotinylated MHC-II/total MHC-II signal ratio)]. Data represent mean fold changes ± SEM of results from four (C) and three (D) independent experiments analyzed by two-way ANOVA with Bonferroni's post hoc multiple-comparison test. **, P
    Streptavidin Hrp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3088 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher streptavidin horseradish
    CD9 regulates MHC-II internalization and recycling in mature MoDCs. (A and B) Immature (A) and LPS-matured (B) WT and CD9 KO MoDCs were incubated with biotinylated MHC-II antibodies for 1 h at 4°C, washed, and incubated for different times at 37°C, and then MHC-II surface expression was detected by flow cytometry after <t>streptavidin</t> labeling in CD11c + cells. Data represent mean fold changes ± SEM of results from three independent experiments performed in triplicate and were analyzed by two-way ANOVA with Bonferroni's post hoc multiple-comparison test. (C and D) Measurement of MHC-II internalization (C) and recycling (D) in mature WT and CD9 KO MoDCs by cell biotinylation assay. After incubation with biotin (blue in the schemes) at 4°C, cells were kept at 37°C (internalization), and the remaining surface biotins were removed by MESNA washing. (D) For recycling experiments, cells were further incubated for different times at 37°C (recycling), and surface biotins removed by MESNA washing. Cell lysates were immunoprecipitated with an anti-MHC-II antibody (M5/114), and biotinylated proteins were detected after membrane incubation with <t>streptavidin-HRP</t> (StrepHRP). Membranes were reprobed with MHC-II antibody for loading measurement. The blots shown are from representative experiments. (C) Only a fractional amount from cells incubated at 4°C in the absence of MESNA (0 min) was loaded. The graph shows the biotinylated MHC-II/total MHC-II signal ratio. The graph shows data determined as follows: [1 − (biotinylated MHC-II/total MHC-II signal ratio)]. Data represent mean fold changes ± SEM of results from four (C) and three (D) independent experiments analyzed by two-way ANOVA with Bonferroni's post hoc multiple-comparison test. **, P
    Streptavidin Horseradish, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher horseradish peroxidase streptavidin
    Expression of protocadherins. GFP was electroporated alone or in combination with protocadherins into K562 cells. a Immunoblotting of cell lysates with anti-GFP shows bands compatible with the expected molecular weights for GFP (27 kDa) and mature PCDHB11-GFP (110 kDa); image representative of three transfections. b Immunoblotting of cell lysates with anti-HA shows bands compatible with the expected molecular weights for mature HA-PCDHB11-GFP (113 kDa) and HA-PCDHGA3 (99 kDa); image representative of two transfections. c Proportion of medium and large clusters was lower in HA-PCDHB11-GFP-transfected than in HA-PCDHGA3-transfected cells, but higher than in control cells ( n = 10-12 images per condition). d Proportion of medium to large cell clusters when normalized by HA-PCDHGA3. For visualization of surface protocadherins, live cells transfected with GFP alone (e) or in combination with HA-PCDHB11-GFP (f) or HA-PCDHGA3 (g) were stained with anti-HA-biotin and <t>streptavidin-Alexa555,</t> then fixed. Blue: DAPI. Green: GFP. Red: surface HA-tagged protocadherins. Scale bar: 10 μm
    Horseradish Peroxidase Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 292 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher strepavidin horseradish peroxidase hrp antibody
    Expression of protocadherins. GFP was electroporated alone or in combination with protocadherins into K562 cells. a Immunoblotting of cell lysates with anti-GFP shows bands compatible with the expected molecular weights for GFP (27 kDa) and mature PCDHB11-GFP (110 kDa); image representative of three transfections. b Immunoblotting of cell lysates with anti-HA shows bands compatible with the expected molecular weights for mature HA-PCDHB11-GFP (113 kDa) and HA-PCDHGA3 (99 kDa); image representative of two transfections. c Proportion of medium and large clusters was lower in HA-PCDHB11-GFP-transfected than in HA-PCDHGA3-transfected cells, but higher than in control cells ( n = 10-12 images per condition). d Proportion of medium to large cell clusters when normalized by HA-PCDHGA3. For visualization of surface protocadherins, live cells transfected with GFP alone (e) or in combination with HA-PCDHB11-GFP (f) or HA-PCDHGA3 (g) were stained with anti-HA-biotin and <t>streptavidin-Alexa555,</t> then fixed. Blue: DAPI. Green: GFP. Red: surface HA-tagged protocadherins. Scale bar: 10 μm
    Strepavidin Horseradish Peroxidase Hrp Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher streptavidin conjugated horseradish peroxidase
    Composition of the P protein complex bound to 5′-vRNA. Nuclear extracts from Vac3P or VacT7 infected HeLa cells were incubated with a biotinylated RNA oligonucleotide containing the conserved 5′ terminal sequence of vRNA bound to <t>streptavidin</t> coated paramagnetic beads. Bound proteins were then analysed by western blot using C-terminal peptide antibodies specific for ( A ) PB1, ( B ) PB2 and ( C ) PA. Lane 1, 5′-vRNA coated SA-beads incubated with Vac3P extract. Lane 2, 5′-vRNA coated SA-beads incubated with VacT7 extract. Lanes 3 and 4, uncoated SA-beads incubated with Vac3P or VacT7 extracts, respectively. Lane 5, total Vac3P extract. Lane 6, total VacT7 extract. Lane 7, purified A/PR/8/34 virus marker.
    Streptavidin Conjugated Horseradish Peroxidase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 609 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Thermo Fisher streptavidin labeled horseradish peroxidase
    Composition of the P protein complex bound to 5′-vRNA. Nuclear extracts from Vac3P or VacT7 infected HeLa cells were incubated with a biotinylated RNA oligonucleotide containing the conserved 5′ terminal sequence of vRNA bound to <t>streptavidin</t> coated paramagnetic beads. Bound proteins were then analysed by western blot using C-terminal peptide antibodies specific for ( A ) PB1, ( B ) PB2 and ( C ) PA. Lane 1, 5′-vRNA coated SA-beads incubated with Vac3P extract. Lane 2, 5′-vRNA coated SA-beads incubated with VacT7 extract. Lanes 3 and 4, uncoated SA-beads incubated with Vac3P or VacT7 extracts, respectively. Lane 5, total Vac3P extract. Lane 6, total VacT7 extract. Lane 7, purified A/PR/8/34 virus marker.
    Streptavidin Labeled Horseradish Peroxidase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher horseradish peroxidase hrp streptavidin
    (A) Effects of anti-α 5 β 1 and anti-β 3 integrin blocking antibodies (Ab) on NIH 3T3 cell adhesion to fibronectin. NIH 3T3 fibroblasts stably expressing wild-type or kinase-inactive EphA8 were detached and incubated for 5 min with either anti-α 5 β 1 or anti-β 3 integrin blocking antibody (5 μg/ml). The cells were then replated on fibronectin-coated coverslips and allowed to adhere for 15 min. Data from three independent experiments are presented as means ± SE. (B) Induction of wild-type EphA8 protein expression in NIH 3T3 fibroblasts. Cells were incubated with 2 μg of doxycycline (Dox) per ml for the indicated periods of time and then lysed, and proteins from the lysates were immunoprecipitated with anti-EphA8 antibody. Immunoprecipitates were separated by 7.5% SDS-PAGE and immunoblotted with anti-EphA8 antibody (top); the same blot was stripped of antibodies and reprobed with antiphosphotyrosine antibody (bottom). (C) Effects of anti-α 5 β 1 and anti-β 3 integrin blocking antibodies on NIH 3T3 cell attachment stimulated by the induced expression of EphA8. Cells were treated with doxycycline for the indicated periods of time, and cell attachment assays were performed after incubation with either anti-α 5 β 1 or anti-β 3 blocking antibodies as described above. (D) Analysis of α 5 β 1 and β 3 integrins expressed in NIH 3T3 cells, concomitant with the induced expression of EphA8. EphA8 expression was induced for the indicated times, then the cells were harvested and cell surface proteins were biotinylated; integrins were immunoprecipitated with anti-α 5 polyclonal antibody, and biotinylated integrins were detected using <t>streptavidin-HRP</t> (top). Cells were lysed in PLC lysis buffer, and protein concentrations were equalized (bottom). Cell lysates were fractionated by SDS-PAGE and then analyzed by immunobloting using anti-β 3 antibody as a probe.
    Horseradish Peroxidase Hrp Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 64 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Thermo Fisher horseradish peroxidase streptavidin conjugate
    AMMP and ELISA target-binding assays. (a) Schematic of the AMMP target-binding assay. An HA-tagged ligand binds to target (Bcl-x L ) immobilized on <t>streptavidin</t> magnetic beads. Anti-HA antibody, labeled with fluorescein, binds to the HA tag and to antifluorescein antibody on the sensor surface. (b) AMMP signal in the target-binding assay is a function of dilution factor and ligand affinity. At the same dilution, Pep1 generates higher signal levels than Pep2, whereas ScPep1 and Pep1ΔHA show background levels of signal. (c) AMMP target-binding assay adjusted for peptide concentration as measured using the HA tag competition assay, demonstrating true relative affinities. (d) Schematic of the ELISA target-binding assay. An HA-tagged ligand binds to biotinylated target (Bcl-x L ) and anti-HA antibody on the ELISA plate. Streptavidin-HRP binds to biotin, resulting in an ELISA signal. (e) Target-binding ELISA assay is much less sensitive than the AMMP assay [described in (b)], and the respective samples behave equivalently: Pep1 generates a higher signal level than Pep2 and ScPep1 and Pep1ΔHA generate no signal over the background. (f) ELISA target-binding assay adjusted for peptide concentration as measured using the HA tag competition assay.
    Horseradish Peroxidase Streptavidin Conjugate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 87/100, based on 151 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher streptavidin hrp conjugate
    GlucB and sshBirA label and biotinylate EVs on the surface ( a, b ) Stable HEK293T cells expressing sshBirA with GlucB or Gluc. ( a ) Live-cell imaging of HEK293T cells stably transduced with GlucB-IRES-GFP or Gluc-IRES-GFP vectors, both with sshBirA-IRES-mCherry. Bar, 100 μm. ( b ) Western blot analysis showing enhanced biotinylation of cells stably expressing GlucB with sshBirA, as compared to GlucB alone. No biotinylation was detected in cells expressing Gluc alone or Gluc with sshBirA. Immunoblotting with anti-Gluc antibodies showed Gluc and GlucB at expected sizes (Gluc: 20 kDa; GlucB: 42 kDa). A low level of biotinylated BAP domain (22 kDa) was also detected in GlucB and GlucB + sshBirA samples. Mock transduced HEK293T were used as a negative control. GAPDH was immunoprobed as a loading control. ( c, d ) Transmission electron micrograph (TEM) demonstrating biotinylation and Gluc labeling of EV-GlucB on the membrane. ( c or anti-Gluc antibody followed by gold-conjugated secondary antibody to visualize GlucB labeling of EVs on the membrane, with EV-Gluc showing no Gluc signal but having the CD63 signal. Bar, 100 nm. ( d ) EVs in suspension were immunolabeled with an anti-biotin antibody followed by 10 nm gold-conjugated secondary antibody and biotinylation of EV-GlucB, but not EV-Gluc surface was detected. ( e ) Dot blot detection of biotinylated EVs. EVs isolated from HEK293T cells stably expressing sshBirA with either GlucB (top) or Gluc (bottom) were dot blotted on nitrocellulose membranes in a dose range followed by probing with <t>streptavidin-HRP</t> and chemiluminescence detection. EV-GlucB showed quantity-dependent biotinylated EVs, whereas EV-Gluc control exhibited no biotinylation background signal. ( f ) Nanoparticle tracking analysis (NTA) of EVs. Similar size distribution between EV-Gluc (peaks: 67, 100 and 177 nm) and EV-GlucB (81, 104 and 175 nm) vesicles was detected.
    Streptavidin Hrp Conjugate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 622 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher horseradish peroxidase streptavidin antibody
    Chronic ethanol feeding increased oxidative stress in adipocytes and adipose tissue. (A) 4-HNE protein adducts were quantified by slot blot analysis in isolated epididymal adipocytes. n=7. (B) Proteins were extracted from rat epididymal adipose tissue, coupled with biotin hydrazide, resolved by SDS-PAGE, transferred to PVDF membrane, and blotted with <t>HRP-streptavidin.</t> HSC70 was used as loading control. (C) Results of densitometry after normalization to HSC70. Values represent means ± SEM. n=4. Values with an asterisk are significantly different from each other, p
    Horseradish Peroxidase Streptavidin Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher streptavidin avidin horseradish peroxidase
    Chronic ethanol feeding increased oxidative stress in adipocytes and adipose tissue. (A) 4-HNE protein adducts were quantified by slot blot analysis in isolated epididymal adipocytes. n=7. (B) Proteins were extracted from rat epididymal adipose tissue, coupled with biotin hydrazide, resolved by SDS-PAGE, transferred to PVDF membrane, and blotted with <t>HRP-streptavidin.</t> HSC70 was used as loading control. (C) Results of densitometry after normalization to HSC70. Values represent means ± SEM. n=4. Values with an asterisk are significantly different from each other, p
    Streptavidin Avidin Horseradish Peroxidase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Thermo Fisher hrp streptavidin
    RA183 binding to RPN13 is stronger than RA190. (A) Chemical structures of RA9, b-AP15, RA190, RA183, and RA183B, in which a biotin moiety (blue) is linked to RA183, and likewise for RA190B. (B) Purified 19S RP (200 ng) was labeled with RA183, RA190, RA183B, or RA190B (20 μM) alone or in the presence of competitor RA183 and RA190 (100 or 200 μM) for 1 h at 37 °C. After labeling, equal aliquots were boiled in sodium dodecyl sulfate (SDS) sample buffer, separated by SDS-polyacrylamide gel electrophoresis (PAGE), transferred to a poly(vinylidene difluoride) (PVDF) membrane, probed with <t>streptavidin</t> peroxidase, and developed with chemiluminescence for the recognition of biotinylated proteins. (C, D) Cell lines from cancers of different tissue origins were lysed in Mammalian Protein Extraction Reagent (M-PER). In addition, Escherichia coli transduced with a plasmid expressing 6His and T7 tag-labeled RPN13 or irrelevant antigen and induced with isopropyl β- d -1-thiogalactopyranoside (0.1 mM, 2 h) were lysed in B-PER. Cell lysates (40 μg of protein) were treated with 5 μM RA183B (L) or not (C) for 45 min at 4 °C and subjected to SDS-PAGE, transferred to PVDF membrane, and probed with horseradish peroxidase <t>(HRP)–streptavidin.</t> The asterisk corresponds to ∼42 kDa RPN13 band.
    Hrp Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 494 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher streptavidin horseradish peroxidase complex
    RA183 binding to RPN13 is stronger than RA190. (A) Chemical structures of RA9, b-AP15, RA190, RA183, and RA183B, in which a biotin moiety (blue) is linked to RA183, and likewise for RA190B. (B) Purified 19S RP (200 ng) was labeled with RA183, RA190, RA183B, or RA190B (20 μM) alone or in the presence of competitor RA183 and RA190 (100 or 200 μM) for 1 h at 37 °C. After labeling, equal aliquots were boiled in sodium dodecyl sulfate (SDS) sample buffer, separated by SDS-polyacrylamide gel electrophoresis (PAGE), transferred to a poly(vinylidene difluoride) (PVDF) membrane, probed with <t>streptavidin</t> peroxidase, and developed with chemiluminescence for the recognition of biotinylated proteins. (C, D) Cell lines from cancers of different tissue origins were lysed in Mammalian Protein Extraction Reagent (M-PER). In addition, Escherichia coli transduced with a plasmid expressing 6His and T7 tag-labeled RPN13 or irrelevant antigen and induced with isopropyl β- d -1-thiogalactopyranoside (0.1 mM, 2 h) were lysed in B-PER. Cell lysates (40 μg of protein) were treated with 5 μM RA183B (L) or not (C) for 45 min at 4 °C and subjected to SDS-PAGE, transferred to PVDF membrane, and probed with horseradish peroxidase <t>(HRP)–streptavidin.</t> The asterisk corresponds to ∼42 kDa RPN13 band.
    Streptavidin Horseradish Peroxidase Complex, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 145 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher hrp conjugated streptavidin
    Effect of glycosylation inhibitors on expression of HA-tagged mCAT-1 in M. dunni cells. (A) Immunoblot analysis of lysates prepared from cells treated for 3 days with the indicated inhibitors: DMM, CST, Sw, DMN (65 μg/ml); 2DG (10 mM); Tu (0.125 ug/ml). (B) Immunoblot of surface biotinylated proteins from DMM-treated and untreated cells. The upper panel was probed with anti-HA; the lower panel shows the same blot stripped and reprobedwith <t>streptavidin-HRP</t> to show that surface biotinylation and protein loading were approximately equal.
    Hrp Conjugated Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1353 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher pierce streptavidin poly horseradish peroxidase
    Effect of glycosylation inhibitors on expression of HA-tagged mCAT-1 in M. dunni cells. (A) Immunoblot analysis of lysates prepared from cells treated for 3 days with the indicated inhibitors: DMM, CST, Sw, DMN (65 μg/ml); 2DG (10 mM); Tu (0.125 ug/ml). (B) Immunoblot of surface biotinylated proteins from DMM-treated and untreated cells. The upper panel was probed with anti-HA; the lower panel shows the same blot stripped and reprobedwith <t>streptavidin-HRP</t> to show that surface biotinylation and protein loading were approximately equal.
    Pierce Streptavidin Poly Horseradish Peroxidase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher streptavidin horseradish perioxidase hrp antibody
    Effect of glycosylation inhibitors on expression of HA-tagged mCAT-1 in M. dunni cells. (A) Immunoblot analysis of lysates prepared from cells treated for 3 days with the indicated inhibitors: DMM, CST, Sw, DMN (65 μg/ml); 2DG (10 mM); Tu (0.125 ug/ml). (B) Immunoblot of surface biotinylated proteins from DMM-treated and untreated cells. The upper panel was probed with anti-HA; the lower panel shows the same blot stripped and reprobedwith <t>streptavidin-HRP</t> to show that surface biotinylation and protein loading were approximately equal.
    Streptavidin Horseradish Perioxidase Hrp Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Effect of glycosylation inhibitors on expression of HA-tagged mCAT-1 in M. dunni cells. (A) Immunoblot analysis of lysates prepared from cells treated for 3 days with the indicated inhibitors: DMM, CST, Sw, DMN (65 μg/ml); 2DG (10 mM); Tu (0.125 ug/ml). (B) Immunoblot of surface biotinylated proteins from DMM-treated and untreated cells. The upper panel was probed with anti-HA; the lower panel shows the same blot stripped and reprobedwith <t>streptavidin-HRP</t> to show that surface biotinylation and protein loading were approximately equal.
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    Effect of glycosylation inhibitors on expression of HA-tagged mCAT-1 in M. dunni cells. (A) Immunoblot analysis of lysates prepared from cells treated for 3 days with the indicated inhibitors: DMM, CST, Sw, DMN (65 μg/ml); 2DG (10 mM); Tu (0.125 ug/ml). (B) Immunoblot of surface biotinylated proteins from DMM-treated and untreated cells. The upper panel was probed with anti-HA; the lower panel shows the same blot stripped and reprobedwith <t>streptavidin-HRP</t> to show that surface biotinylation and protein loading were approximately equal.
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    Effect of glycosylation inhibitors on expression of HA-tagged mCAT-1 in M. dunni cells. (A) Immunoblot analysis of lysates prepared from cells treated for 3 days with the indicated inhibitors: DMM, CST, Sw, DMN (65 μg/ml); 2DG (10 mM); Tu (0.125 ug/ml). (B) Immunoblot of surface biotinylated proteins from DMM-treated and untreated cells. The upper panel was probed with anti-HA; the lower panel shows the same blot stripped and reprobedwith <t>streptavidin-HRP</t> to show that surface biotinylation and protein loading were approximately equal.
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    Metabolic stability of the dimeric and monomeric forms of E-cadherin. (A) Surface proteins of AEcM cells grown in 5-cm-diameter dishes were biotinylated and then chased in regular media. The chase times (in hours) are indicated above the blots. Cells were immunoprecipitated with an excess of anti-myc antibody, and the immunoprecipitates were adjusted to 60 μl with SDS-polyacrylamide gel electrophoresis sample buffer. Five microliters of each immunoprecipitate was loaded. The blots were probed either with <t>streptavidin-HRP</t> (Str-HRP) or anti-myc antibody (Myc). Three different exposure times of the blot developed by Str-HRP are shown. After an exposure time of 10 s [Str-HRP (10)], only the Ec1M protein was visualized, allowing us to estimate the half-life of the monomeric E-cadherin. A longer, 60-s exposure [Str-HRP (60)] visualized coimmunoprecipitated endogenous cadherin (Ec). The blots at the bottom of panel A [Str-HRP (E)] show blots of different exposure times equilibrated on the Ec1M signals, which allowed us to demonstrate that the ratio of Ec1M to endogenous cadherin did not change during the chase periods. Panel B is identical to panel A except that cell lysates before anti-myc immunoprecipitation were subjected to sucrose gradient centrifugation either immediately after the biotinylation (blot 0) or after the 16-h chase (blot 16). The exposure time of blot 0 was shorter than that of blot 16, showing that during the 16-h chase the Ec1M/E-cadherin ratio did not change. Note that immunoprecipitation of Ec1M leads to coimmunoprecipitation of the endogenous E-cadherin (Ec) only in fractions 4 to 6. (C) Surface-biotinylated A-431 cells were dissociated by EGTA and either cocultured for additional 8 h with AEcM cells (lane 1) or cultivated separately and combined after lysis (lane 2). The latter served as a control, showing the absence of interactions between cadherin molecules in solution. The small black bars on the left of the blots indicate the positions of molecular mass markers of 116 and 97.4 kDa.
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    Metabolic stability of the dimeric and monomeric forms of E-cadherin. (A) Surface proteins of AEcM cells grown in 5-cm-diameter dishes were biotinylated and then chased in regular media. The chase times (in hours) are indicated above the blots. Cells were immunoprecipitated with an excess of anti-myc antibody, and the immunoprecipitates were adjusted to 60 μl with SDS-polyacrylamide gel electrophoresis sample buffer. Five microliters of each immunoprecipitate was loaded. The blots were probed either with <t>streptavidin-HRP</t> (Str-HRP) or anti-myc antibody (Myc). Three different exposure times of the blot developed by Str-HRP are shown. After an exposure time of 10 s [Str-HRP (10)], only the Ec1M protein was visualized, allowing us to estimate the half-life of the monomeric E-cadherin. A longer, 60-s exposure [Str-HRP (60)] visualized coimmunoprecipitated endogenous cadherin (Ec). The blots at the bottom of panel A [Str-HRP (E)] show blots of different exposure times equilibrated on the Ec1M signals, which allowed us to demonstrate that the ratio of Ec1M to endogenous cadherin did not change during the chase periods. Panel B is identical to panel A except that cell lysates before anti-myc immunoprecipitation were subjected to sucrose gradient centrifugation either immediately after the biotinylation (blot 0) or after the 16-h chase (blot 16). The exposure time of blot 0 was shorter than that of blot 16, showing that during the 16-h chase the Ec1M/E-cadherin ratio did not change. Note that immunoprecipitation of Ec1M leads to coimmunoprecipitation of the endogenous E-cadherin (Ec) only in fractions 4 to 6. (C) Surface-biotinylated A-431 cells were dissociated by EGTA and either cocultured for additional 8 h with AEcM cells (lane 1) or cultivated separately and combined after lysis (lane 2). The latter served as a control, showing the absence of interactions between cadherin molecules in solution. The small black bars on the left of the blots indicate the positions of molecular mass markers of 116 and 97.4 kDa.
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    RALT binds to SH3 domains. (A) Lysates of 293-RALT transfectants were incubated with the indicated recombinant GST fusion proteins immobilized onto glutathione-agarose beads. Proteins bound to resins were analyzed by immunoblotting with anti-RALT antibodies and the ECL reaction. Lysate in the left lane represents 5% of the input lysate in the binding reaction. (B) GST–GRB-2 fusions expressed from either wt GRB-2 cDNA or cDNA encoding the indicated GRB-2 mutant proteins were purified onto glutathione-agarose beads in similar amounts and used as affinity reagents for the capture of RALT solubilized from 293 transfectants; lysate in the left lane corresponds to 5% input in the binding reaction. Proteins bound to resin were subjected to immunoblot analysis with S1 anti-RALT antibodies. (C) Each of the indicated recombinant proteins (2 μg; purified by affinity chromatography on glutathione-agarose resin) was run on SDS-PAGE gel and transferred to nitrocellulose filters. After the proteins were stained with Ponceau's red to ensure that equal amounts of protein were present in each lane, independent filters were probed with biotin-labeled GST–RALT 1-262 or GST–RALT 263-459; detection was with <t>HRP-conjugated</t> <t>streptavidin</t> followed by the ECL reaction. No specific reactivity was observed with RALT 1-262, and therefore only the filter probed with GST–RALT 263-459 is shown. (D) Lysates from 293 cells transfected with RALT or RALT and GRB-2 expression vectors were analyzed for RALT and GRB-2 expression by immunoblotting (upper two panels). Anti-RALT immunoprecipitates from these lysates were blotted with anti-GRB-2 antibodies along with lysates representing 10% of the input protein in the immunoprecipitation reaction (lower two panels). ECL exposure of the third panel from top was 90 s, while that of the bottom panel was 45 s. WB, Western blotting; Tx, transfection.
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    RALT binds to SH3 domains. (A) Lysates of 293-RALT transfectants were incubated with the indicated recombinant GST fusion proteins immobilized onto glutathione-agarose beads. Proteins bound to resins were analyzed by immunoblotting with anti-RALT antibodies and the ECL reaction. Lysate in the left lane represents 5% of the input lysate in the binding reaction. (B) GST–GRB-2 fusions expressed from either wt GRB-2 cDNA or cDNA encoding the indicated GRB-2 mutant proteins were purified onto glutathione-agarose beads in similar amounts and used as affinity reagents for the capture of RALT solubilized from 293 transfectants; lysate in the left lane corresponds to 5% input in the binding reaction. Proteins bound to resin were subjected to immunoblot analysis with S1 anti-RALT antibodies. (C) Each of the indicated recombinant proteins (2 μg; purified by affinity chromatography on glutathione-agarose resin) was run on SDS-PAGE gel and transferred to nitrocellulose filters. After the proteins were stained with Ponceau's red to ensure that equal amounts of protein were present in each lane, independent filters were probed with biotin-labeled GST–RALT 1-262 or GST–RALT 263-459; detection was with <t>HRP-conjugated</t> <t>streptavidin</t> followed by the ECL reaction. No specific reactivity was observed with RALT 1-262, and therefore only the filter probed with GST–RALT 263-459 is shown. (D) Lysates from 293 cells transfected with RALT or RALT and GRB-2 expression vectors were analyzed for RALT and GRB-2 expression by immunoblotting (upper two panels). Anti-RALT immunoprecipitates from these lysates were blotted with anti-GRB-2 antibodies along with lysates representing 10% of the input protein in the immunoprecipitation reaction (lower two panels). ECL exposure of the third panel from top was 90 s, while that of the bottom panel was 45 s. WB, Western blotting; Tx, transfection.
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    RALT binds to SH3 domains. (A) Lysates of 293-RALT transfectants were incubated with the indicated recombinant GST fusion proteins immobilized onto glutathione-agarose beads. Proteins bound to resins were analyzed by immunoblotting with anti-RALT antibodies and the ECL reaction. Lysate in the left lane represents 5% of the input lysate in the binding reaction. (B) GST–GRB-2 fusions expressed from either wt GRB-2 cDNA or cDNA encoding the indicated GRB-2 mutant proteins were purified onto glutathione-agarose beads in similar amounts and used as affinity reagents for the capture of RALT solubilized from 293 transfectants; lysate in the left lane corresponds to 5% input in the binding reaction. Proteins bound to resin were subjected to immunoblot analysis with S1 anti-RALT antibodies. (C) Each of the indicated recombinant proteins (2 μg; purified by affinity chromatography on glutathione-agarose resin) was run on SDS-PAGE gel and transferred to nitrocellulose filters. After the proteins were stained with Ponceau's red to ensure that equal amounts of protein were present in each lane, independent filters were probed with biotin-labeled GST–RALT 1-262 or GST–RALT 263-459; detection was with <t>HRP-conjugated</t> <t>streptavidin</t> followed by the ECL reaction. No specific reactivity was observed with RALT 1-262, and therefore only the filter probed with GST–RALT 263-459 is shown. (D) Lysates from 293 cells transfected with RALT or RALT and GRB-2 expression vectors were analyzed for RALT and GRB-2 expression by immunoblotting (upper two panels). Anti-RALT immunoprecipitates from these lysates were blotted with anti-GRB-2 antibodies along with lysates representing 10% of the input protein in the immunoprecipitation reaction (lower two panels). ECL exposure of the third panel from top was 90 s, while that of the bottom panel was 45 s. WB, Western blotting; Tx, transfection.
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    RALT binds to SH3 domains. (A) Lysates of 293-RALT transfectants were incubated with the indicated recombinant GST fusion proteins immobilized onto glutathione-agarose beads. Proteins bound to resins were analyzed by immunoblotting with anti-RALT antibodies and the ECL reaction. Lysate in the left lane represents 5% of the input lysate in the binding reaction. (B) GST–GRB-2 fusions expressed from either wt GRB-2 cDNA or cDNA encoding the indicated GRB-2 mutant proteins were purified onto glutathione-agarose beads in similar amounts and used as affinity reagents for the capture of RALT solubilized from 293 transfectants; lysate in the left lane corresponds to 5% input in the binding reaction. Proteins bound to resin were subjected to immunoblot analysis with S1 anti-RALT antibodies. (C) Each of the indicated recombinant proteins (2 μg; purified by affinity chromatography on glutathione-agarose resin) was run on SDS-PAGE gel and transferred to nitrocellulose filters. After the proteins were stained with Ponceau's red to ensure that equal amounts of protein were present in each lane, independent filters were probed with biotin-labeled GST–RALT 1-262 or GST–RALT 263-459; detection was with <t>HRP-conjugated</t> <t>streptavidin</t> followed by the ECL reaction. No specific reactivity was observed with RALT 1-262, and therefore only the filter probed with GST–RALT 263-459 is shown. (D) Lysates from 293 cells transfected with RALT or RALT and GRB-2 expression vectors were analyzed for RALT and GRB-2 expression by immunoblotting (upper two panels). Anti-RALT immunoprecipitates from these lysates were blotted with anti-GRB-2 antibodies along with lysates representing 10% of the input protein in the immunoprecipitation reaction (lower two panels). ECL exposure of the third panel from top was 90 s, while that of the bottom panel was 45 s. WB, Western blotting; Tx, transfection.
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    RALT binds to SH3 domains. (A) Lysates of 293-RALT transfectants were incubated with the indicated recombinant GST fusion proteins immobilized onto glutathione-agarose beads. Proteins bound to resins were analyzed by immunoblotting with anti-RALT antibodies and the ECL reaction. Lysate in the left lane represents 5% of the input lysate in the binding reaction. (B) GST–GRB-2 fusions expressed from either wt GRB-2 cDNA or cDNA encoding the indicated GRB-2 mutant proteins were purified onto glutathione-agarose beads in similar amounts and used as affinity reagents for the capture of RALT solubilized from 293 transfectants; lysate in the left lane corresponds to 5% input in the binding reaction. Proteins bound to resin were subjected to immunoblot analysis with S1 anti-RALT antibodies. (C) Each of the indicated recombinant proteins (2 μg; purified by affinity chromatography on glutathione-agarose resin) was run on SDS-PAGE gel and transferred to nitrocellulose filters. After the proteins were stained with Ponceau's red to ensure that equal amounts of protein were present in each lane, independent filters were probed with biotin-labeled GST–RALT 1-262 or GST–RALT 263-459; detection was with <t>HRP-conjugated</t> <t>streptavidin</t> followed by the ECL reaction. No specific reactivity was observed with RALT 1-262, and therefore only the filter probed with GST–RALT 263-459 is shown. (D) Lysates from 293 cells transfected with RALT or RALT and GRB-2 expression vectors were analyzed for RALT and GRB-2 expression by immunoblotting (upper two panels). Anti-RALT immunoprecipitates from these lysates were blotted with anti-GRB-2 antibodies along with lysates representing 10% of the input protein in the immunoprecipitation reaction (lower two panels). ECL exposure of the third panel from top was 90 s, while that of the bottom panel was 45 s. WB, Western blotting; Tx, transfection.
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    RALT binds to SH3 domains. (A) Lysates of 293-RALT transfectants were incubated with the indicated recombinant GST fusion proteins immobilized onto glutathione-agarose beads. Proteins bound to resins were analyzed by immunoblotting with anti-RALT antibodies and the ECL reaction. Lysate in the left lane represents 5% of the input lysate in the binding reaction. (B) GST–GRB-2 fusions expressed from either wt GRB-2 cDNA or cDNA encoding the indicated GRB-2 mutant proteins were purified onto glutathione-agarose beads in similar amounts and used as affinity reagents for the capture of RALT solubilized from 293 transfectants; lysate in the left lane corresponds to 5% input in the binding reaction. Proteins bound to resin were subjected to immunoblot analysis with S1 anti-RALT antibodies. (C) Each of the indicated recombinant proteins (2 μg; purified by affinity chromatography on glutathione-agarose resin) was run on SDS-PAGE gel and transferred to nitrocellulose filters. After the proteins were stained with Ponceau's red to ensure that equal amounts of protein were present in each lane, independent filters were probed with biotin-labeled GST–RALT 1-262 or GST–RALT 263-459; detection was with <t>HRP-conjugated</t> <t>streptavidin</t> followed by the ECL reaction. No specific reactivity was observed with RALT 1-262, and therefore only the filter probed with GST–RALT 263-459 is shown. (D) Lysates from 293 cells transfected with RALT or RALT and GRB-2 expression vectors were analyzed for RALT and GRB-2 expression by immunoblotting (upper two panels). Anti-RALT immunoprecipitates from these lysates were blotted with anti-GRB-2 antibodies along with lysates representing 10% of the input protein in the immunoprecipitation reaction (lower two panels). ECL exposure of the third panel from top was 90 s, while that of the bottom panel was 45 s. WB, Western blotting; Tx, transfection.
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    Image Search Results


    Interaction of HMGB1 and tail-truncated derivatives with chromatin. ( a ) Schematic of HMGB1. The sequence of the C-terminal intrinsically disordered acidic tail is shown, with the positions of the various truncations. ( b ) Label-transfer for HMGB1 and its truncated products modified with the biotin label-transfer reagent Sulfo-SBED, either alone or after incubation with linker-histone-depleted chromatin. After cross-linking, and cleavage of the reagent, proteins were resolved by SDS/PAGE and transferred to nitrocellulose membrane. The biotin label was detected by probing sequentially with streptavidin and anti-streptavidin (i), and the identity of the H3 band was confirmed by stripping and re-probing the same blot with anti-H3 (ii).

    Journal: Nucleic Acids Research

    Article Title: Characterization of the interaction between HMGB1 and H3--a possible means of positioning HMGB1 in chromatin

    doi: 10.1093/nar/gkt950

    Figure Lengend Snippet: Interaction of HMGB1 and tail-truncated derivatives with chromatin. ( a ) Schematic of HMGB1. The sequence of the C-terminal intrinsically disordered acidic tail is shown, with the positions of the various truncations. ( b ) Label-transfer for HMGB1 and its truncated products modified with the biotin label-transfer reagent Sulfo-SBED, either alone or after incubation with linker-histone-depleted chromatin. After cross-linking, and cleavage of the reagent, proteins were resolved by SDS/PAGE and transferred to nitrocellulose membrane. The biotin label was detected by probing sequentially with streptavidin and anti-streptavidin (i), and the identity of the H3 band was confirmed by stripping and re-probing the same blot with anti-H3 (ii).

    Article Snippet: The biotin label was detected by probing with both streptavidin (HRP-conjugated; Pierce) and HRP-conjugated anti-streptavidin antibody (Abcam; ab7239).

    Techniques: Sequencing, Modification, Incubation, SDS Page, Stripping Membranes

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

    Journal: Molecular and Cellular Biology

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

    doi: 10.1128/MCB.00202-17

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

    Article Snippet: For internalization and recycling assays, biotinylated proteins were revealed using a Fujifilm LAS-4000 system after membrane incubation with streptavidin-HRP (Invitrogen).

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

    Correlation between apoptotic signaling and the generation of large DNAs with decreased generation of excised oligonucleotide repair products. ( A ) Analysis of large DNA fragments and excised oligomers released from genomic DNA following UV irradiation. HeLa cells were exposed to 20 J/m 2 of UVC and incubated for the indicated time points. After extraction of low molecular weight of DNAs from the cells, the DNA molecules were labeled with biotin at 3′-end using terminal deoxynucleotidyl transferase (TdT). The labeled DNA molecules were separated by gel electrophoresis, transferred to a nylon membrane and immobilized by UV cross-linking. The membrane was then incubated with HRP-conjugated streptavidin, and the labeled DNA molecules were detected with a chemiluminescence reagent (upper panel). A small portion of the UV-irradiated cells were lysed and analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. ( B ) Quantification of the excised oligonucleotide repair products and cleaved PARP signals as shown in ( A ). The relative signal intensities were determined by setting the highest signal to 100. The results were quantified and are plotted as mean and standard deviations. ( C ) Quantitative analysis of the excised repair products and the large DNA molecules. ( D ) Wild-type (AA8) and XPG mutant (UV135) CHO cell lines were exposed to UV and processed as described above for the detection of soluble DNAs.

    Journal: Scientific Reports

    Article Title: Simultaneous detection of nucleotide excision repair events and apoptosis-induced DNA fragmentation in genotoxin-treated cells

    doi: 10.1038/s41598-018-20527-6

    Figure Lengend Snippet: Correlation between apoptotic signaling and the generation of large DNAs with decreased generation of excised oligonucleotide repair products. ( A ) Analysis of large DNA fragments and excised oligomers released from genomic DNA following UV irradiation. HeLa cells were exposed to 20 J/m 2 of UVC and incubated for the indicated time points. After extraction of low molecular weight of DNAs from the cells, the DNA molecules were labeled with biotin at 3′-end using terminal deoxynucleotidyl transferase (TdT). The labeled DNA molecules were separated by gel electrophoresis, transferred to a nylon membrane and immobilized by UV cross-linking. The membrane was then incubated with HRP-conjugated streptavidin, and the labeled DNA molecules were detected with a chemiluminescence reagent (upper panel). A small portion of the UV-irradiated cells were lysed and analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. ( B ) Quantification of the excised oligonucleotide repair products and cleaved PARP signals as shown in ( A ). The relative signal intensities were determined by setting the highest signal to 100. The results were quantified and are plotted as mean and standard deviations. ( C ) Quantitative analysis of the excised repair products and the large DNA molecules. ( D ) Wild-type (AA8) and XPG mutant (UV135) CHO cell lines were exposed to UV and processed as described above for the detection of soluble DNAs.

    Article Snippet: The entire blot was probed with streptavidin-HRP conjugates, and the entire image is displayed in each of the figures along with longer exposures of the region of the gel corresponding to the excised products of repair.

    Techniques: Irradiation, Incubation, Molecular Weight, Labeling, Nucleic Acid Electrophoresis, SDS Page, Mutagenesis

    rHSA-P53i and rHSA-PMI bind to MDM2 and MDMX. To detect the interaction between MDM2/MDMX and rHSA fusion proteins, 4 µg each of biotinylated rHSA (lane 1), rHSA-P53i (lane 2), or rHSA-PMI (lane 3) were added to 200 µg of SJSA-1 whole cell lysate. MDM2 or MDMX antibody was added to the lysate followed by pulling down MDM2/MDMX and rHSA complexes using Protein A/G (1:1) resins. Samples were then analyzed by SDS-PAGE and Western blotting using MDM2, MDMX, and Streptavidin-HRP (Strep-HRP) antibodies.

    Journal: PLoS ONE

    Article Title: Human Serum Albumin and p53-Activating Peptide Fusion Protein Is Able to Promote Apoptosis and Deliver Fatty Acid-Modified Molecules

    doi: 10.1371/journal.pone.0080926

    Figure Lengend Snippet: rHSA-P53i and rHSA-PMI bind to MDM2 and MDMX. To detect the interaction between MDM2/MDMX and rHSA fusion proteins, 4 µg each of biotinylated rHSA (lane 1), rHSA-P53i (lane 2), or rHSA-PMI (lane 3) were added to 200 µg of SJSA-1 whole cell lysate. MDM2 or MDMX antibody was added to the lysate followed by pulling down MDM2/MDMX and rHSA complexes using Protein A/G (1:1) resins. Samples were then analyzed by SDS-PAGE and Western blotting using MDM2, MDMX, and Streptavidin-HRP (Strep-HRP) antibodies.

    Article Snippet: Proteins bound to MDM2/MDMX antibody were pulled down using Protein A/G (1:1) resins and samples were analyzed by SDS-PAGE and Western blotting using MDM2, MDMX, and Streptavidin-HRP antibodies (Pierce, 21130 ).

    Techniques: SDS Page, Western Blot

    Expression of protocadherins. GFP was electroporated alone or in combination with protocadherins into K562 cells. a Immunoblotting of cell lysates with anti-GFP shows bands compatible with the expected molecular weights for GFP (27 kDa) and mature PCDHB11-GFP (110 kDa); image representative of three transfections. b Immunoblotting of cell lysates with anti-HA shows bands compatible with the expected molecular weights for mature HA-PCDHB11-GFP (113 kDa) and HA-PCDHGA3 (99 kDa); image representative of two transfections. c Proportion of medium and large clusters was lower in HA-PCDHB11-GFP-transfected than in HA-PCDHGA3-transfected cells, but higher than in control cells ( n = 10-12 images per condition). d Proportion of medium to large cell clusters when normalized by HA-PCDHGA3. For visualization of surface protocadherins, live cells transfected with GFP alone (e) or in combination with HA-PCDHB11-GFP (f) or HA-PCDHGA3 (g) were stained with anti-HA-biotin and streptavidin-Alexa555, then fixed. Blue: DAPI. Green: GFP. Red: surface HA-tagged protocadherins. Scale bar: 10 μm

    Journal: BMC Evolutionary Biology

    Article Title: Functional test of PCDHB11, the most human-specific neuronal surface protein

    doi: 10.1186/s12862-016-0652-x

    Figure Lengend Snippet: Expression of protocadherins. GFP was electroporated alone or in combination with protocadherins into K562 cells. a Immunoblotting of cell lysates with anti-GFP shows bands compatible with the expected molecular weights for GFP (27 kDa) and mature PCDHB11-GFP (110 kDa); image representative of three transfections. b Immunoblotting of cell lysates with anti-HA shows bands compatible with the expected molecular weights for mature HA-PCDHB11-GFP (113 kDa) and HA-PCDHGA3 (99 kDa); image representative of two transfections. c Proportion of medium and large clusters was lower in HA-PCDHB11-GFP-transfected than in HA-PCDHGA3-transfected cells, but higher than in control cells ( n = 10-12 images per condition). d Proportion of medium to large cell clusters when normalized by HA-PCDHGA3. For visualization of surface protocadherins, live cells transfected with GFP alone (e) or in combination with HA-PCDHB11-GFP (f) or HA-PCDHGA3 (g) were stained with anti-HA-biotin and streptavidin-Alexa555, then fixed. Blue: DAPI. Green: GFP. Red: surface HA-tagged protocadherins. Scale bar: 10 μm

    Article Snippet: For GFP detection, membranes were incubated with a rabbit polyclonal antibody (Life Technologies, A-11122) diluted 1:2000 in 50 mM Tris, 100 mM NaCl, 0.1 % Tween 20, pH 7.4, with 3 % bovine serum albumine (Sigma, St. Louis, MO) and a horse-radish peroxidase-conjugated anti-rabbit secondary antibody (Invitrogen, Waltham, MA), while for HA revelation, they were incubated with biotinylated 3 F10 rat monoclonal antibody (Roche Life Science, Indianapolis, IN) diluted 1:500 in the same buffer, and streptavidin-horseradish peroxidase (Invitrogen, Waltham, MA).

    Techniques: Expressing, Transfection, Staining

    Generation and characterization of the peptide-Fc fusion proteins. ( a ) Schematic representation of peptide-Fc construction. The arrow indicates the cleavage site of IL-2 signal sequence. A glycine linker (in blue) was added to further increase the flexibility of the peptide moiety. ( b ) Peptide-Fc fusion expression in HEK293T cells. Subsequent to 48 hours of transfection time, culture supernatants (15 µl each) were analyzed by western blots using biotin-conjugated antihuman Fc antibody followed by streptavidin–HRP. Lanes 1 to 4 correspond to cells transfected with plasmid encoding WN-Fc, LTV-Fc, MY-Fc, and Fc control, respectively. Unt, supernatant from untransfeted cells. ( c ) Secreted peptide-Fc fusion proteins were purified with protein G affinity chromatography from culture supernatants and analyzed by SDS-PAGE under reducing conditions and then stained with Coomassie blue. ( d ) A duplicate gel was transferred to nitrocellulose membrane and processed as in b . Lanes 1 to 4 correspond to WN-Fc, LTV-Fc, MY-Fc, and Fc control fusion proteins, respectively. ( e ) Binding of the peptide-Fc fusion proteins to SKBR3. The cells were incubated with either peptide-Fc fusion proteins (blue histograms) or the Fc control (orange histograms) followed by FITC-conjugated antihuman Fc monoclonal antibody and analysis by flow cytometry. All molecules were tested at 10 µg/ml. In the case of competition experiments, the cells were incubated with the peptide (100 µg/ml) first and then with the corresponding peptide-Fc fusion protein (green histograms). Red histograms represent cells stained with only secondary antibody. Data are from one experiment and are representative of at least 10 independent experiments.

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: Generation of new peptide-Fc fusion proteins that mediate antibody-dependent cellular cytotoxicity against different types of cancer cells

    doi: 10.1038/mtm.2015.43

    Figure Lengend Snippet: Generation and characterization of the peptide-Fc fusion proteins. ( a ) Schematic representation of peptide-Fc construction. The arrow indicates the cleavage site of IL-2 signal sequence. A glycine linker (in blue) was added to further increase the flexibility of the peptide moiety. ( b ) Peptide-Fc fusion expression in HEK293T cells. Subsequent to 48 hours of transfection time, culture supernatants (15 µl each) were analyzed by western blots using biotin-conjugated antihuman Fc antibody followed by streptavidin–HRP. Lanes 1 to 4 correspond to cells transfected with plasmid encoding WN-Fc, LTV-Fc, MY-Fc, and Fc control, respectively. Unt, supernatant from untransfeted cells. ( c ) Secreted peptide-Fc fusion proteins were purified with protein G affinity chromatography from culture supernatants and analyzed by SDS-PAGE under reducing conditions and then stained with Coomassie blue. ( d ) A duplicate gel was transferred to nitrocellulose membrane and processed as in b . Lanes 1 to 4 correspond to WN-Fc, LTV-Fc, MY-Fc, and Fc control fusion proteins, respectively. ( e ) Binding of the peptide-Fc fusion proteins to SKBR3. The cells were incubated with either peptide-Fc fusion proteins (blue histograms) or the Fc control (orange histograms) followed by FITC-conjugated antihuman Fc monoclonal antibody and analysis by flow cytometry. All molecules were tested at 10 µg/ml. In the case of competition experiments, the cells were incubated with the peptide (100 µg/ml) first and then with the corresponding peptide-Fc fusion protein (green histograms). Red histograms represent cells stained with only secondary antibody. Data are from one experiment and are representative of at least 10 independent experiments.

    Article Snippet: Streptavidin–horseradish peroxidase conjugates were obtained from Thermo Scientific (Rockford, IL).

    Techniques: Sequencing, Expressing, Transfection, Western Blot, Plasmid Preparation, Purification, Affinity Chromatography, SDS Page, Staining, Binding Assay, Incubation, Flow Cytometry, Cytometry

    Composition of the P protein complex bound to 5′-vRNA. Nuclear extracts from Vac3P or VacT7 infected HeLa cells were incubated with a biotinylated RNA oligonucleotide containing the conserved 5′ terminal sequence of vRNA bound to streptavidin coated paramagnetic beads. Bound proteins were then analysed by western blot using C-terminal peptide antibodies specific for ( A ) PB1, ( B ) PB2 and ( C ) PA. Lane 1, 5′-vRNA coated SA-beads incubated with Vac3P extract. Lane 2, 5′-vRNA coated SA-beads incubated with VacT7 extract. Lanes 3 and 4, uncoated SA-beads incubated with Vac3P or VacT7 extracts, respectively. Lane 5, total Vac3P extract. Lane 6, total VacT7 extract. Lane 7, purified A/PR/8/34 virus marker.

    Journal: Nucleic Acids Research

    Article Title: Definition of the minimal viral components required for the initiation of unprimed RNA synthesis by influenza virus RNA polymerase

    doi:

    Figure Lengend Snippet: Composition of the P protein complex bound to 5′-vRNA. Nuclear extracts from Vac3P or VacT7 infected HeLa cells were incubated with a biotinylated RNA oligonucleotide containing the conserved 5′ terminal sequence of vRNA bound to streptavidin coated paramagnetic beads. Bound proteins were then analysed by western blot using C-terminal peptide antibodies specific for ( A ) PB1, ( B ) PB2 and ( C ) PA. Lane 1, 5′-vRNA coated SA-beads incubated with Vac3P extract. Lane 2, 5′-vRNA coated SA-beads incubated with VacT7 extract. Lanes 3 and 4, uncoated SA-beads incubated with Vac3P or VacT7 extracts, respectively. Lane 5, total Vac3P extract. Lane 6, total VacT7 extract. Lane 7, purified A/PR/8/34 virus marker.

    Article Snippet: Bound antibodies were detected using biotin conjugated goat, anti-rabbit or anti-mouse IgG secondary antibody as appropriate (Sigma-Aldrich Co.) followed by either streptavidin conjugated alkaline phosphatase/BCIP/NBT development (Sigma-Aldrich Co.) for Figure only; or streptavidin conjugated horseradish peroxidase (Pierce) chemiluminescent development according to the manufacturer’s protocol (BM Chemiluminescence Blotting substrate, Roche Diagnostics Ltd).

    Techniques: Infection, Incubation, Sequencing, Western Blot, Purification, Marker

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

    Journal: The Journal of General Physiology

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

    doi: 10.1085/jgp.200409031

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

    Article Snippet: Depending on the experiment, the nitrocellulose was probed with (a) primary antibody (1/1,000 dilution) followed by horseradish peroxidase–conjugated goat anti–rabbit IgG (1/7,000) (Jackson ImmunoResearch Laboratories) in PBS-T with 1% dry milk or (b) horseradish peroxidase–conjugated streptavidin (Pierce Chemical Co.).

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

    (A) Effects of anti-α 5 β 1 and anti-β 3 integrin blocking antibodies (Ab) on NIH 3T3 cell adhesion to fibronectin. NIH 3T3 fibroblasts stably expressing wild-type or kinase-inactive EphA8 were detached and incubated for 5 min with either anti-α 5 β 1 or anti-β 3 integrin blocking antibody (5 μg/ml). The cells were then replated on fibronectin-coated coverslips and allowed to adhere for 15 min. Data from three independent experiments are presented as means ± SE. (B) Induction of wild-type EphA8 protein expression in NIH 3T3 fibroblasts. Cells were incubated with 2 μg of doxycycline (Dox) per ml for the indicated periods of time and then lysed, and proteins from the lysates were immunoprecipitated with anti-EphA8 antibody. Immunoprecipitates were separated by 7.5% SDS-PAGE and immunoblotted with anti-EphA8 antibody (top); the same blot was stripped of antibodies and reprobed with antiphosphotyrosine antibody (bottom). (C) Effects of anti-α 5 β 1 and anti-β 3 integrin blocking antibodies on NIH 3T3 cell attachment stimulated by the induced expression of EphA8. Cells were treated with doxycycline for the indicated periods of time, and cell attachment assays were performed after incubation with either anti-α 5 β 1 or anti-β 3 blocking antibodies as described above. (D) Analysis of α 5 β 1 and β 3 integrins expressed in NIH 3T3 cells, concomitant with the induced expression of EphA8. EphA8 expression was induced for the indicated times, then the cells were harvested and cell surface proteins were biotinylated; integrins were immunoprecipitated with anti-α 5 polyclonal antibody, and biotinylated integrins were detected using streptavidin-HRP (top). Cells were lysed in PLC lysis buffer, and protein concentrations were equalized (bottom). Cell lysates were fractionated by SDS-PAGE and then analyzed by immunobloting using anti-β 3 antibody as a probe.

    Journal: Molecular and Cellular Biology

    Article Title: The EphA8 Receptor Regulates Integrin Activity through p110? Phosphatidylinositol-3 Kinase in a Tyrosine Kinase Activity-Independent Manner

    doi: 10.1128/MCB.21.14.4579-4597.2001

    Figure Lengend Snippet: (A) Effects of anti-α 5 β 1 and anti-β 3 integrin blocking antibodies (Ab) on NIH 3T3 cell adhesion to fibronectin. NIH 3T3 fibroblasts stably expressing wild-type or kinase-inactive EphA8 were detached and incubated for 5 min with either anti-α 5 β 1 or anti-β 3 integrin blocking antibody (5 μg/ml). The cells were then replated on fibronectin-coated coverslips and allowed to adhere for 15 min. Data from three independent experiments are presented as means ± SE. (B) Induction of wild-type EphA8 protein expression in NIH 3T3 fibroblasts. Cells were incubated with 2 μg of doxycycline (Dox) per ml for the indicated periods of time and then lysed, and proteins from the lysates were immunoprecipitated with anti-EphA8 antibody. Immunoprecipitates were separated by 7.5% SDS-PAGE and immunoblotted with anti-EphA8 antibody (top); the same blot was stripped of antibodies and reprobed with antiphosphotyrosine antibody (bottom). (C) Effects of anti-α 5 β 1 and anti-β 3 integrin blocking antibodies on NIH 3T3 cell attachment stimulated by the induced expression of EphA8. Cells were treated with doxycycline for the indicated periods of time, and cell attachment assays were performed after incubation with either anti-α 5 β 1 or anti-β 3 blocking antibodies as described above. (D) Analysis of α 5 β 1 and β 3 integrins expressed in NIH 3T3 cells, concomitant with the induced expression of EphA8. EphA8 expression was induced for the indicated times, then the cells were harvested and cell surface proteins were biotinylated; integrins were immunoprecipitated with anti-α 5 polyclonal antibody, and biotinylated integrins were detected using streptavidin-HRP (top). Cells were lysed in PLC lysis buffer, and protein concentrations were equalized (bottom). Cell lysates were fractionated by SDS-PAGE and then analyzed by immunobloting using anti-β 3 antibody as a probe.

    Article Snippet: Biotinylated integrins were detected by incubating blots with streptavidin-horseradish peroxidase (HRP) (Pierce).

    Techniques: Blocking Assay, Stable Transfection, Expressing, Incubation, Immunoprecipitation, SDS Page, Cell Attachment Assay, Planar Chromatography, Lysis, Western Blot

    AMMP and ELISA target-binding assays. (a) Schematic of the AMMP target-binding assay. An HA-tagged ligand binds to target (Bcl-x L ) immobilized on streptavidin magnetic beads. Anti-HA antibody, labeled with fluorescein, binds to the HA tag and to antifluorescein antibody on the sensor surface. (b) AMMP signal in the target-binding assay is a function of dilution factor and ligand affinity. At the same dilution, Pep1 generates higher signal levels than Pep2, whereas ScPep1 and Pep1ΔHA show background levels of signal. (c) AMMP target-binding assay adjusted for peptide concentration as measured using the HA tag competition assay, demonstrating true relative affinities. (d) Schematic of the ELISA target-binding assay. An HA-tagged ligand binds to biotinylated target (Bcl-x L ) and anti-HA antibody on the ELISA plate. Streptavidin-HRP binds to biotin, resulting in an ELISA signal. (e) Target-binding ELISA assay is much less sensitive than the AMMP assay [described in (b)], and the respective samples behave equivalently: Pep1 generates a higher signal level than Pep2 and ScPep1 and Pep1ΔHA generate no signal over the background. (f) ELISA target-binding assay adjusted for peptide concentration as measured using the HA tag competition assay.

    Journal: Analytical Chemistry

    Article Title: Robust, Quantitative Analysis of Proteins using Peptide Immunoreagents, in Vitro Translation, and an Ultrasensitive Acoustic Resonant Sensor

    doi: 10.1021/ac500084d

    Figure Lengend Snippet: AMMP and ELISA target-binding assays. (a) Schematic of the AMMP target-binding assay. An HA-tagged ligand binds to target (Bcl-x L ) immobilized on streptavidin magnetic beads. Anti-HA antibody, labeled with fluorescein, binds to the HA tag and to antifluorescein antibody on the sensor surface. (b) AMMP signal in the target-binding assay is a function of dilution factor and ligand affinity. At the same dilution, Pep1 generates higher signal levels than Pep2, whereas ScPep1 and Pep1ΔHA show background levels of signal. (c) AMMP target-binding assay adjusted for peptide concentration as measured using the HA tag competition assay, demonstrating true relative affinities. (d) Schematic of the ELISA target-binding assay. An HA-tagged ligand binds to biotinylated target (Bcl-x L ) and anti-HA antibody on the ELISA plate. Streptavidin-HRP binds to biotin, resulting in an ELISA signal. (e) Target-binding ELISA assay is much less sensitive than the AMMP assay [described in (b)], and the respective samples behave equivalently: Pep1 generates a higher signal level than Pep2 and ScPep1 and Pep1ΔHA generate no signal over the background. (f) ELISA target-binding assay adjusted for peptide concentration as measured using the HA tag competition assay.

    Article Snippet: Plates were washed with wash buffer [1× PBS + 0.1% (v/v) Tween-20] and blocked with 1× PBS + 5% (w/v) BSA for 2 h. In each well, 100 μL of sample and 100 μL of 33 nM biotinylated Bcl-xL were added and incubated for 4 h. Plates were washed, incubated with streptavidin horseradish peroxidase conjugate (Strep-HRP, Thermo Scientific) for 1 h, washed, and incubated with TMB substrate (Thermo Scientific).

    Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Magnetic Beads, Labeling, Concentration Assay, Competitive Binding Assay

    GlucB and sshBirA label and biotinylate EVs on the surface ( a, b ) Stable HEK293T cells expressing sshBirA with GlucB or Gluc. ( a ) Live-cell imaging of HEK293T cells stably transduced with GlucB-IRES-GFP or Gluc-IRES-GFP vectors, both with sshBirA-IRES-mCherry. Bar, 100 μm. ( b ) Western blot analysis showing enhanced biotinylation of cells stably expressing GlucB with sshBirA, as compared to GlucB alone. No biotinylation was detected in cells expressing Gluc alone or Gluc with sshBirA. Immunoblotting with anti-Gluc antibodies showed Gluc and GlucB at expected sizes (Gluc: 20 kDa; GlucB: 42 kDa). A low level of biotinylated BAP domain (22 kDa) was also detected in GlucB and GlucB + sshBirA samples. Mock transduced HEK293T were used as a negative control. GAPDH was immunoprobed as a loading control. ( c, d ) Transmission electron micrograph (TEM) demonstrating biotinylation and Gluc labeling of EV-GlucB on the membrane. ( c or anti-Gluc antibody followed by gold-conjugated secondary antibody to visualize GlucB labeling of EVs on the membrane, with EV-Gluc showing no Gluc signal but having the CD63 signal. Bar, 100 nm. ( d ) EVs in suspension were immunolabeled with an anti-biotin antibody followed by 10 nm gold-conjugated secondary antibody and biotinylation of EV-GlucB, but not EV-Gluc surface was detected. ( e ) Dot blot detection of biotinylated EVs. EVs isolated from HEK293T cells stably expressing sshBirA with either GlucB (top) or Gluc (bottom) were dot blotted on nitrocellulose membranes in a dose range followed by probing with streptavidin-HRP and chemiluminescence detection. EV-GlucB showed quantity-dependent biotinylated EVs, whereas EV-Gluc control exhibited no biotinylation background signal. ( f ) Nanoparticle tracking analysis (NTA) of EVs. Similar size distribution between EV-Gluc (peaks: 67, 100 and 177 nm) and EV-GlucB (81, 104 and 175 nm) vesicles was detected.

    Journal: ACS nano

    Article Title: Dynamic Biodistribution of Extracellular Vesicles In Vivo Using a Multimodal Imaging Reporter

    doi: 10.1021/nn404945r

    Figure Lengend Snippet: GlucB and sshBirA label and biotinylate EVs on the surface ( a, b ) Stable HEK293T cells expressing sshBirA with GlucB or Gluc. ( a ) Live-cell imaging of HEK293T cells stably transduced with GlucB-IRES-GFP or Gluc-IRES-GFP vectors, both with sshBirA-IRES-mCherry. Bar, 100 μm. ( b ) Western blot analysis showing enhanced biotinylation of cells stably expressing GlucB with sshBirA, as compared to GlucB alone. No biotinylation was detected in cells expressing Gluc alone or Gluc with sshBirA. Immunoblotting with anti-Gluc antibodies showed Gluc and GlucB at expected sizes (Gluc: 20 kDa; GlucB: 42 kDa). A low level of biotinylated BAP domain (22 kDa) was also detected in GlucB and GlucB + sshBirA samples. Mock transduced HEK293T were used as a negative control. GAPDH was immunoprobed as a loading control. ( c, d ) Transmission electron micrograph (TEM) demonstrating biotinylation and Gluc labeling of EV-GlucB on the membrane. ( c or anti-Gluc antibody followed by gold-conjugated secondary antibody to visualize GlucB labeling of EVs on the membrane, with EV-Gluc showing no Gluc signal but having the CD63 signal. Bar, 100 nm. ( d ) EVs in suspension were immunolabeled with an anti-biotin antibody followed by 10 nm gold-conjugated secondary antibody and biotinylation of EV-GlucB, but not EV-Gluc surface was detected. ( e ) Dot blot detection of biotinylated EVs. EVs isolated from HEK293T cells stably expressing sshBirA with either GlucB (top) or Gluc (bottom) were dot blotted on nitrocellulose membranes in a dose range followed by probing with streptavidin-HRP and chemiluminescence detection. EV-GlucB showed quantity-dependent biotinylated EVs, whereas EV-Gluc control exhibited no biotinylation background signal. ( f ) Nanoparticle tracking analysis (NTA) of EVs. Similar size distribution between EV-Gluc (peaks: 67, 100 and 177 nm) and EV-GlucB (81, 104 and 175 nm) vesicles was detected.

    Article Snippet: Gluc or GlucB labeled EVs in PBS were spotted onto nitrocellulose membranes at 16, 31, 63, 125, 250, 500 and 1,000 ng protein followed by overnight incubation in 5% BSA fraction V (Invitrogen), immunoblotting with streptavidin-HRP conjugate (Pierce) and chemiluminescence detection of biotinylated EVs with SuperSignal West Pico Chemiluminescent Substrate kit (Pierce) on autoradiography films (Denville Scientific, Metuchen, NJ).

    Techniques: Expressing, Live Cell Imaging, Stable Transfection, Transduction, Western Blot, Negative Control, Transmission Assay, Transmission Electron Microscopy, Labeling, Immunolabeling, Dot Blot, Isolation

    Identification of in vivo binding partners of AKAP95. (A) Schematic representation of Myc-BirA*-AKAP95. (B) Western blot analysis using anti-myc antibody and HRP-labeled streptavidin of lysates from Myc-BirA*-AKAP95 expressing- and control U2OS cells cultured as indicated. (C) Immunolocalization of Myc-BirA*-AKAP95 (anti-myc; red) and protein biotinylation (FITC-labeled streptavidin; green) in stable Myc-BirA*–AKAP95 U2OS cells cultured as indicated. Immunolocalization of AKAP95 in U2OS cells (bottom). Scale bar, 5 μm. (D) Gene ontology analysis and nuclear component classification of proteins identified by AKAP95 BioID. (E) Western blot analysis using indicated antibodies of the samples from Myc-BirA*-AKAP95-expressing and control U2OS cells that were subjected to BioID.

    Journal: Cell Cycle

    Article Title: AKAP95 interacts with nucleoporin TPR in mitosis and is important for the spindle assembly checkpoint

    doi: 10.1080/15384101.2017.1310350

    Figure Lengend Snippet: Identification of in vivo binding partners of AKAP95. (A) Schematic representation of Myc-BirA*-AKAP95. (B) Western blot analysis using anti-myc antibody and HRP-labeled streptavidin of lysates from Myc-BirA*-AKAP95 expressing- and control U2OS cells cultured as indicated. (C) Immunolocalization of Myc-BirA*-AKAP95 (anti-myc; red) and protein biotinylation (FITC-labeled streptavidin; green) in stable Myc-BirA*–AKAP95 U2OS cells cultured as indicated. Immunolocalization of AKAP95 in U2OS cells (bottom). Scale bar, 5 μm. (D) Gene ontology analysis and nuclear component classification of proteins identified by AKAP95 BioID. (E) Western blot analysis using indicated antibodies of the samples from Myc-BirA*-AKAP95-expressing and control U2OS cells that were subjected to BioID.

    Article Snippet: Biotinylated proteins were detected directly using HRP-Streptavidin conjugate (Invitrogen; 43-4323; 1:5000).

    Techniques: In Vivo, Binding Assay, Western Blot, Labeling, Expressing, Cell Culture

    Chronic ethanol feeding increased oxidative stress in adipocytes and adipose tissue. (A) 4-HNE protein adducts were quantified by slot blot analysis in isolated epididymal adipocytes. n=7. (B) Proteins were extracted from rat epididymal adipose tissue, coupled with biotin hydrazide, resolved by SDS-PAGE, transferred to PVDF membrane, and blotted with HRP-streptavidin. HSC70 was used as loading control. (C) Results of densitometry after normalization to HSC70. Values represent means ± SEM. n=4. Values with an asterisk are significantly different from each other, p

    Journal: Alcoholism, Clinical and Experimental Research

    Article Title: Ethanol-induced oxidative stress via the CYP2E1 pathway disrupts adiponectin secretion from adipocytes

    doi: 10.1111/j.1530-0277.2011.01607.x

    Figure Lengend Snippet: Chronic ethanol feeding increased oxidative stress in adipocytes and adipose tissue. (A) 4-HNE protein adducts were quantified by slot blot analysis in isolated epididymal adipocytes. n=7. (B) Proteins were extracted from rat epididymal adipose tissue, coupled with biotin hydrazide, resolved by SDS-PAGE, transferred to PVDF membrane, and blotted with HRP-streptavidin. HSC70 was used as loading control. (C) Results of densitometry after normalization to HSC70. Values represent means ± SEM. n=4. Values with an asterisk are significantly different from each other, p

    Article Snippet: EZ-link biotin hydrazide and poly-HRP streptavidin were purchased from Pierce (Rockford, IL).

    Techniques: Dot Blot, Isolation, SDS Page

    RA183 binding to RPN13 is stronger than RA190. (A) Chemical structures of RA9, b-AP15, RA190, RA183, and RA183B, in which a biotin moiety (blue) is linked to RA183, and likewise for RA190B. (B) Purified 19S RP (200 ng) was labeled with RA183, RA190, RA183B, or RA190B (20 μM) alone or in the presence of competitor RA183 and RA190 (100 or 200 μM) for 1 h at 37 °C. After labeling, equal aliquots were boiled in sodium dodecyl sulfate (SDS) sample buffer, separated by SDS-polyacrylamide gel electrophoresis (PAGE), transferred to a poly(vinylidene difluoride) (PVDF) membrane, probed with streptavidin peroxidase, and developed with chemiluminescence for the recognition of biotinylated proteins. (C, D) Cell lines from cancers of different tissue origins were lysed in Mammalian Protein Extraction Reagent (M-PER). In addition, Escherichia coli transduced with a plasmid expressing 6His and T7 tag-labeled RPN13 or irrelevant antigen and induced with isopropyl β- d -1-thiogalactopyranoside (0.1 mM, 2 h) were lysed in B-PER. Cell lysates (40 μg of protein) were treated with 5 μM RA183B (L) or not (C) for 45 min at 4 °C and subjected to SDS-PAGE, transferred to PVDF membrane, and probed with horseradish peroxidase (HRP)–streptavidin. The asterisk corresponds to ∼42 kDa RPN13 band.

    Journal: ACS Omega

    Article Title: Covalent Rpn13-Binding Inhibitors for the Treatment of Ovarian Cancer

    doi: 10.1021/acsomega.8b01479

    Figure Lengend Snippet: RA183 binding to RPN13 is stronger than RA190. (A) Chemical structures of RA9, b-AP15, RA190, RA183, and RA183B, in which a biotin moiety (blue) is linked to RA183, and likewise for RA190B. (B) Purified 19S RP (200 ng) was labeled with RA183, RA190, RA183B, or RA190B (20 μM) alone or in the presence of competitor RA183 and RA190 (100 or 200 μM) for 1 h at 37 °C. After labeling, equal aliquots were boiled in sodium dodecyl sulfate (SDS) sample buffer, separated by SDS-polyacrylamide gel electrophoresis (PAGE), transferred to a poly(vinylidene difluoride) (PVDF) membrane, probed with streptavidin peroxidase, and developed with chemiluminescence for the recognition of biotinylated proteins. (C, D) Cell lines from cancers of different tissue origins were lysed in Mammalian Protein Extraction Reagent (M-PER). In addition, Escherichia coli transduced with a plasmid expressing 6His and T7 tag-labeled RPN13 or irrelevant antigen and induced with isopropyl β- d -1-thiogalactopyranoside (0.1 mM, 2 h) were lysed in B-PER. Cell lysates (40 μg of protein) were treated with 5 μM RA183B (L) or not (C) for 45 min at 4 °C and subjected to SDS-PAGE, transferred to PVDF membrane, and probed with horseradish peroxidase (HRP)–streptavidin. The asterisk corresponds to ∼42 kDa RPN13 band.

    Article Snippet: Antibodies for Western Blot analysis were obtained by the following commercial sources: anti-p21(WAF1), Bax (Cell Signaling Technology, Danvers, MA), antiubiquitin (SC8017), anti-IkB-α (C-15), anti-ATF-4 (SC-200) (Santa Cruz Biotechnology, Santa Cruz, CA), anti-PARP (BD Pharmingen, San Diego, California), (Stressgen Corp., Victoria, BC, Canada), antitubulin (Sigma, St. Louis, MO), anti-actin, anti-ADRM1 (anti-RPN13) (Sigma-Aldrich), antiubiquitin, Lys48 (05-1307) (Millipore), streptavidin Dynabeads (Invitrogen), HRP–streptavidin (Invitrogen), peroxidase-linked antimouse immunoglobulin G (IgG), and antirabbit IgG (GE Healthcare U.K. Ltd., U.K.) and utilized at the concentration recommended by the manufacturer.

    Techniques: Binding Assay, Purification, Labeling, Polyacrylamide Gel Electrophoresis, Protein Extraction, Transduction, Plasmid Preparation, Expressing, SDS Page

    Effect of glycosylation inhibitors on expression of HA-tagged mCAT-1 in M. dunni cells. (A) Immunoblot analysis of lysates prepared from cells treated for 3 days with the indicated inhibitors: DMM, CST, Sw, DMN (65 μg/ml); 2DG (10 mM); Tu (0.125 ug/ml). (B) Immunoblot of surface biotinylated proteins from DMM-treated and untreated cells. The upper panel was probed with anti-HA; the lower panel shows the same blot stripped and reprobedwith streptavidin-HRP to show that surface biotinylation and protein loading were approximately equal.

    Journal: Retrovirology

    Article Title: Role of receptor polymorphism and glycosylation in syncytium induction and host range variation of ecotropic mouse gammaretroviruses

    doi: 10.1186/1742-4690-5-2

    Figure Lengend Snippet: Effect of glycosylation inhibitors on expression of HA-tagged mCAT-1 in M. dunni cells. (A) Immunoblot analysis of lysates prepared from cells treated for 3 days with the indicated inhibitors: DMM, CST, Sw, DMN (65 μg/ml); 2DG (10 mM); Tu (0.125 ug/ml). (B) Immunoblot of surface biotinylated proteins from DMM-treated and untreated cells. The upper panel was probed with anti-HA; the lower panel shows the same blot stripped and reprobedwith streptavidin-HRP to show that surface biotinylation and protein loading were approximately equal.

    Article Snippet: The membrane was stripped using Pierce Stripping Buffer (catalog no. 21059) and reprobed using HRP-conjugated streptavidin (Pierce; catalog no. 21126).

    Techniques: Expressing

    Identification of an anti-ζ-globin chain mAbs pair for detecting ζ-globin chain. An ELISA plate was coated with 10 μg/mL of the anti-ζ-globin chain mAb clones PL2 or PL3 or an isotype-matched control mAb (AM85B-8B) as indicated. A sandwich ELISA for detecting various concentrations of ζ-globin chains was performed using (A) biotin-labeled mAb PL2 (PL2-biotin) and (B) biotin-labeled mAb PL3 (PL3-biotin). HRP-conjugated streptavidin was used to monitor the antigen-antibody reaction.

    Journal: PLoS ONE

    Article Title: Impact of the detection of ζ-globin chains and hemoglobin Bart’s using immunochromatographic strip tests for α0-thalassemia (--SEA) differential diagnosis

    doi: 10.1371/journal.pone.0223996

    Figure Lengend Snippet: Identification of an anti-ζ-globin chain mAbs pair for detecting ζ-globin chain. An ELISA plate was coated with 10 μg/mL of the anti-ζ-globin chain mAb clones PL2 or PL3 or an isotype-matched control mAb (AM85B-8B) as indicated. A sandwich ELISA for detecting various concentrations of ζ-globin chains was performed using (A) biotin-labeled mAb PL2 (PL2-biotin) and (B) biotin-labeled mAb PL3 (PL3-biotin). HRP-conjugated streptavidin was used to monitor the antigen-antibody reaction.

    Article Snippet: Horseradish peroxidase (HRP)-labeled streptavidin and 3,3’,5,5’-tetramethylbenzidine (TMB) substrate were purchased from Invitrogen (Camarillo, CA, USA).

    Techniques: Enzyme-linked Immunosorbent Assay, Clone Assay, Sandwich ELISA, Labeling

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

    Journal: Nature Communications

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

    doi: 10.1038/ncomms7765

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

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

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

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

    Journal: Biochemistry

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

    doi: 10.1021/bi4014825

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

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

    Techniques: Labeling, Incubation

    Metabolic stability of the dimeric and monomeric forms of E-cadherin. (A) Surface proteins of AEcM cells grown in 5-cm-diameter dishes were biotinylated and then chased in regular media. The chase times (in hours) are indicated above the blots. Cells were immunoprecipitated with an excess of anti-myc antibody, and the immunoprecipitates were adjusted to 60 μl with SDS-polyacrylamide gel electrophoresis sample buffer. Five microliters of each immunoprecipitate was loaded. The blots were probed either with streptavidin-HRP (Str-HRP) or anti-myc antibody (Myc). Three different exposure times of the blot developed by Str-HRP are shown. After an exposure time of 10 s [Str-HRP (10)], only the Ec1M protein was visualized, allowing us to estimate the half-life of the monomeric E-cadherin. A longer, 60-s exposure [Str-HRP (60)] visualized coimmunoprecipitated endogenous cadherin (Ec). The blots at the bottom of panel A [Str-HRP (E)] show blots of different exposure times equilibrated on the Ec1M signals, which allowed us to demonstrate that the ratio of Ec1M to endogenous cadherin did not change during the chase periods. Panel B is identical to panel A except that cell lysates before anti-myc immunoprecipitation were subjected to sucrose gradient centrifugation either immediately after the biotinylation (blot 0) or after the 16-h chase (blot 16). The exposure time of blot 0 was shorter than that of blot 16, showing that during the 16-h chase the Ec1M/E-cadherin ratio did not change. Note that immunoprecipitation of Ec1M leads to coimmunoprecipitation of the endogenous E-cadherin (Ec) only in fractions 4 to 6. (C) Surface-biotinylated A-431 cells were dissociated by EGTA and either cocultured for additional 8 h with AEcM cells (lane 1) or cultivated separately and combined after lysis (lane 2). The latter served as a control, showing the absence of interactions between cadherin molecules in solution. The small black bars on the left of the blots indicate the positions of molecular mass markers of 116 and 97.4 kDa.

    Journal: Molecular and Cellular Biology

    Article Title: Dynamic Interplay between Adhesive and Lateral E-Cadherin Dimers

    doi: 10.1128/MCB.22.21.7449-7458.2002

    Figure Lengend Snippet: Metabolic stability of the dimeric and monomeric forms of E-cadherin. (A) Surface proteins of AEcM cells grown in 5-cm-diameter dishes were biotinylated and then chased in regular media. The chase times (in hours) are indicated above the blots. Cells were immunoprecipitated with an excess of anti-myc antibody, and the immunoprecipitates were adjusted to 60 μl with SDS-polyacrylamide gel electrophoresis sample buffer. Five microliters of each immunoprecipitate was loaded. The blots were probed either with streptavidin-HRP (Str-HRP) or anti-myc antibody (Myc). Three different exposure times of the blot developed by Str-HRP are shown. After an exposure time of 10 s [Str-HRP (10)], only the Ec1M protein was visualized, allowing us to estimate the half-life of the monomeric E-cadherin. A longer, 60-s exposure [Str-HRP (60)] visualized coimmunoprecipitated endogenous cadherin (Ec). The blots at the bottom of panel A [Str-HRP (E)] show blots of different exposure times equilibrated on the Ec1M signals, which allowed us to demonstrate that the ratio of Ec1M to endogenous cadherin did not change during the chase periods. Panel B is identical to panel A except that cell lysates before anti-myc immunoprecipitation were subjected to sucrose gradient centrifugation either immediately after the biotinylation (blot 0) or after the 16-h chase (blot 16). The exposure time of blot 0 was shorter than that of blot 16, showing that during the 16-h chase the Ec1M/E-cadherin ratio did not change. Note that immunoprecipitation of Ec1M leads to coimmunoprecipitation of the endogenous E-cadherin (Ec) only in fractions 4 to 6. (C) Surface-biotinylated A-431 cells were dissociated by EGTA and either cocultured for additional 8 h with AEcM cells (lane 1) or cultivated separately and combined after lysis (lane 2). The latter served as a control, showing the absence of interactions between cadherin molecules in solution. The small black bars on the left of the blots indicate the positions of molecular mass markers of 116 and 97.4 kDa.

    Article Snippet: Biotinylated proteins were visualized with streptavidin-horseradish peroxidase (HRP) conjugate (Pierce) in conjunction with ECL (Boehringer Mannheim, Indianapolis, Ind.).

    Techniques: Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Gradient Centrifugation, Lysis

    RALT binds to SH3 domains. (A) Lysates of 293-RALT transfectants were incubated with the indicated recombinant GST fusion proteins immobilized onto glutathione-agarose beads. Proteins bound to resins were analyzed by immunoblotting with anti-RALT antibodies and the ECL reaction. Lysate in the left lane represents 5% of the input lysate in the binding reaction. (B) GST–GRB-2 fusions expressed from either wt GRB-2 cDNA or cDNA encoding the indicated GRB-2 mutant proteins were purified onto glutathione-agarose beads in similar amounts and used as affinity reagents for the capture of RALT solubilized from 293 transfectants; lysate in the left lane corresponds to 5% input in the binding reaction. Proteins bound to resin were subjected to immunoblot analysis with S1 anti-RALT antibodies. (C) Each of the indicated recombinant proteins (2 μg; purified by affinity chromatography on glutathione-agarose resin) was run on SDS-PAGE gel and transferred to nitrocellulose filters. After the proteins were stained with Ponceau's red to ensure that equal amounts of protein were present in each lane, independent filters were probed with biotin-labeled GST–RALT 1-262 or GST–RALT 263-459; detection was with HRP-conjugated streptavidin followed by the ECL reaction. No specific reactivity was observed with RALT 1-262, and therefore only the filter probed with GST–RALT 263-459 is shown. (D) Lysates from 293 cells transfected with RALT or RALT and GRB-2 expression vectors were analyzed for RALT and GRB-2 expression by immunoblotting (upper two panels). Anti-RALT immunoprecipitates from these lysates were blotted with anti-GRB-2 antibodies along with lysates representing 10% of the input protein in the immunoprecipitation reaction (lower two panels). ECL exposure of the third panel from top was 90 s, while that of the bottom panel was 45 s. WB, Western blotting; Tx, transfection.

    Journal: Molecular and Cellular Biology

    Article Title: Inhibition of ErbB-2 Mitogenic and Transforming Activity by RALT, a Mitogen-Induced Signal Transducer Which Binds to the ErbB-2 Kinase Domain †

    doi:

    Figure Lengend Snippet: RALT binds to SH3 domains. (A) Lysates of 293-RALT transfectants were incubated with the indicated recombinant GST fusion proteins immobilized onto glutathione-agarose beads. Proteins bound to resins were analyzed by immunoblotting with anti-RALT antibodies and the ECL reaction. Lysate in the left lane represents 5% of the input lysate in the binding reaction. (B) GST–GRB-2 fusions expressed from either wt GRB-2 cDNA or cDNA encoding the indicated GRB-2 mutant proteins were purified onto glutathione-agarose beads in similar amounts and used as affinity reagents for the capture of RALT solubilized from 293 transfectants; lysate in the left lane corresponds to 5% input in the binding reaction. Proteins bound to resin were subjected to immunoblot analysis with S1 anti-RALT antibodies. (C) Each of the indicated recombinant proteins (2 μg; purified by affinity chromatography on glutathione-agarose resin) was run on SDS-PAGE gel and transferred to nitrocellulose filters. After the proteins were stained with Ponceau's red to ensure that equal amounts of protein were present in each lane, independent filters were probed with biotin-labeled GST–RALT 1-262 or GST–RALT 263-459; detection was with HRP-conjugated streptavidin followed by the ECL reaction. No specific reactivity was observed with RALT 1-262, and therefore only the filter probed with GST–RALT 263-459 is shown. (D) Lysates from 293 cells transfected with RALT or RALT and GRB-2 expression vectors were analyzed for RALT and GRB-2 expression by immunoblotting (upper two panels). Anti-RALT immunoprecipitates from these lysates were blotted with anti-GRB-2 antibodies along with lysates representing 10% of the input protein in the immunoprecipitation reaction (lower two panels). ECL exposure of the third panel from top was 90 s, while that of the bottom panel was 45 s. WB, Western blotting; Tx, transfection.

    Article Snippet: RALT proteins captured by the filter were detected by horseradish peroxidase (HRP)-conjugated streptavidin (Pierce) (0.1 μg/ml) followed by enhanced chemiluminescence (ECL).

    Techniques: Incubation, Recombinant, Binding Assay, Mutagenesis, Purification, Affinity Chromatography, SDS Page, Staining, Labeling, Transfection, Expressing, Immunoprecipitation, Western Blot