streptavidin hrp  (Thermo Fisher)


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    Structured Review

    Thermo Fisher streptavidin hrp
    Schematic diagram showed non-radioactive metabolic incorporation followed by azide-biotin or azide-Alex555 labeling, and biotin signals of proteins were detected by <t>streptavidin-HRP</t> by western blot. HPG is incorporated into newly synthesized proteins by metabolism and protein synthesis and the triazole conjugation between newly alkyne proteins labeled HPG and azide labeled either biotin or Alex555 via CuSO4 catalysis (A) . (B-a) The detection of biotin signals from extracted total proteins labeled by labeling reaction. Normal culture medium was changed to replace DMEM free of L-methionine supplemented with HPG after pulse 4 hr, and proteins were extracted in each of group at various time points including 0, 4, 24 and 72 hr. (B-b) Biotin signals of total proteins were detected. 1: Normal culture condition group; 2: HPG plus anisomycin group; 3: HPG group. (B-c,d,e) Biotin signals of Bcl-2, MMP-9 and IgG were individually detected in the immunoprecipitate pulled down by primary antibodies via siRNA post-transfection followed by non-radioactive metabolic labeling. (B-f) Radioactive isotope 35 S-methonine incorporated into synthesized IgG purified by immunoprecipitation was detected by autoradiography. 1: 35 S-methonine treated human choriocarcinoma cell line BeWo group and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 2: cycloheximide plus 35 S-methonine treated BeWo group then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 3: 35 S-methonine treated skin fibroblast and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins.
    Streptavidin Hrp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 162 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 162 article reviews
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    streptavidin hrp - by Bioz Stars, 2020-04
    99/100 stars

    Images

    1) Product Images from "A morphologic and semi-quantitative technique to analyze synthesis and release of specific proteins in cells"

    Article Title: A morphologic and semi-quantitative technique to analyze synthesis and release of specific proteins in cells

    Journal: BMC Cell Biology

    doi: 10.1186/s12860-014-0045-1

    Schematic diagram showed non-radioactive metabolic incorporation followed by azide-biotin or azide-Alex555 labeling, and biotin signals of proteins were detected by streptavidin-HRP by western blot. HPG is incorporated into newly synthesized proteins by metabolism and protein synthesis and the triazole conjugation between newly alkyne proteins labeled HPG and azide labeled either biotin or Alex555 via CuSO4 catalysis (A) . (B-a) The detection of biotin signals from extracted total proteins labeled by labeling reaction. Normal culture medium was changed to replace DMEM free of L-methionine supplemented with HPG after pulse 4 hr, and proteins were extracted in each of group at various time points including 0, 4, 24 and 72 hr. (B-b) Biotin signals of total proteins were detected. 1: Normal culture condition group; 2: HPG plus anisomycin group; 3: HPG group. (B-c,d,e) Biotin signals of Bcl-2, MMP-9 and IgG were individually detected in the immunoprecipitate pulled down by primary antibodies via siRNA post-transfection followed by non-radioactive metabolic labeling. (B-f) Radioactive isotope 35 S-methonine incorporated into synthesized IgG purified by immunoprecipitation was detected by autoradiography. 1: 35 S-methonine treated human choriocarcinoma cell line BeWo group and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 2: cycloheximide plus 35 S-methonine treated BeWo group then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 3: 35 S-methonine treated skin fibroblast and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins.
    Figure Legend Snippet: Schematic diagram showed non-radioactive metabolic incorporation followed by azide-biotin or azide-Alex555 labeling, and biotin signals of proteins were detected by streptavidin-HRP by western blot. HPG is incorporated into newly synthesized proteins by metabolism and protein synthesis and the triazole conjugation between newly alkyne proteins labeled HPG and azide labeled either biotin or Alex555 via CuSO4 catalysis (A) . (B-a) The detection of biotin signals from extracted total proteins labeled by labeling reaction. Normal culture medium was changed to replace DMEM free of L-methionine supplemented with HPG after pulse 4 hr, and proteins were extracted in each of group at various time points including 0, 4, 24 and 72 hr. (B-b) Biotin signals of total proteins were detected. 1: Normal culture condition group; 2: HPG plus anisomycin group; 3: HPG group. (B-c,d,e) Biotin signals of Bcl-2, MMP-9 and IgG were individually detected in the immunoprecipitate pulled down by primary antibodies via siRNA post-transfection followed by non-radioactive metabolic labeling. (B-f) Radioactive isotope 35 S-methonine incorporated into synthesized IgG purified by immunoprecipitation was detected by autoradiography. 1: 35 S-methonine treated human choriocarcinoma cell line BeWo group and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 2: cycloheximide plus 35 S-methonine treated BeWo group then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 3: 35 S-methonine treated skin fibroblast and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins.

    Techniques Used: Labeling, Western Blot, Synthesized, Conjugation Assay, Transfection, Purification, Immunoprecipitation, Autoradiography

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

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

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.541250

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

    Techniques Used: Electron Microscopy, Generated, Produced

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

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

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00202-17

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

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

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

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

    Journal: Veterinary Research

    doi: 10.1051/vetres/2010048

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

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

    5) Product Images from "Detecting N-myristoylation and S-acylation of host and pathogen proteins in plants using click chemistry"

    Article Title: Detecting N-myristoylation and S-acylation of host and pathogen proteins in plants using click chemistry

    Journal: Plant Methods

    doi: 10.1186/s13007-016-0138-2

    Fatty acid modifications of proteins involved in plant immunity. a Arabidopsis protoplasts were transformed with HA epitope-tagged FLS2 wild-type (WT) or an fls2 mutant encoding C830S, C831S. Protoplasts were treated with 10 μM Alk14, incubated for 6 h, and cells collected. Total protein was extracted, FLS2 proteins immunoprecipitated using anti-HA resin, and click chemistry performed. Incorporated Alk14 was visualized by fluorescence imaging and total protein was detected by anti-HA western blotting. b Transgenic Arabidopsis plants conditionally expressing avrPto were treated with 20 μM dexamethasone to induce transgene expression. Leaves were infiltrated twice with 10 μM Alk12, 6 h after induction and 6 h before sampling. Tissue was collected 30 h after induction and total protein extracted. AvrPto was immunoprecipitated using anti-AvrPto resin and a biotin tag added using click chemistry. Streptavidin-HRP western blotting was used to detect incorporation of Alk12. Anti-AvrPto western blotting was used to verify equal amounts of protein in all samples. c Nicotiana benthamiana leaves were infiltrated with Agrobacterium strains carrying avrPto - YFP fusion constructs encoding the WT protein or a G2A mutant. 10 μM Alk12 was infiltrated twice, 24 h after Agrobacterium infiltration and 6 h before sampling. Tissue was collected 48 h after transformation and total protein extracted. AvrPto proteins were immunoprecipitated using anti-GFP resin and a biotin tag attached using click chemistry. Incorporated Alk12 was detected by streptavidin-HRP western blotting. The anti-GFP western blot shows relative protein levels. d Nicotiana benthamiana was used to transiently express Pto - YFP fusions encoding the WT protein or a G2A mutant. 10 μM Alk12 was infiltrated twice, 24 h after Agrobacterium infiltration and 6 h before sampling. Tissue was collected 48 h after transformation, total protein extracted, and Pto proteins immunoprecipitated using anti-GFP resin. A biotin tag was attached using click chemistry and incorporation of Alk12 was detected by streptavidin-HRP western blotting. Protein levels were visualized by anti-GFP western blotting
    Figure Legend Snippet: Fatty acid modifications of proteins involved in plant immunity. a Arabidopsis protoplasts were transformed with HA epitope-tagged FLS2 wild-type (WT) or an fls2 mutant encoding C830S, C831S. Protoplasts were treated with 10 μM Alk14, incubated for 6 h, and cells collected. Total protein was extracted, FLS2 proteins immunoprecipitated using anti-HA resin, and click chemistry performed. Incorporated Alk14 was visualized by fluorescence imaging and total protein was detected by anti-HA western blotting. b Transgenic Arabidopsis plants conditionally expressing avrPto were treated with 20 μM dexamethasone to induce transgene expression. Leaves were infiltrated twice with 10 μM Alk12, 6 h after induction and 6 h before sampling. Tissue was collected 30 h after induction and total protein extracted. AvrPto was immunoprecipitated using anti-AvrPto resin and a biotin tag added using click chemistry. Streptavidin-HRP western blotting was used to detect incorporation of Alk12. Anti-AvrPto western blotting was used to verify equal amounts of protein in all samples. c Nicotiana benthamiana leaves were infiltrated with Agrobacterium strains carrying avrPto - YFP fusion constructs encoding the WT protein or a G2A mutant. 10 μM Alk12 was infiltrated twice, 24 h after Agrobacterium infiltration and 6 h before sampling. Tissue was collected 48 h after transformation and total protein extracted. AvrPto proteins were immunoprecipitated using anti-GFP resin and a biotin tag attached using click chemistry. Incorporated Alk12 was detected by streptavidin-HRP western blotting. The anti-GFP western blot shows relative protein levels. d Nicotiana benthamiana was used to transiently express Pto - YFP fusions encoding the WT protein or a G2A mutant. 10 μM Alk12 was infiltrated twice, 24 h after Agrobacterium infiltration and 6 h before sampling. Tissue was collected 48 h after transformation, total protein extracted, and Pto proteins immunoprecipitated using anti-GFP resin. A biotin tag was attached using click chemistry and incorporation of Alk12 was detected by streptavidin-HRP western blotting. Protein levels were visualized by anti-GFP western blotting

    Techniques Used: Transformation Assay, Mutagenesis, Incubation, Immunoprecipitation, Fluorescence, Imaging, Western Blot, Transgenic Assay, Expressing, Sampling, Construct

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

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

    Journal: eLife

    doi: 10.7554/eLife.29224

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

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

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

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

    Journal: Virology

    doi: 10.1016/j.virol.2007.01.015

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

    Techniques Used: Concentration Assay, Binding Assay

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

    Techniques Used: Concentration Assay, Binding Assay

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

    Techniques Used: Concentration Assay, Binding Assay

    8) Product Images from "The splicing regulator PTBP2 is an AID interacting protein and promotes binding of AID to switch region DNA"

    Article Title: The splicing regulator PTBP2 is an AID interacting protein and promotes binding of AID to switch region DNA

    Journal: Nature immunology

    doi: 10.1038/ni.1977

    AID interacts with PTBP2. (a) Schematic representation of the AID expression construct (biotagDM-AID). The lysine that is biotinylated by BirA is indicated with an asterisk. The H56R,E58Q mutation inactivates the DNA deaminase activity of AID. (b) Protein extracts derived from stimulated CH12 BirA or CH12 BirA/biotagDM-AID cells were analyzed on immunoblots with AID antibodies. (c) Cell extracts from CH12 BirA or CH12 BirA/biotagDM-AID were incubated with streptavidin-agarose beads and bound proteins analyzed by immunoblotting with AID antibodies (upper) or streptavidin-coupled to horseradish-peroxidase (SA-HRP, lower). (d) DM-AID binds to S μ . Cross-linked DNA protein complexes from unstimulated or CIT-stimulated CH12 BirA/biotagDM-AID cells were subjected to modified ChIP in which steptavidin-agarose replaced antibodies used in conventional ChIP. Three-fold dilutions of DNA bound to streptavidin agarose were analyzed by PCR for the presence of S μ or the μ promoter. (e-f) Whole cell extracts derived from anti-CD40+IL-4-stimulated wild-type or AID-deficient mouse splenic B cells were immunoprecipitated with AID ( e ) or PTBP2 ( f ) antibodies and the immunoprecipitates were probed with anti-PTBP2 or anti-AID, respectively on immunoblots. E1 and E2 are two elutions of bound proteins. The data are representative of two independent experiments.
    Figure Legend Snippet: AID interacts with PTBP2. (a) Schematic representation of the AID expression construct (biotagDM-AID). The lysine that is biotinylated by BirA is indicated with an asterisk. The H56R,E58Q mutation inactivates the DNA deaminase activity of AID. (b) Protein extracts derived from stimulated CH12 BirA or CH12 BirA/biotagDM-AID cells were analyzed on immunoblots with AID antibodies. (c) Cell extracts from CH12 BirA or CH12 BirA/biotagDM-AID were incubated with streptavidin-agarose beads and bound proteins analyzed by immunoblotting with AID antibodies (upper) or streptavidin-coupled to horseradish-peroxidase (SA-HRP, lower). (d) DM-AID binds to S μ . Cross-linked DNA protein complexes from unstimulated or CIT-stimulated CH12 BirA/biotagDM-AID cells were subjected to modified ChIP in which steptavidin-agarose replaced antibodies used in conventional ChIP. Three-fold dilutions of DNA bound to streptavidin agarose were analyzed by PCR for the presence of S μ or the μ promoter. (e-f) Whole cell extracts derived from anti-CD40+IL-4-stimulated wild-type or AID-deficient mouse splenic B cells were immunoprecipitated with AID ( e ) or PTBP2 ( f ) antibodies and the immunoprecipitates were probed with anti-PTBP2 or anti-AID, respectively on immunoblots. E1 and E2 are two elutions of bound proteins. The data are representative of two independent experiments.

    Techniques Used: Expressing, Construct, Mutagenesis, Activity Assay, Derivative Assay, Western Blot, Incubation, Modification, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Immunoprecipitation

    9) Product Images from "Protein Complex Interactor Analysis and Differential Activity of KDM3 Subfamily Members Towards H3K9 Methylation"

    Article Title: Protein Complex Interactor Analysis and Differential Activity of KDM3 Subfamily Members Towards H3K9 Methylation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0060549

    SCAI is a specific interactor candidate of KDM3B. (A) SCAI protein sequence with the peptides identified by MS highlighted in red. The amino acids marked in green indicate trypsin cleavage sites. SCAI sequence coverage by MS was 51%. (B) Reciprocal co-immunoprecipitation of SCAI and KDM3B. V5-SCAI was either co-expressed with Avi-KDM3A or Avi-KDM3B. Reciprocal co-immunoprecipitations using V5- antibodies or streptavidin-coated beads were performed and the immunoprecipitated proteins from each immunoprecipitation were separated on SDS gels. A V5-antibody and streptavidin-HRP were used to detect SCAI and KDM3A or KDM3B, respectively. Only KDM3B but not KDM3A co-precipitated with and was able to precipitate V5-SCAI, respectively. (C) Sub-cellular co-localization of KDM3B and SCAI in HEK293T cells. Avi-KDM3B and V5-SCAI were co-expressed in HEK293T cells and detected by immunoreagents against their respective tags (b and c). The two proteins were found to co-localize in the nucleus (d).
    Figure Legend Snippet: SCAI is a specific interactor candidate of KDM3B. (A) SCAI protein sequence with the peptides identified by MS highlighted in red. The amino acids marked in green indicate trypsin cleavage sites. SCAI sequence coverage by MS was 51%. (B) Reciprocal co-immunoprecipitation of SCAI and KDM3B. V5-SCAI was either co-expressed with Avi-KDM3A or Avi-KDM3B. Reciprocal co-immunoprecipitations using V5- antibodies or streptavidin-coated beads were performed and the immunoprecipitated proteins from each immunoprecipitation were separated on SDS gels. A V5-antibody and streptavidin-HRP were used to detect SCAI and KDM3A or KDM3B, respectively. Only KDM3B but not KDM3A co-precipitated with and was able to precipitate V5-SCAI, respectively. (C) Sub-cellular co-localization of KDM3B and SCAI in HEK293T cells. Avi-KDM3B and V5-SCAI were co-expressed in HEK293T cells and detected by immunoreagents against their respective tags (b and c). The two proteins were found to co-localize in the nucleus (d).

    Techniques Used: Sequencing, Mass Spectrometry, Immunoprecipitation

    10) Product Images from "Novel Molecular Multilevel Targeted Antitumor Agents"

    Article Title: Novel Molecular Multilevel Targeted Antitumor Agents

    Journal: Cancer translational medicine

    doi: 10.4103/ctm.ctm_12_17

    (A,B) Flow cytometry for IL-13RA2 in U-251 GBM cells. Isotype control (A) and (B) receptor detection at various timepoints. Highly purified IL-13.E13K-D2-NLS-cys (C) and IL-13.E13K-D2-LLS-cys (D) . (E) Immunoblot of non-biotinylated (lane 1) and biotinylated (lane 2) IL-13.E13K-D2-LLS-cys probed with streptavidin-HRP. (F,G) Internalization of biotinylated IL-13.E13K-D2-LLS-cys (1 µM) in U-251 MG cells. The cells were analyzed using anti-streptavidin Alexa Fluor 555 (red) by fluorescence microscopy. Two different fields are shown in two column panels. (H) U-251-MG cells were treated with biotin-labeled IL-13.E13K-D2-LLS-cys (1 µM) and cells were stained for the nuclei and the protein. DIC, differential interference contrast. (I) Subcellular localization of IL-13.E13K-D2-LLS-cys was monitored using Z-stack analysis. (J) Internalization and intracellular distribution of IL-13.E13K-D2-LLS-cys. U-251 MG cells were incubated for 8 hrs with 1 µM biotin-conjugated IL-13.E13K-D2-LLS-cys (red) with co-staining of LAMP-1 protein (green).
    Figure Legend Snippet: (A,B) Flow cytometry for IL-13RA2 in U-251 GBM cells. Isotype control (A) and (B) receptor detection at various timepoints. Highly purified IL-13.E13K-D2-NLS-cys (C) and IL-13.E13K-D2-LLS-cys (D) . (E) Immunoblot of non-biotinylated (lane 1) and biotinylated (lane 2) IL-13.E13K-D2-LLS-cys probed with streptavidin-HRP. (F,G) Internalization of biotinylated IL-13.E13K-D2-LLS-cys (1 µM) in U-251 MG cells. The cells were analyzed using anti-streptavidin Alexa Fluor 555 (red) by fluorescence microscopy. Two different fields are shown in two column panels. (H) U-251-MG cells were treated with biotin-labeled IL-13.E13K-D2-LLS-cys (1 µM) and cells were stained for the nuclei and the protein. DIC, differential interference contrast. (I) Subcellular localization of IL-13.E13K-D2-LLS-cys was monitored using Z-stack analysis. (J) Internalization and intracellular distribution of IL-13.E13K-D2-LLS-cys. U-251 MG cells were incubated for 8 hrs with 1 µM biotin-conjugated IL-13.E13K-D2-LLS-cys (red) with co-staining of LAMP-1 protein (green).

    Techniques Used: Flow Cytometry, Cytometry, Purification, Fluorescence, Microscopy, Labeling, Staining, Incubation

    (A) Purified recombinant IL-13-D2-KK2 protein. SDS-PAGE stained with Coommassie blue. (B) Immunoblot of biotinylated IL-13-D2-KK2 probed with Streptavidin-HRP. (C) Cell internalization of biotinylated IL-13-D2-KK2. The protein was analyzed by using anti-streptavidin Alexa Fluor 555 and fluorescent microscopy. (D) U-251 cells were treated with biotinylated IL-13-D2-KK2 for 8 hrs and the co-localization of biotinylated IL-13-D2-KK2 with mitochondrial ATP-synthase enzyme was analyzed by confocal microscopy. (E) IL-13-D2-KK2 effect on GBM cell lines U-251 MG, G48a and T98G analyzed using phase contrast microscopy. (F) GBM cells were treated with IL-13-D2-KK2 for 72 hrs and the cytotoxicity was measured by an MTS assay.
    Figure Legend Snippet: (A) Purified recombinant IL-13-D2-KK2 protein. SDS-PAGE stained with Coommassie blue. (B) Immunoblot of biotinylated IL-13-D2-KK2 probed with Streptavidin-HRP. (C) Cell internalization of biotinylated IL-13-D2-KK2. The protein was analyzed by using anti-streptavidin Alexa Fluor 555 and fluorescent microscopy. (D) U-251 cells were treated with biotinylated IL-13-D2-KK2 for 8 hrs and the co-localization of biotinylated IL-13-D2-KK2 with mitochondrial ATP-synthase enzyme was analyzed by confocal microscopy. (E) IL-13-D2-KK2 effect on GBM cell lines U-251 MG, G48a and T98G analyzed using phase contrast microscopy. (F) GBM cells were treated with IL-13-D2-KK2 for 72 hrs and the cytotoxicity was measured by an MTS assay.

    Techniques Used: Purification, Recombinant, SDS Page, Staining, Microscopy, Confocal Microscopy, MTS Assay

    11) Product Images from "Selenium-Based S-Adenosylmethionine Analog Reveals the Mammalian Seven-Beta-Strand Methyltransferase METTL10 to Be an EF1A1 Lysine Methyltransferase"

    Article Title: Selenium-Based S-Adenosylmethionine Analog Reveals the Mammalian Seven-Beta-Strand Methyltransferase METTL10 to Be an EF1A1 Lysine Methyltransferase

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0105394

    ProSeAM, a synthetic SAM analog, has a wide spectrum of reactivity for histones and non-histone substrates. A, Schematic overview for analyzing lysine methylation. A synthetic cofactor was used to transfer an alkyne moiety to the ε-amino group of lysine by KMTs (1). The modified proteins were tagged with biotin via CuAAC reaction (2). Tagged-proteins in the crude lysates were pulled down with affinity beads (3), and the precipitants were further analyzed with a LC-MS apparatus (4). B, Chemical structure of SAM (1), propargylated SAM (2) and ProSeAM (3). C, H3 peptide (1-21 a.a.) and ProSeAM was incubated with or without GST-G9a at 20°C for 2 h, then the peptide was analyzed by MALDI-TOF MS. D, full-length Histone H3 (1 µg) and ProSeAM (500 µM) were incubated with indicated KMTs (0.5 µg) for 2 h at 20°C. The histones were separated by SDS-PAGE, transferred to a nitrocellulose membrane and probed with streptavidin-HRP (top) or anti-Histone H3 antibody (bottom). E, The non-histone substrates His-HSP90 and His-HSP70 (1 µg) were incubated with His-SMYD2 and His-METTL21A (1 µg), respectively. After the reaction, proteins were separated by SDS-PAGE (right). Their modifications were detected by western blotting with streptavidin-HRP as in Fig. 1D. *and ** showed automodification of SMYD2 and METTL21A, respectively (left). F, His-HSP70 (WT and K561R) were incubated with or without His-METTL21A in the presence of ProSeAM for 2 h at 20°C. Modified proteins were biotinylated and detected with streptavidin-HRP (top) or anti-HSP70 antibody for the loading control (bottom).
    Figure Legend Snippet: ProSeAM, a synthetic SAM analog, has a wide spectrum of reactivity for histones and non-histone substrates. A, Schematic overview for analyzing lysine methylation. A synthetic cofactor was used to transfer an alkyne moiety to the ε-amino group of lysine by KMTs (1). The modified proteins were tagged with biotin via CuAAC reaction (2). Tagged-proteins in the crude lysates were pulled down with affinity beads (3), and the precipitants were further analyzed with a LC-MS apparatus (4). B, Chemical structure of SAM (1), propargylated SAM (2) and ProSeAM (3). C, H3 peptide (1-21 a.a.) and ProSeAM was incubated with or without GST-G9a at 20°C for 2 h, then the peptide was analyzed by MALDI-TOF MS. D, full-length Histone H3 (1 µg) and ProSeAM (500 µM) were incubated with indicated KMTs (0.5 µg) for 2 h at 20°C. The histones were separated by SDS-PAGE, transferred to a nitrocellulose membrane and probed with streptavidin-HRP (top) or anti-Histone H3 antibody (bottom). E, The non-histone substrates His-HSP90 and His-HSP70 (1 µg) were incubated with His-SMYD2 and His-METTL21A (1 µg), respectively. After the reaction, proteins were separated by SDS-PAGE (right). Their modifications were detected by western blotting with streptavidin-HRP as in Fig. 1D. *and ** showed automodification of SMYD2 and METTL21A, respectively (left). F, His-HSP70 (WT and K561R) were incubated with or without His-METTL21A in the presence of ProSeAM for 2 h at 20°C. Modified proteins were biotinylated and detected with streptavidin-HRP (top) or anti-HSP70 antibody for the loading control (bottom).

    Techniques Used: Methylation, Modification, Liquid Chromatography with Mass Spectroscopy, Incubation, Mass Spectrometry, SDS Page, Western Blot

    Proteomic identification of substrates for seven-beta-strand MTases. A, Schematic protocol for proteomic identification. HEK293T cell lysates were added to either propargylic Se-adenosyl- l -selenomethionine (ProSeAM) alone (1) or ProSeAM plus recombinant KMT (lysate:enzyme ratio was 10∶1) (2). After the in vitro reaction, labeled proteins were tagged with biotin and then precipitated with streptavidin beads. The precipitants were then digested with trypsin, and the trypsinized protein fragments were analyzed by LC-MS/MS. B, ProSeAM competes with SAM in the labeling reaction. HEK293T cell lysates were incubated with ProSeAM (250 µM) in the presence or absence of the indicated amount of SAM (0 to 2.5 mM). Modified proteins were biotinylated and detected with streptavidin-HRP (top). Equal protein loading was confirmed by western blotting with anti-α-tubulin antibody (bottom). C, western blot of labeled proteins. A 5% input of precipitated proteins without ProSeAM (1), with ProSeAM alone (2), with ProSeAM plus GST-G9a (3), with ProSeAM plus His-METTL21A (4) or with ProSeAM plus His-METTL10 was separately analyzed with western blotting with streptavidin-HRP (top) prior to the MS analysis, to compare the labeled proteins. Equal protein loading was confirmed by western blotting with anti-α-tubulin antibody (bottom). D, Doughnut chart of the subcellular distribution of proteins labeled with ProSeAM. HEK293T lysates alone (lane 1 in Fig. 2C) and HEK293T lysates with ProSeAM (lane 2 in Fig. 2C) were analyzed as described in A and Experimental procedures (n = 3). In total, 318 proteins were identified as ProSeAM-labeled proteins. E, List of METTL21A substrates. HEK293T cell lysates and ProSeAM were incubated with or without METTL21A (lane 2 and lane 4 in Fig. 2C), and analyzed as above. Molecular weight, peptide area (reflecting the quantity of detected protein), and fold enrichment of the peptide area are listed: ND, not determined because the substrate was detected only in the condition for lane 4 of B. The total numbers of identified proteins, 2-fold increase (compared to control in each experiment), and overlapped identified numbers of 3 independent experiments are listed in Table S2 .
    Figure Legend Snippet: Proteomic identification of substrates for seven-beta-strand MTases. A, Schematic protocol for proteomic identification. HEK293T cell lysates were added to either propargylic Se-adenosyl- l -selenomethionine (ProSeAM) alone (1) or ProSeAM plus recombinant KMT (lysate:enzyme ratio was 10∶1) (2). After the in vitro reaction, labeled proteins were tagged with biotin and then precipitated with streptavidin beads. The precipitants were then digested with trypsin, and the trypsinized protein fragments were analyzed by LC-MS/MS. B, ProSeAM competes with SAM in the labeling reaction. HEK293T cell lysates were incubated with ProSeAM (250 µM) in the presence or absence of the indicated amount of SAM (0 to 2.5 mM). Modified proteins were biotinylated and detected with streptavidin-HRP (top). Equal protein loading was confirmed by western blotting with anti-α-tubulin antibody (bottom). C, western blot of labeled proteins. A 5% input of precipitated proteins without ProSeAM (1), with ProSeAM alone (2), with ProSeAM plus GST-G9a (3), with ProSeAM plus His-METTL21A (4) or with ProSeAM plus His-METTL10 was separately analyzed with western blotting with streptavidin-HRP (top) prior to the MS analysis, to compare the labeled proteins. Equal protein loading was confirmed by western blotting with anti-α-tubulin antibody (bottom). D, Doughnut chart of the subcellular distribution of proteins labeled with ProSeAM. HEK293T lysates alone (lane 1 in Fig. 2C) and HEK293T lysates with ProSeAM (lane 2 in Fig. 2C) were analyzed as described in A and Experimental procedures (n = 3). In total, 318 proteins were identified as ProSeAM-labeled proteins. E, List of METTL21A substrates. HEK293T cell lysates and ProSeAM were incubated with or without METTL21A (lane 2 and lane 4 in Fig. 2C), and analyzed as above. Molecular weight, peptide area (reflecting the quantity of detected protein), and fold enrichment of the peptide area are listed: ND, not determined because the substrate was detected only in the condition for lane 4 of B. The total numbers of identified proteins, 2-fold increase (compared to control in each experiment), and overlapped identified numbers of 3 independent experiments are listed in Table S2 .

    Techniques Used: Recombinant, In Vitro, Labeling, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Incubation, Modification, Western Blot, Molecular Weight

    12) Product Images from "Bisulfite-free, single base-resolution analysis of 5-hydroxymethylcytosine in genomic DNA by chemical-mediated mismatch single base-resolution analysis of 5-hydroxymethylcytosine in genomic DNA by chemical-mediated mismatch †Electronic supplementary information (ESI) available. See DOI: 10.1039/c8sc0427"

    Article Title: Bisulfite-free, single base-resolution analysis of 5-hydroxymethylcytosine in genomic DNA by chemical-mediated mismatch single base-resolution analysis of 5-hydroxymethylcytosine in genomic DNA by chemical-mediated mismatch †Electronic supplementary information (ESI) available. See DOI: 10.1039/c8sc0427

    Journal: Chemical Science

    doi: 10.1039/c8sc04272a

    Polyacrylamide gel electrophoresis analysis and dot-blot assay. (a) Polyacrylamide gel electrophoresis analysis of the protected 5fC. Lane 1: ODN-5fC without treatment; lane 2: ODN-5fC protected with hydroxylamine; lane 3: ODN-5fC protected with hydroxylamine and then treated with azi-BP. (b) Dot-blot assay of the streptavidin–HRP detection of oxidized 5hmC labeled with azi-BP and DBCO-PEG4-biotin. Dot 1: 80 bp ds ODN-5hmC without treatment; dot 2: 80 bp ds ODN-5hmC ligated with an adapter and oxidized by KRuO 4 and then labeled with azi-BP and DBCO-PEG4-biotin; dot 3: 80 bp ds ODN-5fC; dot 4: 80 bp ds ODN-5fC protected with hydroxylamine and then incubated with azi-BP and DBCO-PEG4-biotin; dot 5: 80 bp ds ODN-5fC treated with azi-BP and DBCO-PEG4-biotin. Only the biotin labeled DNA can produce a dot. And after methylene blue incubation, we can verify the existence of DNA for every dot.
    Figure Legend Snippet: Polyacrylamide gel electrophoresis analysis and dot-blot assay. (a) Polyacrylamide gel electrophoresis analysis of the protected 5fC. Lane 1: ODN-5fC without treatment; lane 2: ODN-5fC protected with hydroxylamine; lane 3: ODN-5fC protected with hydroxylamine and then treated with azi-BP. (b) Dot-blot assay of the streptavidin–HRP detection of oxidized 5hmC labeled with azi-BP and DBCO-PEG4-biotin. Dot 1: 80 bp ds ODN-5hmC without treatment; dot 2: 80 bp ds ODN-5hmC ligated with an adapter and oxidized by KRuO 4 and then labeled with azi-BP and DBCO-PEG4-biotin; dot 3: 80 bp ds ODN-5fC; dot 4: 80 bp ds ODN-5fC protected with hydroxylamine and then incubated with azi-BP and DBCO-PEG4-biotin; dot 5: 80 bp ds ODN-5fC treated with azi-BP and DBCO-PEG4-biotin. Only the biotin labeled DNA can produce a dot. And after methylene blue incubation, we can verify the existence of DNA for every dot.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Dot Blot, Labeling, Incubation

    13) Product Images from "Fbxl19 recruitment to CpG islands is required for Rnf20-mediated H2B mono-ubiquitination"

    Article Title: Fbxl19 recruitment to CpG islands is required for Rnf20-mediated H2B mono-ubiquitination

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx310

    Fbxl19 is implicated in mono-ubiquitination of H2B. ( A ) A bar graph presenting knockdown (KD) efficiency of Fbxl19 measured by qRT-PCR. Error bars indicate standard error of the mean ( n = 3). ( B and C ) Western blots showing decreased levels of H2Bub1 upon KD (B) and KO (C) of Fbxl19. ( D ) A bar graph presenting overexpression (OE) level of Fbxl19 measured by qRT-PCR. Error bar indicates standard error of the mean ( n = 3). ( E ) Western blots showing induced level of H2Bub1 upon OE of Fbxl19. OE level of Fbxl19 was detected by HRP-conjugated streptavidin. WT indicates wild-type ES cells. NS indicates non-specific band.
    Figure Legend Snippet: Fbxl19 is implicated in mono-ubiquitination of H2B. ( A ) A bar graph presenting knockdown (KD) efficiency of Fbxl19 measured by qRT-PCR. Error bars indicate standard error of the mean ( n = 3). ( B and C ) Western blots showing decreased levels of H2Bub1 upon KD (B) and KO (C) of Fbxl19. ( D ) A bar graph presenting overexpression (OE) level of Fbxl19 measured by qRT-PCR. Error bar indicates standard error of the mean ( n = 3). ( E ) Western blots showing induced level of H2Bub1 upon OE of Fbxl19. OE level of Fbxl19 was detected by HRP-conjugated streptavidin. WT indicates wild-type ES cells. NS indicates non-specific band.

    Techniques Used: Quantitative RT-PCR, Western Blot, Over Expression

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

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0010184

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

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

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

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

    15) Product Images from "Development of camelid single chain antibodies against Shiga toxin type 2 (Stx2) with therapeutic potential against Hemolytic Uremic Syndrome (HUS)"

    Article Title: Development of camelid single chain antibodies against Shiga toxin type 2 (Stx2) with therapeutic potential against Hemolytic Uremic Syndrome (HUS)

    Journal: Scientific Reports

    doi: 10.1038/srep24913

    In vitro activity of 2vb27 VHH under different formats. ( A ) Serially diluted purified 2vb27, (2vb27) 2 or (2vb27) 2 -SA were tested on the Vero cell neutralization assay as detailed in Materials and Methods. Each sample was tested by quadruplicate and is represented as mean ± SEM. ( B ) Binding of (2vb27) 2 -SA to human seroalbumin (SA). Nickel coated Maxisorp microtiter plates were incubated with serially diluted (2vb27) 2 -SA. Biotinylated human SA was added and binding was detected with Streptavidin-HRP. Reaction was developed with TMB and absorbance was read at 450 nm.
    Figure Legend Snippet: In vitro activity of 2vb27 VHH under different formats. ( A ) Serially diluted purified 2vb27, (2vb27) 2 or (2vb27) 2 -SA were tested on the Vero cell neutralization assay as detailed in Materials and Methods. Each sample was tested by quadruplicate and is represented as mean ± SEM. ( B ) Binding of (2vb27) 2 -SA to human seroalbumin (SA). Nickel coated Maxisorp microtiter plates were incubated with serially diluted (2vb27) 2 -SA. Biotinylated human SA was added and binding was detected with Streptavidin-HRP. Reaction was developed with TMB and absorbance was read at 450 nm.

    Techniques Used: In Vitro, Activity Assay, Purification, Neutralization, Binding Assay, Incubation

    16) Product Images from "Glutaredoxin 1 regulates cigarette smoke-mediated lung inflammation through differential modulation of I?B kinases in mice: impact on histone acetylation"

    Article Title: Glutaredoxin 1 regulates cigarette smoke-mediated lung inflammation through differential modulation of I?B kinases in mice: impact on histone acetylation

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    doi: 10.1152/ajplung.00426.2009

    Inactivation of IKKβ and accumulation of phosphorylated IKKα in Glrx1 −/− mice in response to CS exposure. A : lung homogenates were subjected to immunoprecipitation with IKKα and IKKβ antibodies, and immunoblotted with streptavidin-conjugated HRP ( top ) and IKKα and IKKβ ( lower ) antibodies. B : IKKα and IKKβ immunoprecipitates separated under nonreducing conditions, and then immunoblotted with anti-GSH ( top ) and IKKα and IKKβ ( lower ) antibodies. C : immunoprecipitated IKKα and IKKβ were analyzed for phosphorylation of serine residues using specific phospho-serine antibody, respectively. Gel pictures shown are representative of at least 3 separate experiments.
    Figure Legend Snippet: Inactivation of IKKβ and accumulation of phosphorylated IKKα in Glrx1 −/− mice in response to CS exposure. A : lung homogenates were subjected to immunoprecipitation with IKKα and IKKβ antibodies, and immunoblotted with streptavidin-conjugated HRP ( top ) and IKKα and IKKβ ( lower ) antibodies. B : IKKα and IKKβ immunoprecipitates separated under nonreducing conditions, and then immunoblotted with anti-GSH ( top ) and IKKα and IKKβ ( lower ) antibodies. C : immunoprecipitated IKKα and IKKβ were analyzed for phosphorylation of serine residues using specific phospho-serine antibody, respectively. Gel pictures shown are representative of at least 3 separate experiments.

    Techniques Used: Mouse Assay, Immunoprecipitation

    17) Product Images from "Efficient proximity labeling in living cells and organisms with TurboID"

    Article Title: Efficient proximity labeling in living cells and organisms with TurboID

    Journal: Nature biotechnology

    doi: 10.1038/nbt.4201

    Characterization of TurboID and miniTurbo in mammalian cells ( a ) Comparison of TurboID and miniTurbo to three other promiscuous ligases (BioID 5 , BioID2 26 , and BASU 27 ) in the cytosol of HEK 293T cells. Here, 500 μM exogenous biotin was used for labeling, whereas 50 μM was used in Supplementary Figure 6c-e . Streptavidin-HRP blotting detects promiscuously biotinylated proteins, and anti-V5 blotting detects ligase expression. U, untransfected. Asterisks denote ligase self-biotinylation bands. This experiment was performed twice with similar results. ( b ) Quantitation of experiment in (a). For shorter timepoints (-biotin and 10 min), we used a longer-exposure image of the same blot, shown in Supplementary Figure 6a ; for longer timepoints (1, 6, 18 hr), we used a shorter-exposure image of the blot in (a), shown in Supplementary Figure 6b . Quantitation performed as in Figure 1g . Grey dots indicate quantitation of signal intensity from each replicate, colored bars indicate mean signal intensity calculated from the two replicates. ( c ) Comparison of promiscuous ligases in multiple HEK organelles. Each ligase was fused to a peptide targeting sequence (see Supplementary Table 8 ) directing them to the locations indicated in the scheme at right. BioID samples were treated with 50 μM biotin for 18 hours. TurboID and miniTurbo samples were labeled for 10 minutes with 50 (+) or 500 (++) μM biotin. U, untransfected. Asterisks denote ligase self-biotinylation. This experiment was performed five times for nuclear constructs, three for mitochondrial constructs, four times for ER membrane constructs, and twice for ER lumen constructs with similar results. ( d ) Mass spectrometry-based proteomic experiment comparing TurboID and BioID on the ER membrane (ERM), facing cytosol. Experimental design and labeling conditions. Ligase fusion constructs were stably expressed in HEK 239T. BioID samples were treated with 50 μM biotin for 18 hours, while TurboID samples were treated with 500 μM biotin for 10 minutes or 1 hr. After labeling, cells were lysed and biotinylated proteins were enriched with streptavidin beads, digested to peptides, and conjugated to TMT (tandem mass tag) labels. All 11 samples were then combined and analyzed by LC-MS/MS. This experiment was performed once with two replicates per condition. ( e ) Specificity analysis for proteomic datasets derived from experiment in (d). Size of each ERM proteome at top. Bars show percentage of each proteome with prior secretory pathway annotation, according to GOCC, Phobius, human protein atlas, human plasma proteome database, and literature (see Methods and Supplementary Table 2 Tab 4 ). ( f ) Same as (e), except for each ERM proteome, we analyze the subset with ER, Golgi, or plasma membrane annotation. Annotations from GOCC were assigned in the priority order: ER > Golgi > plasma membrane (see Methods and Supplementary Table 2 Tab 5 ). ( g ) Breakdown of ER proteins enriched by TurboID and BioID, by transmembrane or soluble. Soluble proteins were further divided into luminal or cytosol-facing. Annotations obtained from GOCC, UniProt, TMHMM, and literature (see Methods and Supplementary Table 2 Tab 6 ). ( h ) Characterization of nuclear and mitochondrial matrix proteomes obtained via BioID (18 hour), TurboID (10 min), and miniTurbo (10 min)-catalyzed labeling. Proteome sizes across top. Bars show fraction of each nuclear (left) or mitochondrial (right) proteome with prior nuclear or mitochondrial annotation, according to GOCC, MitoCarta, or literature (see Methods and Supplementary Table 3 Tab 1, Supplementary Table 4, Tab 1 ). Design of proteomic experiment shown in Supplementary Figure 10a , proteomic lists in Supplementary Tables 6-7 ; further analysis of proteome data in Supplementary Figure 10 .
    Figure Legend Snippet: Characterization of TurboID and miniTurbo in mammalian cells ( a ) Comparison of TurboID and miniTurbo to three other promiscuous ligases (BioID 5 , BioID2 26 , and BASU 27 ) in the cytosol of HEK 293T cells. Here, 500 μM exogenous biotin was used for labeling, whereas 50 μM was used in Supplementary Figure 6c-e . Streptavidin-HRP blotting detects promiscuously biotinylated proteins, and anti-V5 blotting detects ligase expression. U, untransfected. Asterisks denote ligase self-biotinylation bands. This experiment was performed twice with similar results. ( b ) Quantitation of experiment in (a). For shorter timepoints (-biotin and 10 min), we used a longer-exposure image of the same blot, shown in Supplementary Figure 6a ; for longer timepoints (1, 6, 18 hr), we used a shorter-exposure image of the blot in (a), shown in Supplementary Figure 6b . Quantitation performed as in Figure 1g . Grey dots indicate quantitation of signal intensity from each replicate, colored bars indicate mean signal intensity calculated from the two replicates. ( c ) Comparison of promiscuous ligases in multiple HEK organelles. Each ligase was fused to a peptide targeting sequence (see Supplementary Table 8 ) directing them to the locations indicated in the scheme at right. BioID samples were treated with 50 μM biotin for 18 hours. TurboID and miniTurbo samples were labeled for 10 minutes with 50 (+) or 500 (++) μM biotin. U, untransfected. Asterisks denote ligase self-biotinylation. This experiment was performed five times for nuclear constructs, three for mitochondrial constructs, four times for ER membrane constructs, and twice for ER lumen constructs with similar results. ( d ) Mass spectrometry-based proteomic experiment comparing TurboID and BioID on the ER membrane (ERM), facing cytosol. Experimental design and labeling conditions. Ligase fusion constructs were stably expressed in HEK 239T. BioID samples were treated with 50 μM biotin for 18 hours, while TurboID samples were treated with 500 μM biotin for 10 minutes or 1 hr. After labeling, cells were lysed and biotinylated proteins were enriched with streptavidin beads, digested to peptides, and conjugated to TMT (tandem mass tag) labels. All 11 samples were then combined and analyzed by LC-MS/MS. This experiment was performed once with two replicates per condition. ( e ) Specificity analysis for proteomic datasets derived from experiment in (d). Size of each ERM proteome at top. Bars show percentage of each proteome with prior secretory pathway annotation, according to GOCC, Phobius, human protein atlas, human plasma proteome database, and literature (see Methods and Supplementary Table 2 Tab 4 ). ( f ) Same as (e), except for each ERM proteome, we analyze the subset with ER, Golgi, or plasma membrane annotation. Annotations from GOCC were assigned in the priority order: ER > Golgi > plasma membrane (see Methods and Supplementary Table 2 Tab 5 ). ( g ) Breakdown of ER proteins enriched by TurboID and BioID, by transmembrane or soluble. Soluble proteins were further divided into luminal or cytosol-facing. Annotations obtained from GOCC, UniProt, TMHMM, and literature (see Methods and Supplementary Table 2 Tab 6 ). ( h ) Characterization of nuclear and mitochondrial matrix proteomes obtained via BioID (18 hour), TurboID (10 min), and miniTurbo (10 min)-catalyzed labeling. Proteome sizes across top. Bars show fraction of each nuclear (left) or mitochondrial (right) proteome with prior nuclear or mitochondrial annotation, according to GOCC, MitoCarta, or literature (see Methods and Supplementary Table 3 Tab 1, Supplementary Table 4, Tab 1 ). Design of proteomic experiment shown in Supplementary Figure 10a , proteomic lists in Supplementary Tables 6-7 ; further analysis of proteome data in Supplementary Figure 10 .

    Techniques Used: Labeling, Expressing, Quantitation Assay, Sequencing, Construct, Mass Spectrometry, Stable Transfection, Liquid Chromatography with Mass Spectroscopy, Derivative Assay

    Directed evolution of TurboID ( a ) Proximity-dependent biotinylation catalyzed by promiscuous biotin ligases. Ligases catalyze the formation of biotin-5′-AMP anhydride, which diffuses out of the active site to biotinylate proximal endogenous proteins on nucleophilic residues such as lysine. ( b ) Yeast display-based selection scheme. A > 10 7 library of ligase variants is displayed on the yeast surface as a fusion to mating protein Aga2p. All ligases have a C-terminal myc epitope tag. Biotin and ATP are added to the yeast library for between 10 minutes and 24 hours. Ligase-catalyzed promiscuous biotinylation is detected by staining with streptavidin-phycoerythrin and ligase expression is detected by staining with anti-myc antibody. Two-dimensional FACS sorting enables enrichment of cells displaying a high ratio of streptavidin to myc staining. ( c ) Tyramide signal amplification (TSA) 32 improves biotin detection sensitivity on the yeast surface. In the top row, the three indicated yeast samples (G1 is the winning ligase mutant from the first generation of evolution) were labeled with exogenous biotin for 18 hours then stained for FACS as in (b). The y-axis shows biotinylation extent, and the x-axis quantifies ligase expression level. In the second row, after 18 hours of biotin incubation, yeast were stained with streptavidin-HRP, reacted with biotin-phenol 2 , 32 to create additional biotinylation sites, then stained with streptavidin-phycoerythrin and anti-myc antibody before FACS. The third row omits biotin. Percentage of cells in upper right quadrant (Q2/(Q2+Q4)) shown in top right of each graph. This experiment was performed once, but each yeast sample has been analyzed under identical conditions at least twice in separate experiments with similar results. ( d ) E. coli biotin ligase structure (PDB: 2EWN) with sites mutated in TurboID (left) and miniTurbo (right) shown in red. The N-terminal domain (aa1-63) is also removed from miniTurbo. A non-hydrolyzable analog of biotin-5′-AMP, biotinol-5′-AMP, is shown in yellow stick. ( e ) FACS plots summarizing progress of directed evolution. G1-G3 are the winning clones from generations 1-3 of directed evolution. G3Δ has its N-terminal domain (aa1-63) deleted. Omit biotin samples were grown in biotin-deficient media (see Methods ) for the entire induction period (~18-24 hr). This experiment was performed twice with similar results, except G3Δ omit biotin, which was performed once. ( f ) Comparison of ligase variants in the HEK cytosol showing that TurboID and miniTurbo are much more active than BioID, as well as the starting template and various intermediate clones from the evolution. Indicated ligases were expressed as NES (nuclear export signal) fusions in the HEK cytosol. 50 μM exogenous biotin was added for 3 hours, then whole cell lysates were analyzed by streptavidin blotting. Ligase expression detected by anti-V5 blotting. U, untransfected. S, BirA-R118S. Asterisks indicate ligase self-biotinylation. BioID labeling for 18 hours (50 μM biotin) shown for comparison in the last lane. This experiment was performed twice with similar results. ( g ) Quantitation of streptavidin blot data in (f) and from a 30 minute labeling experiment shown in Supplementary Figure 4b . Quantitation excludes self-biotinylation band. Sum intensity of each lane is divided by the sum intensity of the ligase expression band; ratios are normalized to that of BioID/18 hours, which is set to 1.0. Grey dots indicate quantitation of signal intensity from each replicate, colored bars indicate mean signal intensity calculated from the two replicates.
    Figure Legend Snippet: Directed evolution of TurboID ( a ) Proximity-dependent biotinylation catalyzed by promiscuous biotin ligases. Ligases catalyze the formation of biotin-5′-AMP anhydride, which diffuses out of the active site to biotinylate proximal endogenous proteins on nucleophilic residues such as lysine. ( b ) Yeast display-based selection scheme. A > 10 7 library of ligase variants is displayed on the yeast surface as a fusion to mating protein Aga2p. All ligases have a C-terminal myc epitope tag. Biotin and ATP are added to the yeast library for between 10 minutes and 24 hours. Ligase-catalyzed promiscuous biotinylation is detected by staining with streptavidin-phycoerythrin and ligase expression is detected by staining with anti-myc antibody. Two-dimensional FACS sorting enables enrichment of cells displaying a high ratio of streptavidin to myc staining. ( c ) Tyramide signal amplification (TSA) 32 improves biotin detection sensitivity on the yeast surface. In the top row, the three indicated yeast samples (G1 is the winning ligase mutant from the first generation of evolution) were labeled with exogenous biotin for 18 hours then stained for FACS as in (b). The y-axis shows biotinylation extent, and the x-axis quantifies ligase expression level. In the second row, after 18 hours of biotin incubation, yeast were stained with streptavidin-HRP, reacted with biotin-phenol 2 , 32 to create additional biotinylation sites, then stained with streptavidin-phycoerythrin and anti-myc antibody before FACS. The third row omits biotin. Percentage of cells in upper right quadrant (Q2/(Q2+Q4)) shown in top right of each graph. This experiment was performed once, but each yeast sample has been analyzed under identical conditions at least twice in separate experiments with similar results. ( d ) E. coli biotin ligase structure (PDB: 2EWN) with sites mutated in TurboID (left) and miniTurbo (right) shown in red. The N-terminal domain (aa1-63) is also removed from miniTurbo. A non-hydrolyzable analog of biotin-5′-AMP, biotinol-5′-AMP, is shown in yellow stick. ( e ) FACS plots summarizing progress of directed evolution. G1-G3 are the winning clones from generations 1-3 of directed evolution. G3Δ has its N-terminal domain (aa1-63) deleted. Omit biotin samples were grown in biotin-deficient media (see Methods ) for the entire induction period (~18-24 hr). This experiment was performed twice with similar results, except G3Δ omit biotin, which was performed once. ( f ) Comparison of ligase variants in the HEK cytosol showing that TurboID and miniTurbo are much more active than BioID, as well as the starting template and various intermediate clones from the evolution. Indicated ligases were expressed as NES (nuclear export signal) fusions in the HEK cytosol. 50 μM exogenous biotin was added for 3 hours, then whole cell lysates were analyzed by streptavidin blotting. Ligase expression detected by anti-V5 blotting. U, untransfected. S, BirA-R118S. Asterisks indicate ligase self-biotinylation. BioID labeling for 18 hours (50 μM biotin) shown for comparison in the last lane. This experiment was performed twice with similar results. ( g ) Quantitation of streptavidin blot data in (f) and from a 30 minute labeling experiment shown in Supplementary Figure 4b . Quantitation excludes self-biotinylation band. Sum intensity of each lane is divided by the sum intensity of the ligase expression band; ratios are normalized to that of BioID/18 hours, which is set to 1.0. Grey dots indicate quantitation of signal intensity from each replicate, colored bars indicate mean signal intensity calculated from the two replicates.

    Techniques Used: Selection, Staining, Expressing, FACS, Amplification, Mutagenesis, Labeling, Incubation, Clone Assay, Quantitation Assay

    TurboID and miniTurbo in flies, worms, and other species ( a ) Comparison of ligases in yeast. EBY100 S. cerevisiae expressing BioID, TurboID, or miniTurbo in the cytosol were treated with 50 μM biotin for 18 hours. Whole cell lysates were blotted with streptavidin-HRP to visualize biotinylated proteins, and anti-V5 antibody to visualize ligase expression. U, untransfected. Asterisks denote ligase self-biotinylation. Bands in untransfected lane are endogenous naturally-biotinylated proteins. This experiment was performed twice with similar results. ( b ) Comparison of ligases in E. coli. Ligases, fused at their N-terminal ends to His6-maltose binding protein, were expressed in the cytosol of BL21 E. coli and 50 μM exogenous biotin was added for 18 hours. Whole cell lysates were analyzed as in (a). This experiment was performed twice with similar results. ( c ) – ( g ) Comparison of ligases in flies. ( c ) Scheme for tissue-specific expression of ligases in the wing disc of D. melanogaster . ptc-Gal4 induces ligase expression in a strip of cells within the wing imaginal disc that borders the anterior/posterior compartments. ( d ) Imaging of larval wing discs after 5 days of growth on biotin-containing food. Biotinylated proteins are detected by staining with streptavidin-AlexaFluor555, and ligase expression is detected by anti-V5 staining. Panels show the pouch region of the wing disc, indicated by the dashed line in (c). Scale bar, 40 μm. Each experimental condition has at least three technical replicates; one representative image is shown. This experiment was independently repeated two times with similar results. ( e ) Quantitation of streptavidin-AlexaFluor555 signal intensities in (d). Error bars, s.e.m. Average fold-change shown as text above bars. Sample size values (n) from left column to right: 5, 6, 3. ( f ) Scheme for ubiquitous expression of ligases in flies, at all developmental timepoints, via the act-Gal4 driver. ( g ) Western blotting of fly lysates prepared as in (f). Biotinylated proteins detected by blotting with streptavidin-HRP, ligase expression detected by anti-V5 blotting. In control sample, act-Gal4 drives expression of UAS-luciferase. Bands in control lanes are endogenous naturally-biotinylated proteins. This experiment was performed twice with similar results. ( h ) – ( k ) Comparison of ligases in worms. ( h ) Scheme for tissue-specific expression of ligases in C. elegans intestine via ges-1p promoter. Transgenic strains are fed either biotin-producing E. coli OP50 (biotin+), or biotin-auxotrophic E. coli MG1655bioB:kan (biotin-). Promoter ges-1p drives ligase expression approximately 150 minutes after the first cell cleavage. ( i ) Adult worms prepared as in (h) were shifted to 25°C for one generation, then lysed and analyzed by Western blotting. Control worms (N2) do not express ligase. Anti-HA antibody detects ligase expression. Streptavidin-IRDye detects biotinylated proteins. This experiment was performed five times (n = 5). In biotin+ conditions, BioID biotinylation activity was undetectable and TurboID gave robust biotinylation signal (n = 5/5). Despite high activity detected by immunofluorescence in embryos, we only detected some low level of biotinylation by miniTurbo in adults (n = 2/5), likely due to its low expression levels. ( j ) Representative images of bean stage worm embryos (stage 1) from (h). See Supplementary Figure 15a for representative images of comma stage worm embryos (stage 2). Embryos were fixed and stained with streptavidin-AF488 to detect biotinylated proteins, and anti-HA antibody to detect ligase expression. Intestine is outlined by a white dotted line. Scale bar, 10 μm. Quantitation of multiple replicates shown in (k). ( k ) Quantitation of streptavidin-AF488 signal acquired from IF staining of embryonic stages 1 and 2 shown in (j) and Supplementary Figure 15a . Mean streptavidin pixel intensities for each embryo assessed are plotted for BioID (B), TurboID (T), and miniTurbo (mT). Two independent transgenic lines for BioID and TurboID and one for miniTurbo were assessed. Number of embryos imaged (n) from left to right: 26, 18, 11, 16, 25, 8, 19, 23, 14, 14, 23, 9. Statistical significance via Mann-Whitney U test (two-sided). ***p ≤ 0.0001, **p ≤ 0.001, *p ≤ 0.01. Pink asterisks indicate significance of pairwise comparisons between biotin- and corresponding biotin+ treated embryos. Mean (reported in Supplementary Figure 15b ) is shown as a black horizontal line for each condition, and error bars indicate s.e.m. Note that the streptavidin-AF488 pixel intensities for miniTurbo are an underrepresentation of the signal as camera exposure settings were lowered to avoid pixel saturation (see Methods ). See Supplementary Figure 15 for more details.
    Figure Legend Snippet: TurboID and miniTurbo in flies, worms, and other species ( a ) Comparison of ligases in yeast. EBY100 S. cerevisiae expressing BioID, TurboID, or miniTurbo in the cytosol were treated with 50 μM biotin for 18 hours. Whole cell lysates were blotted with streptavidin-HRP to visualize biotinylated proteins, and anti-V5 antibody to visualize ligase expression. U, untransfected. Asterisks denote ligase self-biotinylation. Bands in untransfected lane are endogenous naturally-biotinylated proteins. This experiment was performed twice with similar results. ( b ) Comparison of ligases in E. coli. Ligases, fused at their N-terminal ends to His6-maltose binding protein, were expressed in the cytosol of BL21 E. coli and 50 μM exogenous biotin was added for 18 hours. Whole cell lysates were analyzed as in (a). This experiment was performed twice with similar results. ( c ) – ( g ) Comparison of ligases in flies. ( c ) Scheme for tissue-specific expression of ligases in the wing disc of D. melanogaster . ptc-Gal4 induces ligase expression in a strip of cells within the wing imaginal disc that borders the anterior/posterior compartments. ( d ) Imaging of larval wing discs after 5 days of growth on biotin-containing food. Biotinylated proteins are detected by staining with streptavidin-AlexaFluor555, and ligase expression is detected by anti-V5 staining. Panels show the pouch region of the wing disc, indicated by the dashed line in (c). Scale bar, 40 μm. Each experimental condition has at least three technical replicates; one representative image is shown. This experiment was independently repeated two times with similar results. ( e ) Quantitation of streptavidin-AlexaFluor555 signal intensities in (d). Error bars, s.e.m. Average fold-change shown as text above bars. Sample size values (n) from left column to right: 5, 6, 3. ( f ) Scheme for ubiquitous expression of ligases in flies, at all developmental timepoints, via the act-Gal4 driver. ( g ) Western blotting of fly lysates prepared as in (f). Biotinylated proteins detected by blotting with streptavidin-HRP, ligase expression detected by anti-V5 blotting. In control sample, act-Gal4 drives expression of UAS-luciferase. Bands in control lanes are endogenous naturally-biotinylated proteins. This experiment was performed twice with similar results. ( h ) – ( k ) Comparison of ligases in worms. ( h ) Scheme for tissue-specific expression of ligases in C. elegans intestine via ges-1p promoter. Transgenic strains are fed either biotin-producing E. coli OP50 (biotin+), or biotin-auxotrophic E. coli MG1655bioB:kan (biotin-). Promoter ges-1p drives ligase expression approximately 150 minutes after the first cell cleavage. ( i ) Adult worms prepared as in (h) were shifted to 25°C for one generation, then lysed and analyzed by Western blotting. Control worms (N2) do not express ligase. Anti-HA antibody detects ligase expression. Streptavidin-IRDye detects biotinylated proteins. This experiment was performed five times (n = 5). In biotin+ conditions, BioID biotinylation activity was undetectable and TurboID gave robust biotinylation signal (n = 5/5). Despite high activity detected by immunofluorescence in embryos, we only detected some low level of biotinylation by miniTurbo in adults (n = 2/5), likely due to its low expression levels. ( j ) Representative images of bean stage worm embryos (stage 1) from (h). See Supplementary Figure 15a for representative images of comma stage worm embryos (stage 2). Embryos were fixed and stained with streptavidin-AF488 to detect biotinylated proteins, and anti-HA antibody to detect ligase expression. Intestine is outlined by a white dotted line. Scale bar, 10 μm. Quantitation of multiple replicates shown in (k). ( k ) Quantitation of streptavidin-AF488 signal acquired from IF staining of embryonic stages 1 and 2 shown in (j) and Supplementary Figure 15a . Mean streptavidin pixel intensities for each embryo assessed are plotted for BioID (B), TurboID (T), and miniTurbo (mT). Two independent transgenic lines for BioID and TurboID and one for miniTurbo were assessed. Number of embryos imaged (n) from left to right: 26, 18, 11, 16, 25, 8, 19, 23, 14, 14, 23, 9. Statistical significance via Mann-Whitney U test (two-sided). ***p ≤ 0.0001, **p ≤ 0.001, *p ≤ 0.01. Pink asterisks indicate significance of pairwise comparisons between biotin- and corresponding biotin+ treated embryos. Mean (reported in Supplementary Figure 15b ) is shown as a black horizontal line for each condition, and error bars indicate s.e.m. Note that the streptavidin-AF488 pixel intensities for miniTurbo are an underrepresentation of the signal as camera exposure settings were lowered to avoid pixel saturation (see Methods ). See Supplementary Figure 15 for more details.

    Techniques Used: Expressing, Binding Assay, Stripping Membranes, Imaging, Staining, Quantitation Assay, Activated Clotting Time Assay, Western Blot, Luciferase, Transgenic Assay, Activity Assay, Immunofluorescence, MANN-WHITNEY

    18) Product Images from "Development of Adenosine Deaminase-Specific IgY Antibodies: Diagnostic and Inhibitory Application"

    Article Title: Development of Adenosine Deaminase-Specific IgY Antibodies: Diagnostic and Inhibitory Application

    Journal: Applied Biochemistry and Biotechnology

    doi: 10.1007/s12010-017-2626-x

    Sandwich-type ELISA assay for calf adenosine deaminase detection. The plate was coated with anti-cADA affinity-purified IgY antibodies and control IgY antibodies (2.5 μg/ml) followed by incubation with cADA at concentrations ranging from 500 to 0.05 ng/ml. For detection, affinity-purified anti-cADA IgY biotin-labeled antibodies were used at a concentration of 2.5 μg/ml. The complexes were detected with streptavidin conjugate with HRP (1: 5000 dilution). Symbols represent mean ± SD from two independent experiments performed in duplicate for each point and are expressed as the OD 490 * values obtained after subtraction of the background values
    Figure Legend Snippet: Sandwich-type ELISA assay for calf adenosine deaminase detection. The plate was coated with anti-cADA affinity-purified IgY antibodies and control IgY antibodies (2.5 μg/ml) followed by incubation with cADA at concentrations ranging from 500 to 0.05 ng/ml. For detection, affinity-purified anti-cADA IgY biotin-labeled antibodies were used at a concentration of 2.5 μg/ml. The complexes were detected with streptavidin conjugate with HRP (1: 5000 dilution). Symbols represent mean ± SD from two independent experiments performed in duplicate for each point and are expressed as the OD 490 * values obtained after subtraction of the background values

    Techniques Used: Enzyme-linked Immunosorbent Assay, Affinity Purification, Incubation, Labeling, Concentration Assay

    19) Product Images from "Activity-Based Labeling of Matrix Metalloproteinases in Living Vertebrate Embryos"

    Article Title: Activity-Based Labeling of Matrix Metalloproteinases in Living Vertebrate Embryos

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0043434

    Hx-BP labels active MMPs in vitro . 135 ng of human recombinant MMP-2 (hrMMP-2) was incubated with 0.5 nmol of biotinylated HxBP, with or without activation by APMA and with or without UV crosslinking, then resolved by SDS-PAGE and blotted to PVDF membrane. Streptavidin-HRP detection of biotinylated hrMMP-2 is dependent on both the activation status of the protease, and on exposure to UV light (h v ).
    Figure Legend Snippet: Hx-BP labels active MMPs in vitro . 135 ng of human recombinant MMP-2 (hrMMP-2) was incubated with 0.5 nmol of biotinylated HxBP, with or without activation by APMA and with or without UV crosslinking, then resolved by SDS-PAGE and blotted to PVDF membrane. Streptavidin-HRP detection of biotinylated hrMMP-2 is dependent on both the activation status of the protease, and on exposure to UV light (h v ).

    Techniques Used: In Vitro, Recombinant, Incubation, Activation Assay, SDS Page

    HxBP labels its target proteins in vivo . Live Xenopus tadpoles were injected with HxBP-alkyne, allowed to recover, exposed to UV light, and then sacrificed and protein extracts of the tails were subjected to click chemistry to biotinylate HxBP-tagged proteins. In panel A, biotinylated proteins were affinity purified by avidin chromatography, resolved by SDS-PAGE and detected by silver staining. A single protein with a mobility consistent with xMMP2 is detectable in the lane containing the HxBP-labeled proteome of the larvae that were induced to express xMMP2 by treatment with T3, and then biotinylated by click chemistry. In panel B, tail homogenates were split and run in parallel on a gelatin zymography gel (left) and for blotting to PVDF (right). Blots were probed with streptavidin-HRP, and revealed a single labeled protein at the same mobility as the gelatinolytic xMMP2 band on the zymograph.
    Figure Legend Snippet: HxBP labels its target proteins in vivo . Live Xenopus tadpoles were injected with HxBP-alkyne, allowed to recover, exposed to UV light, and then sacrificed and protein extracts of the tails were subjected to click chemistry to biotinylate HxBP-tagged proteins. In panel A, biotinylated proteins were affinity purified by avidin chromatography, resolved by SDS-PAGE and detected by silver staining. A single protein with a mobility consistent with xMMP2 is detectable in the lane containing the HxBP-labeled proteome of the larvae that were induced to express xMMP2 by treatment with T3, and then biotinylated by click chemistry. In panel B, tail homogenates were split and run in parallel on a gelatin zymography gel (left) and for blotting to PVDF (right). Blots were probed with streptavidin-HRP, and revealed a single labeled protein at the same mobility as the gelatinolytic xMMP2 band on the zymograph.

    Techniques Used: In Vivo, Injection, Affinity Purification, Avidin-Biotin Assay, Chromatography, SDS Page, Silver Staining, Labeling, Zymography

    20) Product Images from "Oolemmal proteomics - identification of highly abundant heat shock proteins and molecular chaperones in the mature mouse egg and their localization on the plasma membrane"

    Article Title: Oolemmal proteomics - identification of highly abundant heat shock proteins and molecular chaperones in the mature mouse egg and their localization on the plasma membrane

    Journal: Reproductive biology and endocrinology : RB & E

    doi: 10.1186/1477-7827-1-27

    2D Electrophoresis, biotinylation and avidin blotting identifies surface-labeled oolemmal proteins. 2D electrophoresis of 800 zona-free mouse egg proteins, surface labeled with 2 mg/ml Sulfo-NHS biotin, and separated by 2D electrophoresis. Blotted with streptavidin-HRP and visualized by ECL (A). Corresponding 2D electrophoretic gel of 800 unlabeled zona-free mouse eggs visualized by silver staining. Labelled black arrows indicate previously identified protein spots (B). Horizontal axis of each shows isoelectric point and vertical axis shows molecular weight (kDa).
    Figure Legend Snippet: 2D Electrophoresis, biotinylation and avidin blotting identifies surface-labeled oolemmal proteins. 2D electrophoresis of 800 zona-free mouse egg proteins, surface labeled with 2 mg/ml Sulfo-NHS biotin, and separated by 2D electrophoresis. Blotted with streptavidin-HRP and visualized by ECL (A). Corresponding 2D electrophoretic gel of 800 unlabeled zona-free mouse eggs visualized by silver staining. Labelled black arrows indicate previously identified protein spots (B). Horizontal axis of each shows isoelectric point and vertical axis shows molecular weight (kDa).

    Techniques Used: Two-Dimensional Gel Electrophoresis, Avidin-Biotin Assay, Labeling, Silver Staining, Molecular Weight

    21) Product Images from "The PINK1 p.I368N mutation affects protein stability and ubiquitin kinase activity"

    Article Title: The PINK1 p.I368N mutation affects protein stability and ubiquitin kinase activity

    Journal: Molecular Neurodegeneration

    doi: 10.1186/s13024-017-0174-z

    PINK1 p.I368N is a kinase dead mutant that fails to activate PARKIN and the mitochondrial quality control. a HeLa cells were simultaneously transfected with PINK1 siRNA and V5 empty vector, PINK1 WT or p.I368N mutant. Cells were left untreated or incubated with 10 μM CCCP for 4 h. Cell lysates were analyzed by WB, probed with anti-V5, PINK1 and p-Ser65-Ub antibodies. GAPDH was used as a loading control. Auto-phosphorylated PINK1 (anti-V5) (asterisk) was detected on phos-tag gels only in lysates from PINK1-V5 WT transfected, CCCP treated cells. In PINK1-V5 p.I368N transfected cells, CCCP-induced phosphorylation of Ub was largely abolished. b IP of PINK1-V5 using V5 antibody followed by in vitro kinase assay. HeLa cells were transfected with the V5 empty vector, PINK1-V5 WT or p.I368N and treated with or without CCCP for 2 h. Washed immunoprecipitates were incubated with N-terminally biotinylated mono-Ub and 500 μM ATP in phosphorylation buffer at 37 °C for 24 h. Total and phosphorylated Ub were analyzed by WB using streptavidin-HRP and p-Ser65-Ub antibody, respectively. c PARKIN translocation to damaged mitochondria measured by HCI. HeLa cells stably overexpressing GFP-tagged PARKIN were simultaneously transfected with PINK1 (or control) siRNA and with V5 empty vector, PINK1-V5 WT or p.I368N mutant, as indicated, and with mCherry as a selection marker. Cells were left untreated or treated with CCCP for 2 h and GFP-PARKIN translocation was measured in mCherry-positive cells. Data represents the mean of two independent experiments run with each six replicate wells. Statistical significance was assessed by two-way ANOVA with Tukey’s post hoc; **, p
    Figure Legend Snippet: PINK1 p.I368N is a kinase dead mutant that fails to activate PARKIN and the mitochondrial quality control. a HeLa cells were simultaneously transfected with PINK1 siRNA and V5 empty vector, PINK1 WT or p.I368N mutant. Cells were left untreated or incubated with 10 μM CCCP for 4 h. Cell lysates were analyzed by WB, probed with anti-V5, PINK1 and p-Ser65-Ub antibodies. GAPDH was used as a loading control. Auto-phosphorylated PINK1 (anti-V5) (asterisk) was detected on phos-tag gels only in lysates from PINK1-V5 WT transfected, CCCP treated cells. In PINK1-V5 p.I368N transfected cells, CCCP-induced phosphorylation of Ub was largely abolished. b IP of PINK1-V5 using V5 antibody followed by in vitro kinase assay. HeLa cells were transfected with the V5 empty vector, PINK1-V5 WT or p.I368N and treated with or without CCCP for 2 h. Washed immunoprecipitates were incubated with N-terminally biotinylated mono-Ub and 500 μM ATP in phosphorylation buffer at 37 °C for 24 h. Total and phosphorylated Ub were analyzed by WB using streptavidin-HRP and p-Ser65-Ub antibody, respectively. c PARKIN translocation to damaged mitochondria measured by HCI. HeLa cells stably overexpressing GFP-tagged PARKIN were simultaneously transfected with PINK1 (or control) siRNA and with V5 empty vector, PINK1-V5 WT or p.I368N mutant, as indicated, and with mCherry as a selection marker. Cells were left untreated or treated with CCCP for 2 h and GFP-PARKIN translocation was measured in mCherry-positive cells. Data represents the mean of two independent experiments run with each six replicate wells. Statistical significance was assessed by two-way ANOVA with Tukey’s post hoc; **, p

    Techniques Used: Mutagenesis, Transfection, Plasmid Preparation, Incubation, Western Blot, In Vitro, Kinase Assay, Translocation Assay, Stable Transfection, Selection, Marker

    22) Product Images from "PARP3 is a sensor of nicked nucleosomes and monoribosylates histone H2BGlu2"

    Article Title: PARP3 is a sensor of nicked nucleosomes and monoribosylates histone H2BGlu2

    Journal: Nature Communications

    doi: 10.1038/ncomms12404

    The PARP3 DNA-binding interface is required for PARP3 stimulation and accumulation at chromosome DNA damage. ( a ) Wild-type or the indicated mutant full-length cPARP3 (300 nM) was incubated for 20 min at room temp with biotin-NAD + (12.5 μM) and 200 nM of oligonucleotide duplex harbouring either a 5′-phosphorylated nick or 5′-phosphorylated DSB with 3′-overhang. Reaction products were separated by SDS–PAGE, blotted, and detected with streptavidin-HRP. Autoribosylated cPARP3 was quantified and plotted relative to that generated in reactions containing nicked duplex and wild type cPARP3. Data are the mean (±s.e.m.) from three independent experiments. ( b ) Time-course of wild-type or mutant cPARP3 incubated from 0 to 30 min in the same conditions as above. ( c ) Recombinant wild-type or mutant cPARP3 (0–0.8 μM) was incubated with a 3′-fluorescein isothiocyanate (FITC)-labeled oligonucleotide duplex harbouring a 5′-phosphorylated nick (100 nM), and protein-DNA complexes detected by EMSA. ( d ) Recruitment of wild-type and mutant human PARP3-GFP to sites of UVA-laser DNA damage in human U2-OS cells. (left) Representative images of WT and mutant PARP3-GFP before treatment (Unt) and 1 min after laser damage. (top right) Quantification of GFP accumulation at sites of laser damage (% increase over initial level). Data are the mean (±s.e.m.) of 25 or more cells per sample. The hPARP3 WGR mutations were Y83A, W101L and R103N and H384A/E514A in the catalytic domain (denoted ‘CM'). ( e , top) PARP3 −/− DT40 cells stably transfected with either empty vector (vector) or vector encoding wild-type hPARP3 (WT) or the mutant derivatives Y83A, W101L and R103N were treated on ice with γ-rays (20 Gy) and incubated for the indicated times to allow repair. DNA strand breaks were quantified (tail moment) by alkaline comet assays. The inset is a western blot showing the expression level of wild type and mutant hPARP3 in PARP3 −/− DT40 cells. (bottom) The above DT40 cell lines were treated with the indicated doses of γ-rays and survival quantified in clonogenic assays. Data are the mean (±s.e.m.) of three independent experiments. Where not visible, error bars are smaller than the symbols. HRP, horseradish peroxidase.
    Figure Legend Snippet: The PARP3 DNA-binding interface is required for PARP3 stimulation and accumulation at chromosome DNA damage. ( a ) Wild-type or the indicated mutant full-length cPARP3 (300 nM) was incubated for 20 min at room temp with biotin-NAD + (12.5 μM) and 200 nM of oligonucleotide duplex harbouring either a 5′-phosphorylated nick or 5′-phosphorylated DSB with 3′-overhang. Reaction products were separated by SDS–PAGE, blotted, and detected with streptavidin-HRP. Autoribosylated cPARP3 was quantified and plotted relative to that generated in reactions containing nicked duplex and wild type cPARP3. Data are the mean (±s.e.m.) from three independent experiments. ( b ) Time-course of wild-type or mutant cPARP3 incubated from 0 to 30 min in the same conditions as above. ( c ) Recombinant wild-type or mutant cPARP3 (0–0.8 μM) was incubated with a 3′-fluorescein isothiocyanate (FITC)-labeled oligonucleotide duplex harbouring a 5′-phosphorylated nick (100 nM), and protein-DNA complexes detected by EMSA. ( d ) Recruitment of wild-type and mutant human PARP3-GFP to sites of UVA-laser DNA damage in human U2-OS cells. (left) Representative images of WT and mutant PARP3-GFP before treatment (Unt) and 1 min after laser damage. (top right) Quantification of GFP accumulation at sites of laser damage (% increase over initial level). Data are the mean (±s.e.m.) of 25 or more cells per sample. The hPARP3 WGR mutations were Y83A, W101L and R103N and H384A/E514A in the catalytic domain (denoted ‘CM'). ( e , top) PARP3 −/− DT40 cells stably transfected with either empty vector (vector) or vector encoding wild-type hPARP3 (WT) or the mutant derivatives Y83A, W101L and R103N were treated on ice with γ-rays (20 Gy) and incubated for the indicated times to allow repair. DNA strand breaks were quantified (tail moment) by alkaline comet assays. The inset is a western blot showing the expression level of wild type and mutant hPARP3 in PARP3 −/− DT40 cells. (bottom) The above DT40 cell lines were treated with the indicated doses of γ-rays and survival quantified in clonogenic assays. Data are the mean (±s.e.m.) of three independent experiments. Where not visible, error bars are smaller than the symbols. HRP, horseradish peroxidase.

    Techniques Used: Binding Assay, Mutagenesis, Incubation, TNKS1 Histone Ribosylation Assay, SDS Page, Generated, Recombinant, Labeling, Stable Transfection, Transfection, Plasmid Preparation, Western Blot, Expressing

    PARP3 monoribosylates H2B in damaged chromatin. ( a , left) 10μg of the chicken chromatin employed in these experiments was fractionated by SDS–PAGE and stained with Coomassie blue. (right) One microgram of soluble MNase-treated chicken chromatin or 50-mer oligonucleotide duplex (200 nM) harbouring a nick with 3′-P/5′-OH termini was mock-treated (0) or treated with 1, 0.5 or 0.25 U T4 PNK to restore 3′-OH/5′-P termini. These DNA substrates were then incubated with 100 nM hPARP3 and 12.5 μM biotin-NAD + for 30 min and biotinylated products separated by 15% SDS–PAGE and detected with streptavidin-HRP. ( b ) 1 μg chicken chromatin or the indicated recombinant histone was incubated with 100 nM hPARP3 in the presence of 300 nM 32 P-NAD + or 12.5 μM biotin-NAD and oligonucleotide harbouring either a DSB (middle) or SSB (right) and the reaction products fractionated by 15% SDS–PAGE and detected by autoradiography or streptavidin-HRP. (left) An aliquot of the chicken chromatin and recombinant histones was fractionated by SDS–PAGE and stained with Coomassie blue. ( c , left) Aliquots of recombinant histone standards were fractionated separately or together as an octamer on triton-acid urea gels and analysed by staining with Coomassie blue. (right) The products of the PARP3 ribosylation reactions conducted in b were fractionated on triton-acid urea gels and analysed by autoradiography. HRP, horseradish peroxidase.
    Figure Legend Snippet: PARP3 monoribosylates H2B in damaged chromatin. ( a , left) 10μg of the chicken chromatin employed in these experiments was fractionated by SDS–PAGE and stained with Coomassie blue. (right) One microgram of soluble MNase-treated chicken chromatin or 50-mer oligonucleotide duplex (200 nM) harbouring a nick with 3′-P/5′-OH termini was mock-treated (0) or treated with 1, 0.5 or 0.25 U T4 PNK to restore 3′-OH/5′-P termini. These DNA substrates were then incubated with 100 nM hPARP3 and 12.5 μM biotin-NAD + for 30 min and biotinylated products separated by 15% SDS–PAGE and detected with streptavidin-HRP. ( b ) 1 μg chicken chromatin or the indicated recombinant histone was incubated with 100 nM hPARP3 in the presence of 300 nM 32 P-NAD + or 12.5 μM biotin-NAD and oligonucleotide harbouring either a DSB (middle) or SSB (right) and the reaction products fractionated by 15% SDS–PAGE and detected by autoradiography or streptavidin-HRP. (left) An aliquot of the chicken chromatin and recombinant histones was fractionated by SDS–PAGE and stained with Coomassie blue. ( c , left) Aliquots of recombinant histone standards were fractionated separately or together as an octamer on triton-acid urea gels and analysed by staining with Coomassie blue. (right) The products of the PARP3 ribosylation reactions conducted in b were fractionated on triton-acid urea gels and analysed by autoradiography. HRP, horseradish peroxidase.

    Techniques Used: SDS Page, Staining, Incubation, TNKS1 Histone Ribosylation Assay, Recombinant, Autoradiography

    23) Product Images from "Activity of the SPCA1 calcium pump couples sphingomyelin synthesis to sorting of secretory proteins in the trans-Golgi network"

    Article Title: Activity of the SPCA1 calcium pump couples sphingomyelin synthesis to sorting of secretory proteins in the trans-Golgi network

    Journal: Developmental cell

    doi: 10.1016/j.devcel.2018.10.012

    SPCA1 associates with sphingolipid in Golgi membrane. (A) SMS1, SPCA1, and pacSph localize to the TGN. Antisera to SPCA1, p230 (TGN), or GM130 ( cis Golgi) were used to detect each protein by immunofluorescence microscopy in gene edited HeLa cells. To detect endogenous SMS1, a SMS1-SNAP tag fusion protein (constructed by genome editing) was labeled with SNAP-Cell 647-SiR. To visualize sphingolipids in situ , sphingosine-1-phosphate lyase deficient ( SGPL1- ). Scale bars, 10 μm. Insets in the merged images show a higher magnification view of the Golgi region. (B) Schematic diagram of protocol used to test for UV-induced crosslinking of SPCA1 and pacSph. (C) SPCA1 and pacSph can be crosslinked. The left panel is an anti-GFP immunoblot showing GFP-SPCA1 that was immunopurified from the UV treated and untreated samples. In the right hand blot, the same samples were probed with streptavidin-HRP to detect pacSph crosslinked to SPCA1. (D) .
    Figure Legend Snippet: SPCA1 associates with sphingolipid in Golgi membrane. (A) SMS1, SPCA1, and pacSph localize to the TGN. Antisera to SPCA1, p230 (TGN), or GM130 ( cis Golgi) were used to detect each protein by immunofluorescence microscopy in gene edited HeLa cells. To detect endogenous SMS1, a SMS1-SNAP tag fusion protein (constructed by genome editing) was labeled with SNAP-Cell 647-SiR. To visualize sphingolipids in situ , sphingosine-1-phosphate lyase deficient ( SGPL1- ). Scale bars, 10 μm. Insets in the merged images show a higher magnification view of the Golgi region. (B) Schematic diagram of protocol used to test for UV-induced crosslinking of SPCA1 and pacSph. (C) SPCA1 and pacSph can be crosslinked. The left panel is an anti-GFP immunoblot showing GFP-SPCA1 that was immunopurified from the UV treated and untreated samples. In the right hand blot, the same samples were probed with streptavidin-HRP to detect pacSph crosslinked to SPCA1. (D) .

    Techniques Used: Immunofluorescence, Microscopy, Construct, Labeling, In Situ

    24) Product Images from "Fbxl19 recruitment to CpG islands is required for Rnf20-mediated H2B mono-ubiquitination"

    Article Title: Fbxl19 recruitment to CpG islands is required for Rnf20-mediated H2B mono-ubiquitination

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx310

    Fbxl19 is implicated in mono-ubiquitination of H2B. ( A ) A bar graph presenting knockdown (KD) efficiency of Fbxl19 measured by qRT-PCR. Error bars indicate standard error of the mean ( n = 3). ( B and C ) Western blots showing decreased levels of H2Bub1 upon KD (B) and KO (C) of Fbxl19. ( D ) A bar graph presenting overexpression (OE) level of Fbxl19 measured by qRT-PCR. Error bar indicates standard error of the mean ( n = 3). ( E ) Western blots showing induced level of H2Bub1 upon OE of Fbxl19. OE level of Fbxl19 was detected by HRP-conjugated streptavidin. WT indicates wild-type ES cells. NS indicates non-specific band.
    Figure Legend Snippet: Fbxl19 is implicated in mono-ubiquitination of H2B. ( A ) A bar graph presenting knockdown (KD) efficiency of Fbxl19 measured by qRT-PCR. Error bars indicate standard error of the mean ( n = 3). ( B and C ) Western blots showing decreased levels of H2Bub1 upon KD (B) and KO (C) of Fbxl19. ( D ) A bar graph presenting overexpression (OE) level of Fbxl19 measured by qRT-PCR. Error bar indicates standard error of the mean ( n = 3). ( E ) Western blots showing induced level of H2Bub1 upon OE of Fbxl19. OE level of Fbxl19 was detected by HRP-conjugated streptavidin. WT indicates wild-type ES cells. NS indicates non-specific band.

    Techniques Used: Quantitative RT-PCR, Western Blot, Over Expression

    25) Product Images from "The Cytoplasmic Tail of the T Cell Receptor CD3 ε Subunit Contains a Phospholipid-Binding Motif that Regulates T Cell Functions 1"

    Article Title: The Cytoplasmic Tail of the T Cell Receptor CD3 ε Subunit Contains a Phospholipid-Binding Motif that Regulates T Cell Functions 1

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

    doi: 10.4049/jimmunol.0900404

    The CD3 ε subunit contains a basic-rich domain that complexes charged phospholipids. A , The amino acid sequence of the cytoplasmic tail of CD3 ε and the individual subdomains, termed the BRS, PRS, and ITAM, are shown. Asterisks denote the positively charged lysine or arginine residues in the BRS. In the lower section, PIP-Strips were probed with GST-fusion proteins consisting of the entire cytoplasmic tail of CD3 ε , the individual subdomains of CD3 ε (BRS, PRS, or ITAM), or with fusion proteins that contained mutations of the BRS (BRS-Substitute and BRS-Truncate). Binding was detected by anti-GST western blotting. B , Sucrose-loaded liposomes, consisting of the indicated phospholipids, were incubated with GST ( lanes 2–5 ) or GST-BRS ( lanes 7–10 ). Protein binding in the pellet fraction was assessed by anti-GST immunoblotting ( upper panel ). Lanes 1 and 6 , GST or GST-BRS were resolved as m.w. controls. Supernatants were also blotted to verify equal loading ( lower panel ). C , PC (◆), or a 20:80 ratio of PC to PI(4)P (□) or PI(4,5)P 2 (△), were coated onto 96-well plates and incubated with biotinylated peptides containing the BRS or the phosphorylated ITAM of CD3 ζ . Peptide binding was assessed with streptavidin-HRP using ELISA-based assays. The assay was performed in triplicate.
    Figure Legend Snippet: The CD3 ε subunit contains a basic-rich domain that complexes charged phospholipids. A , The amino acid sequence of the cytoplasmic tail of CD3 ε and the individual subdomains, termed the BRS, PRS, and ITAM, are shown. Asterisks denote the positively charged lysine or arginine residues in the BRS. In the lower section, PIP-Strips were probed with GST-fusion proteins consisting of the entire cytoplasmic tail of CD3 ε , the individual subdomains of CD3 ε (BRS, PRS, or ITAM), or with fusion proteins that contained mutations of the BRS (BRS-Substitute and BRS-Truncate). Binding was detected by anti-GST western blotting. B , Sucrose-loaded liposomes, consisting of the indicated phospholipids, were incubated with GST ( lanes 2–5 ) or GST-BRS ( lanes 7–10 ). Protein binding in the pellet fraction was assessed by anti-GST immunoblotting ( upper panel ). Lanes 1 and 6 , GST or GST-BRS were resolved as m.w. controls. Supernatants were also blotted to verify equal loading ( lower panel ). C , PC (◆), or a 20:80 ratio of PC to PI(4)P (□) or PI(4,5)P 2 (△), were coated onto 96-well plates and incubated with biotinylated peptides containing the BRS or the phosphorylated ITAM of CD3 ζ . Peptide binding was assessed with streptavidin-HRP using ELISA-based assays. The assay was performed in triplicate.

    Techniques Used: Sequencing, Binding Assay, Western Blot, Incubation, Protein Binding, Enzyme-linked Immunosorbent Assay

    The membrane-proximal location of the BRS is essential for thymocyte development. A , CD3 ε was immunoprecipitated from C57BL/6 ( lane 1 ), BRS-Displace ( lane 2 ), or CD3 ε -deficient ( lane 3 ) thymocytes. Precipitates were first immunoblotted using anti-CD3 δ ( upper panel ) or CD3 γ ( middle panel ) polyclonal antisera, then reprobed using anti-CD3 ε ( lower panel and data not shown). B , Thymocytes were stained using biotinylated anti-TCR β . The cells were then lysed, and TCR β was immunoprecipitated using immobilized streptavidin beads. Whole cell lysates ( lower panel ) or TCR β precipitates ( upper panel ) were resolved and probed with CD3 ε antisera. C , Cell surface proteins on thymocytes were biotinylated. CD3 ε was immunoprecipitated, and the precipitates were immunoblotted with streptavidin-HRP to detect surface-biotinylated proteins. D , RAG-deficient or BRS-Displace mice were injected i.p. with PBS or anti-CD3 ε mAbs. Seven days postinjection, the thymocytes were enumerated and analyzed for the expression of CD4 and CD8 by flow cytometry. RAG −/− : n = 2 mice per group. BRS-Displace: n = 3 mice per group.
    Figure Legend Snippet: The membrane-proximal location of the BRS is essential for thymocyte development. A , CD3 ε was immunoprecipitated from C57BL/6 ( lane 1 ), BRS-Displace ( lane 2 ), or CD3 ε -deficient ( lane 3 ) thymocytes. Precipitates were first immunoblotted using anti-CD3 δ ( upper panel ) or CD3 γ ( middle panel ) polyclonal antisera, then reprobed using anti-CD3 ε ( lower panel and data not shown). B , Thymocytes were stained using biotinylated anti-TCR β . The cells were then lysed, and TCR β was immunoprecipitated using immobilized streptavidin beads. Whole cell lysates ( lower panel ) or TCR β precipitates ( upper panel ) were resolved and probed with CD3 ε antisera. C , Cell surface proteins on thymocytes were biotinylated. CD3 ε was immunoprecipitated, and the precipitates were immunoblotted with streptavidin-HRP to detect surface-biotinylated proteins. D , RAG-deficient or BRS-Displace mice were injected i.p. with PBS or anti-CD3 ε mAbs. Seven days postinjection, the thymocytes were enumerated and analyzed for the expression of CD4 and CD8 by flow cytometry. RAG −/− : n = 2 mice per group. BRS-Displace: n = 3 mice per group.

    Techniques Used: Immunoprecipitation, Staining, Mouse Assay, Injection, Expressing, Flow Cytometry, Cytometry

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

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

    Journal: Veterinary Research

    doi: 10.1051/vetres/2010048

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

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

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

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

    Journal: Protein expression and purification

    doi: 10.1016/j.pep.2009.10.011

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

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

    28) Product Images from "Mechanisms of CPT1C-Dependent AMPAR Trafficking Enhancement"

    Article Title: Mechanisms of CPT1C-Dependent AMPAR Trafficking Enhancement

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2018.00275

    CPT1C acts as depalmitoylating enzyme of GluA1. (A) Palmitoylation levels detected with Acyl-Biotin Exchange (ABE) assay of GluA1 alone (GFP), or together with CPT1C-GFP or CPT1C(H470A) in HEK293AD-GluA1 expressing cells transfected with DHHC3/GODZ palmitoylating enzyme. The biotinylated GluA1 immunoprecipitates subsequent to the ABE assay for all conditions were subjected to SDS-PAGE. Palmitoylation of GluA1 subunit is detected only in plus-hydroxylamine (+HAM) samples (three lanes from the left). −HAM samples control non-specific incorporation of biotin (three lanes from the right). GluA1 palmitoylation levels (right top) were detected by Western blotting with streptavidin-HRP (palmitoylation). The total amount of immunoprecipitated GluA1 was detected by Western blotting with anti-GluA1-NT antibody (anti-GluA1, bottom) after stripping the membranes. (B) Quantification of palmitoylation levels for GluA1 alone (GFP), together with CPT1C or CPT1C(H470A) in HEK293AD cells constitutively expressing GluA1. Ratio of palmitoylated GluA1 to total GluA1 is shown as mean and S.E.M. (** p
    Figure Legend Snippet: CPT1C acts as depalmitoylating enzyme of GluA1. (A) Palmitoylation levels detected with Acyl-Biotin Exchange (ABE) assay of GluA1 alone (GFP), or together with CPT1C-GFP or CPT1C(H470A) in HEK293AD-GluA1 expressing cells transfected with DHHC3/GODZ palmitoylating enzyme. The biotinylated GluA1 immunoprecipitates subsequent to the ABE assay for all conditions were subjected to SDS-PAGE. Palmitoylation of GluA1 subunit is detected only in plus-hydroxylamine (+HAM) samples (three lanes from the left). −HAM samples control non-specific incorporation of biotin (three lanes from the right). GluA1 palmitoylation levels (right top) were detected by Western blotting with streptavidin-HRP (palmitoylation). The total amount of immunoprecipitated GluA1 was detected by Western blotting with anti-GluA1-NT antibody (anti-GluA1, bottom) after stripping the membranes. (B) Quantification of palmitoylation levels for GluA1 alone (GFP), together with CPT1C or CPT1C(H470A) in HEK293AD cells constitutively expressing GluA1. Ratio of palmitoylated GluA1 to total GluA1 is shown as mean and S.E.M. (** p

    Techniques Used: Expressing, Transfection, SDS Page, Western Blot, Immunoprecipitation, Stripping Membranes

    29) Product Images from "B7-H4 Modulates Regulatory CD4+ T Cell Induction and Function via Ligation of a Semaphorin 3a/Plexin A4/Neuropilin-1 Complex"

    Article Title: B7-H4 Modulates Regulatory CD4+ T Cell Induction and Function via Ligation of a Semaphorin 3a/Plexin A4/Neuropilin-1 Complex

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

    doi: 10.4049/jimmunol.1700811

    hB7-H4Ig binds Sema3a + mouse Tregs. The amount of hB7-H4Ig biotin-bound was detected via streptavidin–HRP, and the OD (450 nm) was determined. SJL/J mice ( n = 5 per group) were primed with PLP 139–151 /CFA and treated with PBS, Control Ig, or hB7-H4Ig (100 μg/dose; 3 times per wk; 2 wk) via i.p. injection. At 2 h after the first injection and 2 h after the sixth injection, serum samples were collected, and the concentration of ( A ) total Sema3a and ( B ) Sema3a bound to hB7-H4Ig was assessed via ELISA. ( C ) Total lymph node cells from unprimed SJL-Foxp3/GFP mice ( n = 5) were collected, and hB7-H4Ig binding on various Treg populations was assessed. Live CD4 + /CD25 + /Foxp3-GFP + single cells were gated into the PlxnA4 versus Nrp-1 contour plots for each of the fluorescence minus one (FMO), Control Ig, and hB7-H4Ig binding FACS samples. The expression of Sema3a versus hB7-H4Ig binding was assessed on the PlxnA4 + /Nrp-1 − , PlxnA4 + /Nrp-1 + , PlxnA4 − /Nrp-1 − , and PlxnA4 − /Nrp-1 + populations. One representative experiment of three is presented. Asterisks indicate a statistically significant difference in the level of Sema3a and Sema3a/B7-H4Ig complex present following hB7-H4Ig treatment in comparison with cells cultured in the presence of Control Ig treatment. * p
    Figure Legend Snippet: hB7-H4Ig binds Sema3a + mouse Tregs. The amount of hB7-H4Ig biotin-bound was detected via streptavidin–HRP, and the OD (450 nm) was determined. SJL/J mice ( n = 5 per group) were primed with PLP 139–151 /CFA and treated with PBS, Control Ig, or hB7-H4Ig (100 μg/dose; 3 times per wk; 2 wk) via i.p. injection. At 2 h after the first injection and 2 h after the sixth injection, serum samples were collected, and the concentration of ( A ) total Sema3a and ( B ) Sema3a bound to hB7-H4Ig was assessed via ELISA. ( C ) Total lymph node cells from unprimed SJL-Foxp3/GFP mice ( n = 5) were collected, and hB7-H4Ig binding on various Treg populations was assessed. Live CD4 + /CD25 + /Foxp3-GFP + single cells were gated into the PlxnA4 versus Nrp-1 contour plots for each of the fluorescence minus one (FMO), Control Ig, and hB7-H4Ig binding FACS samples. The expression of Sema3a versus hB7-H4Ig binding was assessed on the PlxnA4 + /Nrp-1 − , PlxnA4 + /Nrp-1 + , PlxnA4 − /Nrp-1 − , and PlxnA4 − /Nrp-1 + populations. One representative experiment of three is presented. Asterisks indicate a statistically significant difference in the level of Sema3a and Sema3a/B7-H4Ig complex present following hB7-H4Ig treatment in comparison with cells cultured in the presence of Control Ig treatment. * p

    Techniques Used: Mouse Assay, Plasmid Purification, Injection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Binding Assay, Fluorescence, FACS, Expressing, Cell Culture

    30) Product Images from "Activity of the SPCA1 calcium pump couples sphingomyelin synthesis to sorting of secretory proteins in the trans-Golgi network"

    Article Title: Activity of the SPCA1 calcium pump couples sphingomyelin synthesis to sorting of secretory proteins in the trans-Golgi network

    Journal: Developmental cell

    doi: 10.1016/j.devcel.2018.10.012

    SPCA1 associates with sphingolipid in Golgi membrane. (A) SMS1, SPCA1, and pacSph localize to the TGN. Antisera to SPCA1, p230 (TGN), or GM130 ( cis Golgi) were used to detect each protein by immunofluorescence microscopy in gene edited HeLa cells. To detect endogenous SMS1, a SMS1-SNAP tag fusion protein (constructed by genome editing) was labeled with SNAP-Cell 647-SiR. To visualize sphingolipids in situ , sphingosine-1-phosphate lyase deficient ( SGPL1- ). Scale bars, 10 μm. Insets in the merged images show a higher magnification view of the Golgi region. (B) Schematic diagram of protocol used to test for UV-induced crosslinking of SPCA1 and pacSph. (C) SPCA1 and pacSph can be crosslinked. The left panel is an anti-GFP immunoblot showing GFP-SPCA1 that was immunopurified from the UV treated and untreated samples. In the right hand blot, the same samples were probed with streptavidin-HRP to detect pacSph crosslinked to SPCA1. (D) .
    Figure Legend Snippet: SPCA1 associates with sphingolipid in Golgi membrane. (A) SMS1, SPCA1, and pacSph localize to the TGN. Antisera to SPCA1, p230 (TGN), or GM130 ( cis Golgi) were used to detect each protein by immunofluorescence microscopy in gene edited HeLa cells. To detect endogenous SMS1, a SMS1-SNAP tag fusion protein (constructed by genome editing) was labeled with SNAP-Cell 647-SiR. To visualize sphingolipids in situ , sphingosine-1-phosphate lyase deficient ( SGPL1- ). Scale bars, 10 μm. Insets in the merged images show a higher magnification view of the Golgi region. (B) Schematic diagram of protocol used to test for UV-induced crosslinking of SPCA1 and pacSph. (C) SPCA1 and pacSph can be crosslinked. The left panel is an anti-GFP immunoblot showing GFP-SPCA1 that was immunopurified from the UV treated and untreated samples. In the right hand blot, the same samples were probed with streptavidin-HRP to detect pacSph crosslinked to SPCA1. (D) .

    Techniques Used: Immunofluorescence, Microscopy, Construct, Labeling, In Situ

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

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

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.541250

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

    Techniques Used: Electron Microscopy, Generated, Produced

    32) Product Images from "Selenium-Based S-Adenosylmethionine Analog Reveals the Mammalian Seven-Beta-Strand Methyltransferase METTL10 to Be an EF1A1 Lysine Methyltransferase"

    Article Title: Selenium-Based S-Adenosylmethionine Analog Reveals the Mammalian Seven-Beta-Strand Methyltransferase METTL10 to Be an EF1A1 Lysine Methyltransferase

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0105394

    ProSeAM, a synthetic SAM analog, has a wide spectrum of reactivity for histones and non-histone substrates. A, Schematic overview for analyzing lysine methylation. A synthetic cofactor was used to transfer an alkyne moiety to the ε-amino group of lysine by KMTs (1). The modified proteins were tagged with biotin via CuAAC reaction (2). Tagged-proteins in the crude lysates were pulled down with affinity beads (3), and the precipitants were further analyzed with a LC-MS apparatus (4). B, Chemical structure of SAM (1), propargylated SAM (2) and ProSeAM (3). C, H3 peptide (1-21 a.a.) and ProSeAM was incubated with or without GST-G9a at 20°C for 2 h, then the peptide was analyzed by MALDI-TOF MS. D, full-length Histone H3 (1 µg) and ProSeAM (500 µM) were incubated with indicated KMTs (0.5 µg) for 2 h at 20°C. The histones were separated by SDS-PAGE, transferred to a nitrocellulose membrane and probed with streptavidin-HRP (top) or anti-Histone H3 antibody (bottom). E, The non-histone substrates His-HSP90 and His-HSP70 (1 µg) were incubated with His-SMYD2 and His-METTL21A (1 µg), respectively. After the reaction, proteins were separated by SDS-PAGE (right). Their modifications were detected by western blotting with streptavidin-HRP as in Fig. 1D. *and ** showed automodification of SMYD2 and METTL21A, respectively (left). F, His-HSP70 (WT and K561R) were incubated with or without His-METTL21A in the presence of ProSeAM for 2 h at 20°C. Modified proteins were biotinylated and detected with streptavidin-HRP (top) or anti-HSP70 antibody for the loading control (bottom).
    Figure Legend Snippet: ProSeAM, a synthetic SAM analog, has a wide spectrum of reactivity for histones and non-histone substrates. A, Schematic overview for analyzing lysine methylation. A synthetic cofactor was used to transfer an alkyne moiety to the ε-amino group of lysine by KMTs (1). The modified proteins were tagged with biotin via CuAAC reaction (2). Tagged-proteins in the crude lysates were pulled down with affinity beads (3), and the precipitants were further analyzed with a LC-MS apparatus (4). B, Chemical structure of SAM (1), propargylated SAM (2) and ProSeAM (3). C, H3 peptide (1-21 a.a.) and ProSeAM was incubated with or without GST-G9a at 20°C for 2 h, then the peptide was analyzed by MALDI-TOF MS. D, full-length Histone H3 (1 µg) and ProSeAM (500 µM) were incubated with indicated KMTs (0.5 µg) for 2 h at 20°C. The histones were separated by SDS-PAGE, transferred to a nitrocellulose membrane and probed with streptavidin-HRP (top) or anti-Histone H3 antibody (bottom). E, The non-histone substrates His-HSP90 and His-HSP70 (1 µg) were incubated with His-SMYD2 and His-METTL21A (1 µg), respectively. After the reaction, proteins were separated by SDS-PAGE (right). Their modifications were detected by western blotting with streptavidin-HRP as in Fig. 1D. *and ** showed automodification of SMYD2 and METTL21A, respectively (left). F, His-HSP70 (WT and K561R) were incubated with or without His-METTL21A in the presence of ProSeAM for 2 h at 20°C. Modified proteins were biotinylated and detected with streptavidin-HRP (top) or anti-HSP70 antibody for the loading control (bottom).

    Techniques Used: Methylation, Modification, Liquid Chromatography with Mass Spectroscopy, Incubation, Mass Spectrometry, SDS Page, Western Blot

    Proteomic identification of substrates for seven-beta-strand MTases. A, Schematic protocol for proteomic identification. HEK293T cell lysates were added to either propargylic Se-adenosyl- l -selenomethionine (ProSeAM) alone (1) or ProSeAM plus recombinant KMT (lysate:enzyme ratio was 10∶1) (2). After the in vitro reaction, labeled proteins were tagged with biotin and then precipitated with streptavidin beads. The precipitants were then digested with trypsin, and the trypsinized protein fragments were analyzed by LC-MS/MS. B, ProSeAM competes with SAM in the labeling reaction. HEK293T cell lysates were incubated with ProSeAM (250 µM) in the presence or absence of the indicated amount of SAM (0 to 2.5 mM). Modified proteins were biotinylated and detected with streptavidin-HRP (top). Equal protein loading was confirmed by western blotting with anti-α-tubulin antibody (bottom). C, western blot of labeled proteins. A 5% input of precipitated proteins without ProSeAM (1), with ProSeAM alone (2), with ProSeAM plus GST-G9a (3), with ProSeAM plus His-METTL21A (4) or with ProSeAM plus His-METTL10 was separately analyzed with western blotting with streptavidin-HRP (top) prior to the MS analysis, to compare the labeled proteins. Equal protein loading was confirmed by western blotting with anti-α-tubulin antibody (bottom). D, Doughnut chart of the subcellular distribution of proteins labeled with ProSeAM. HEK293T lysates alone (lane 1 in Fig. 2C) and HEK293T lysates with ProSeAM (lane 2 in Fig. 2C) were analyzed as described in A and Experimental procedures (n = 3). In total, 318 proteins were identified as ProSeAM-labeled proteins. E, List of METTL21A substrates. HEK293T cell lysates and ProSeAM were incubated with or without METTL21A (lane 2 and lane 4 in Fig. 2C), and analyzed as above. Molecular weight, peptide area (reflecting the quantity of detected protein), and fold enrichment of the peptide area are listed: ND, not determined because the substrate was detected only in the condition for lane 4 of B. The total numbers of identified proteins, 2-fold increase (compared to control in each experiment), and overlapped identified numbers of 3 independent experiments are listed in Table S2 .
    Figure Legend Snippet: Proteomic identification of substrates for seven-beta-strand MTases. A, Schematic protocol for proteomic identification. HEK293T cell lysates were added to either propargylic Se-adenosyl- l -selenomethionine (ProSeAM) alone (1) or ProSeAM plus recombinant KMT (lysate:enzyme ratio was 10∶1) (2). After the in vitro reaction, labeled proteins were tagged with biotin and then precipitated with streptavidin beads. The precipitants were then digested with trypsin, and the trypsinized protein fragments were analyzed by LC-MS/MS. B, ProSeAM competes with SAM in the labeling reaction. HEK293T cell lysates were incubated with ProSeAM (250 µM) in the presence or absence of the indicated amount of SAM (0 to 2.5 mM). Modified proteins were biotinylated and detected with streptavidin-HRP (top). Equal protein loading was confirmed by western blotting with anti-α-tubulin antibody (bottom). C, western blot of labeled proteins. A 5% input of precipitated proteins without ProSeAM (1), with ProSeAM alone (2), with ProSeAM plus GST-G9a (3), with ProSeAM plus His-METTL21A (4) or with ProSeAM plus His-METTL10 was separately analyzed with western blotting with streptavidin-HRP (top) prior to the MS analysis, to compare the labeled proteins. Equal protein loading was confirmed by western blotting with anti-α-tubulin antibody (bottom). D, Doughnut chart of the subcellular distribution of proteins labeled with ProSeAM. HEK293T lysates alone (lane 1 in Fig. 2C) and HEK293T lysates with ProSeAM (lane 2 in Fig. 2C) were analyzed as described in A and Experimental procedures (n = 3). In total, 318 proteins were identified as ProSeAM-labeled proteins. E, List of METTL21A substrates. HEK293T cell lysates and ProSeAM were incubated with or without METTL21A (lane 2 and lane 4 in Fig. 2C), and analyzed as above. Molecular weight, peptide area (reflecting the quantity of detected protein), and fold enrichment of the peptide area are listed: ND, not determined because the substrate was detected only in the condition for lane 4 of B. The total numbers of identified proteins, 2-fold increase (compared to control in each experiment), and overlapped identified numbers of 3 independent experiments are listed in Table S2 .

    Techniques Used: Recombinant, In Vitro, Labeling, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Incubation, Modification, Western Blot, Molecular Weight

    33) Product Images from "Variability of cholesterol accessibility in human red blood cells measured using a bacterial cholesterol-binding toxin"

    Article Title: Variability of cholesterol accessibility in human red blood cells measured using a bacterial cholesterol-binding toxin

    Journal: eLife

    doi: 10.7554/eLife.23355

    Effects of cell surface proteins on fALOD4 binding to RBCs. ( A ) Effect of proteolysis of RBC surface membrane proteins on fALOD4 binding to RBCs. RBCs isolated from a healthy individual were labeled with biotin and then treated with increasing amounts of pronase as indicated. An aliquot of the treated RBCs was used for fALOD4 binding assays and the remainder was subjected to 10% SDS/PAGE and probed with streptavidin-HRP (0.22 µg/mL) as described in the Materials and methods. fALOD4 binding values were normalized to the untreated sample. Data points represent the mean of three independent measurements. Error bars represent the SEM. The experiment was repeated three times and the results were similar. ( B ) Relationship between fALOD4 binding and ABO blood group antigens (upper panel) and Rhesus blood group (Rh antigen) (lower panel). RBC fALOD4 binding was determined in triplicate using RBCs from 178 White blood donors as described in the Materials and methods. Each fALOD4 binding measurement was performed in triplicate and values were normalized to the reference blood sample. Boxes represent the 25th and 75th percentiles and whiskers represent the minimum and maximum measurements. *p
    Figure Legend Snippet: Effects of cell surface proteins on fALOD4 binding to RBCs. ( A ) Effect of proteolysis of RBC surface membrane proteins on fALOD4 binding to RBCs. RBCs isolated from a healthy individual were labeled with biotin and then treated with increasing amounts of pronase as indicated. An aliquot of the treated RBCs was used for fALOD4 binding assays and the remainder was subjected to 10% SDS/PAGE and probed with streptavidin-HRP (0.22 µg/mL) as described in the Materials and methods. fALOD4 binding values were normalized to the untreated sample. Data points represent the mean of three independent measurements. Error bars represent the SEM. The experiment was repeated three times and the results were similar. ( B ) Relationship between fALOD4 binding and ABO blood group antigens (upper panel) and Rhesus blood group (Rh antigen) (lower panel). RBC fALOD4 binding was determined in triplicate using RBCs from 178 White blood donors as described in the Materials and methods. Each fALOD4 binding measurement was performed in triplicate and values were normalized to the reference blood sample. Boxes represent the 25th and 75th percentiles and whiskers represent the minimum and maximum measurements. *p

    Techniques Used: Binding Assay, Isolation, Labeling, SDS Page

    34) Product Images from "Proteomic Analysis of Virus-Host Interactions in an Infectious Context Using Recombinant Viruses *"

    Article Title: Proteomic Analysis of Virus-Host Interactions in an Infectious Context Using Recombinant Viruses *

    Journal: Molecular & Cellular Proteomics : MCP

    doi: 10.1074/mcp.M110.007443

    MV-V and MV-N exhibit very distinct lists of cellular binding partners. A , Schematic representation of rMV2/N-STrEP and rMV2/CH-STrEP genomes that encode for MV-N and CH with a C-terminal One-STrEP tag (N-STrEP and CH-STrEP, respectively). B , N-STrEP and CH-STrEP expression was validated by Western blot using anti-N antibodies or Streptavidin-HRP conjugates. C , Single-step growth curves of rMV2/N-STrEP and rMV2/CH-STrEP. D , Purified protein complexes analysis for N-STrEP and CH-STrEP by SDS-PAGE followed by silver staining. E , Overlap between protein sets identified as MV-V or MV-N interacting partners.
    Figure Legend Snippet: MV-V and MV-N exhibit very distinct lists of cellular binding partners. A , Schematic representation of rMV2/N-STrEP and rMV2/CH-STrEP genomes that encode for MV-N and CH with a C-terminal One-STrEP tag (N-STrEP and CH-STrEP, respectively). B , N-STrEP and CH-STrEP expression was validated by Western blot using anti-N antibodies or Streptavidin-HRP conjugates. C , Single-step growth curves of rMV2/N-STrEP and rMV2/CH-STrEP. D , Purified protein complexes analysis for N-STrEP and CH-STrEP by SDS-PAGE followed by silver staining. E , Overlap between protein sets identified as MV-V or MV-N interacting partners.

    Techniques Used: Binding Assay, Strep-tag, Expressing, Western Blot, Purification, SDS Page, Silver Staining

    Characterization of recombinant viruses expressing STrEP-V or STrEP-CH proteins. A , Vero cells were infected with the native MV Schwarz strain, or the rMV2/STrEP-CH expressing the One-STrEP-tagged CH protein, or the rMV2/STrEP-V expressing the One-STrEP-tagged MV-V protein. Expression of native and One-STrEP-tagged MV-V proteins was determined by Western blot using anti-V monoclonal antibodies ( top panel). Expression of One-STrEP-tagged CH or MV-V proteins was determined by Western blot using Streptavidin-HRP conjugates ( lower panel). B , Fluorescent microscopy showing efficiency of CH protein expression in HEK293T 24 h postinfection by rMV2/STrEP-CH. Fluorescence images were taken with a 10 × objective. White scale bar correspond to 100 μm. C , Single-step growth curves obtained for rMV2/STrEP-V and rMV2/STrEP-CH. MV Schwarz strain was used as a control. Vero cells in 6-well dishes were infected with MV Schwarz, rMV2/STrEP-V, or rMV2/STrEP-CH at an MOI of 0.1. Cell-associated virions were recovered at each time point, and titers were determined using the TCID50 method.
    Figure Legend Snippet: Characterization of recombinant viruses expressing STrEP-V or STrEP-CH proteins. A , Vero cells were infected with the native MV Schwarz strain, or the rMV2/STrEP-CH expressing the One-STrEP-tagged CH protein, or the rMV2/STrEP-V expressing the One-STrEP-tagged MV-V protein. Expression of native and One-STrEP-tagged MV-V proteins was determined by Western blot using anti-V monoclonal antibodies ( top panel). Expression of One-STrEP-tagged CH or MV-V proteins was determined by Western blot using Streptavidin-HRP conjugates ( lower panel). B , Fluorescent microscopy showing efficiency of CH protein expression in HEK293T 24 h postinfection by rMV2/STrEP-CH. Fluorescence images were taken with a 10 × objective. White scale bar correspond to 100 μm. C , Single-step growth curves obtained for rMV2/STrEP-V and rMV2/STrEP-CH. MV Schwarz strain was used as a control. Vero cells in 6-well dishes were infected with MV Schwarz, rMV2/STrEP-V, or rMV2/STrEP-CH at an MOI of 0.1. Cell-associated virions were recovered at each time point, and titers were determined using the TCID50 method.

    Techniques Used: Recombinant, Expressing, Infection, Western Blot, Microscopy, Fluorescence

    35) Product Images from "In vivo assessment of two endothelialization approaches on bioprosthetic valves for the treatment of chronic deep venous insufficiency"

    Article Title: In vivo assessment of two endothelialization approaches on bioprosthetic valves for the treatment of chronic deep venous insufficiency

    Journal: Journal of biomedical materials research. Part B, Applied biomaterials

    doi: 10.1002/jbm.b.33507

    Biotinylation of anti-KDR antibody. (a) Ponceau stain indicates the total protein of the heavy and light chains for both the unmodified anti-KDR antibody (column 1) and the biotinylated anti-KDR antibody (column 2). (b) Streptavidin HRP preferentially bound to the heavy chain of the biotinylated anti-KDR antibody. Activity of the biotinylated antibodies was confirmed with staining of EOCs. (c) The biotinylated anti-KDR antibody adhered to fixed EOCs as detected by AlexaFluor 568-conjugated streptavidin (top) with DAPI indicating total cells (bottom). Inset contains negative control of unmodified anti-KDR stained EOCs. Scale bars = 100 μm.
    Figure Legend Snippet: Biotinylation of anti-KDR antibody. (a) Ponceau stain indicates the total protein of the heavy and light chains for both the unmodified anti-KDR antibody (column 1) and the biotinylated anti-KDR antibody (column 2). (b) Streptavidin HRP preferentially bound to the heavy chain of the biotinylated anti-KDR antibody. Activity of the biotinylated antibodies was confirmed with staining of EOCs. (c) The biotinylated anti-KDR antibody adhered to fixed EOCs as detected by AlexaFluor 568-conjugated streptavidin (top) with DAPI indicating total cells (bottom). Inset contains negative control of unmodified anti-KDR stained EOCs. Scale bars = 100 μm.

    Techniques Used: Staining, Activity Assay, Negative Control

    36) Product Images from "AMPAR interacting protein CPT1C enhances surface expression of GluA1-containing receptors"

    Article Title: AMPAR interacting protein CPT1C enhances surface expression of GluA1-containing receptors

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2014.00469

    GluA1 palmitoylation state is not altered by CPT1C and does not affect the interaction with GluA1. (A) Palmitoylation levels of GluA1 alone (GluA1) and together with CPT1C-GFP (GluA1+CPT1C), in transfected tsA201 cells, detected by means of Acyl-Biotin Exchange (ABE). The thiol-biotinylated immunoprecipitates of GluA1 following the ABE assay for both transfected conditions were subjected to SDS-PAGE. Palmitoylation of GluA1 can only be detected in plus-hydroxilamine (+HAM) samples. Minus -HAM samples control non-specific incorporation of biotin. GluA1 palmitoylation levels (top) were detected by Western blotting with streptavidin-HRP (palmitoylation). After stripping the membranes the total amount of immunoprecipitated GluA1 was detected by Western blotting with anti-GluA1-NT antibody (anti-GluA1, bottom). (B) Quantification of palmitoylation levels for GluA1 alone (open circles) or GluA1 plus CPT1C (filled circles) in tsA201 cells. Ratio of palmitoylated GluA1 to total GluA1 for each single experiment is shown together with mean (discontinuous horizontal lines) and SEM (continuous vertical lines) ( p > 0.05; Mann–Whitney U -test; n = 8 for both). (C) Co-IP of the membranous fraction of tsA201 cells co-expressing GluA1 wild type or non-palmitoylable mutants—GluA1(C585S), GluA1(C811S), and GluA1(C585,811S)—together with CPT1C-GFP. The interaction between CPT1C and GluA1 is not dependent on palmitoylation of C585 or C811 residues. As negative controls GluA1 was co-expressed with an empty plasmid expressing GFP alone (first lanes of the boxes) and CPT1C-GFP was co-expressed with an empty pDsRed (second lanes from the boxes). Transfected cells were lysed and membranes were solubilized as described in Figure 1 and methods. An input sample collected prior to immunoprecipitation of these extracts is shown as “INPUT.” Inputs and immunoprecipitated samples were separated and Western Blotted as described in Figure 1 . Immunoprecipitations were replicated three times.
    Figure Legend Snippet: GluA1 palmitoylation state is not altered by CPT1C and does not affect the interaction with GluA1. (A) Palmitoylation levels of GluA1 alone (GluA1) and together with CPT1C-GFP (GluA1+CPT1C), in transfected tsA201 cells, detected by means of Acyl-Biotin Exchange (ABE). The thiol-biotinylated immunoprecipitates of GluA1 following the ABE assay for both transfected conditions were subjected to SDS-PAGE. Palmitoylation of GluA1 can only be detected in plus-hydroxilamine (+HAM) samples. Minus -HAM samples control non-specific incorporation of biotin. GluA1 palmitoylation levels (top) were detected by Western blotting with streptavidin-HRP (palmitoylation). After stripping the membranes the total amount of immunoprecipitated GluA1 was detected by Western blotting with anti-GluA1-NT antibody (anti-GluA1, bottom). (B) Quantification of palmitoylation levels for GluA1 alone (open circles) or GluA1 plus CPT1C (filled circles) in tsA201 cells. Ratio of palmitoylated GluA1 to total GluA1 for each single experiment is shown together with mean (discontinuous horizontal lines) and SEM (continuous vertical lines) ( p > 0.05; Mann–Whitney U -test; n = 8 for both). (C) Co-IP of the membranous fraction of tsA201 cells co-expressing GluA1 wild type or non-palmitoylable mutants—GluA1(C585S), GluA1(C811S), and GluA1(C585,811S)—together with CPT1C-GFP. The interaction between CPT1C and GluA1 is not dependent on palmitoylation of C585 or C811 residues. As negative controls GluA1 was co-expressed with an empty plasmid expressing GFP alone (first lanes of the boxes) and CPT1C-GFP was co-expressed with an empty pDsRed (second lanes from the boxes). Transfected cells were lysed and membranes were solubilized as described in Figure 1 and methods. An input sample collected prior to immunoprecipitation of these extracts is shown as “INPUT.” Inputs and immunoprecipitated samples were separated and Western Blotted as described in Figure 1 . Immunoprecipitations were replicated three times.

    Techniques Used: Transfection, SDS Page, Western Blot, Stripping Membranes, Immunoprecipitation, MANN-WHITNEY, Co-Immunoprecipitation Assay, Expressing, Plasmid Preparation

    37) Product Images from "Phospholipase D2 Modulates the Secretory Pathway in RBL-2H3 Mast Cells"

    Article Title: Phospholipase D2 Modulates the Secretory Pathway in RBL-2H3 Mast Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0139888

    ManNAz is incorporated in the biosynthetic secretory pathway in RBL-2H3 mast cells. (A) After 3 days of ManNAz incubation, cells were lysed and blotted with streptavidin-HRP. In the control lane RBL-2H3 cell lysate was cultured in the absence of ManNAz. (B) ManNAz was incorporated in all cell lines, with a higher degree in PLD2CA and PLD2CI cells. P = 0.0148. (C) The cell lysates were applied to polyacrylamide gels and stained with Coomassie blue to verify equal protein loading. (D) RBL-2H3 cells were incubated with ManNAz for 1 or 3 days. ManNAz-Alexa 488 was localized at the plasma membrane (arrows). The right column shows one plane of the confocal image, to better visualize ManNAz-Alexa 488 on the membrane (Bars: 10μm). (E) RBL-2H3 cells were incubated with ManNAz for 1 or 3 days. To visualize ManNAz subcellular localization cells were permeabilized. ManNAz-Alexa 488 can be seen in a juxtanuclear region and inside cytoplasmic vesicles (arrows). The right column shows one plane of the confocal image, to better visualize ManNAz-Alexa 488 inside the cell (Bars: 10μm). (F) ManNAz-Alexa 488 colocalizes with GD1b derived gangliosides labeled with anti-mouse IgG conjugated to Alexa 594 on the plasma membrane (Bars: 10μm).
    Figure Legend Snippet: ManNAz is incorporated in the biosynthetic secretory pathway in RBL-2H3 mast cells. (A) After 3 days of ManNAz incubation, cells were lysed and blotted with streptavidin-HRP. In the control lane RBL-2H3 cell lysate was cultured in the absence of ManNAz. (B) ManNAz was incorporated in all cell lines, with a higher degree in PLD2CA and PLD2CI cells. P = 0.0148. (C) The cell lysates were applied to polyacrylamide gels and stained with Coomassie blue to verify equal protein loading. (D) RBL-2H3 cells were incubated with ManNAz for 1 or 3 days. ManNAz-Alexa 488 was localized at the plasma membrane (arrows). The right column shows one plane of the confocal image, to better visualize ManNAz-Alexa 488 on the membrane (Bars: 10μm). (E) RBL-2H3 cells were incubated with ManNAz for 1 or 3 days. To visualize ManNAz subcellular localization cells were permeabilized. ManNAz-Alexa 488 can be seen in a juxtanuclear region and inside cytoplasmic vesicles (arrows). The right column shows one plane of the confocal image, to better visualize ManNAz-Alexa 488 inside the cell (Bars: 10μm). (F) ManNAz-Alexa 488 colocalizes with GD1b derived gangliosides labeled with anti-mouse IgG conjugated to Alexa 594 on the plasma membrane (Bars: 10μm).

    Techniques Used: Incubation, Cell Culture, Staining, Derivative Assay, Labeling

    38) Product Images from "An improved smaller biotin ligase for BioID proximity labeling"

    Article Title: An improved smaller biotin ligase for BioID proximity labeling

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E15-12-0844

    Promiscuous biotinylation by BioID2. (A) The dimensions of E. coli (left; PDB ID 1BIA) and A. aeolicus (right; PDB ID 2EAY) biotin ligases based on prior structural analyses. The catalytic (yellow), ATP-binding (green), and DNA-binding (red) domains. (B) BioID and BioID2 were fused with LaA and expressed in HEK293 cells. Fusion proteins were detected by specific antibodies against BioID or BioID2, respectively (red). Biotinylated proteins were labeled with streptavidin (green). DNA was labeled with Hoechst dye 33258 (blue). Scale bar, 10 μm. (C) Proteins biotinylated by BioID-LaA, BioID2-LaA, BioID-only, or BioID2-only were detected with HRP-conjugated streptavidin after SDS–PAGE separation. Expression of either promiscuous ligase leads to biotinylation of endogenous proteins (left). Fusion proteins were detected with anti-myc antibodies (right). (D) BioID-human Sun2 or BioID2-human Sun2 were transiently expressed in NIH3T3 cells. Fusion proteins were detected using an anti-Sun2 antibody incapable of detecting murine Sun2. Scale bar, 10 μm. (E) The NE/ER ratio of the mean intensity of BioID-human Sun2 or BioID2-human Sun2 detected with anti-human Sun2. Values are mean ± SEM. We measured 48 nuclei/condition.
    Figure Legend Snippet: Promiscuous biotinylation by BioID2. (A) The dimensions of E. coli (left; PDB ID 1BIA) and A. aeolicus (right; PDB ID 2EAY) biotin ligases based on prior structural analyses. The catalytic (yellow), ATP-binding (green), and DNA-binding (red) domains. (B) BioID and BioID2 were fused with LaA and expressed in HEK293 cells. Fusion proteins were detected by specific antibodies against BioID or BioID2, respectively (red). Biotinylated proteins were labeled with streptavidin (green). DNA was labeled with Hoechst dye 33258 (blue). Scale bar, 10 μm. (C) Proteins biotinylated by BioID-LaA, BioID2-LaA, BioID-only, or BioID2-only were detected with HRP-conjugated streptavidin after SDS–PAGE separation. Expression of either promiscuous ligase leads to biotinylation of endogenous proteins (left). Fusion proteins were detected with anti-myc antibodies (right). (D) BioID-human Sun2 or BioID2-human Sun2 were transiently expressed in NIH3T3 cells. Fusion proteins were detected using an anti-Sun2 antibody incapable of detecting murine Sun2. Scale bar, 10 μm. (E) The NE/ER ratio of the mean intensity of BioID-human Sun2 or BioID2-human Sun2 detected with anti-human Sun2. Values are mean ± SEM. We measured 48 nuclei/condition.

    Techniques Used: Binding Assay, Labeling, SDS Page, Expressing

    An extended flexible linker increases the number of candidates detected by Nup43-BioID2. (A) Linear model of Nup43-BioID2 and Nup43-Linker-BioID2 fusion proteins. An extended flexible linker consisting of 13 repeats of GGGGS predicted to provide an ∼25-nm extension was inserted between the Nup43 bait and BioID2 ligase. (B) Expression of Nup43-BioID2 or Nup43-Linker-BioID2 led to biotinylation of endogenous proteins at the NPC. NPCs were labeled using an anti-Nup153 antibody (red). Biotinylated proteins were detected with streptavidin (green). DNA was labeled with Hoechst dye 33258 (blue). Images were taken at the surface of the NE by confocal microscopy. Scale bar, 10 μm. (C) Proteins biotinylated by Nup43-BioID2 and Nup43-Linker-BioID2 were detected with HRP-conjugated streptavidin (top). Fusion proteins were labeled with anti-HA antibody (bottom). (D) Nup107–Nup160 complex candidates identified by both Nup43-BioID2 (middle) and Nup43-Linker-BioID2 (right) are labeled gray. Uniquely detected candidates are colored in green, and fusion proteins are indicated with blue. (E) For the entire NPC, Nups identified by both Nup43-BioID2 (left) and Nup43-Linker-BioID2 (right) are labeled gray. Uniquely detected candidates are colored in green, and fusion proteins are indicated with patterned blue.
    Figure Legend Snippet: An extended flexible linker increases the number of candidates detected by Nup43-BioID2. (A) Linear model of Nup43-BioID2 and Nup43-Linker-BioID2 fusion proteins. An extended flexible linker consisting of 13 repeats of GGGGS predicted to provide an ∼25-nm extension was inserted between the Nup43 bait and BioID2 ligase. (B) Expression of Nup43-BioID2 or Nup43-Linker-BioID2 led to biotinylation of endogenous proteins at the NPC. NPCs were labeled using an anti-Nup153 antibody (red). Biotinylated proteins were detected with streptavidin (green). DNA was labeled with Hoechst dye 33258 (blue). Images were taken at the surface of the NE by confocal microscopy. Scale bar, 10 μm. (C) Proteins biotinylated by Nup43-BioID2 and Nup43-Linker-BioID2 were detected with HRP-conjugated streptavidin (top). Fusion proteins were labeled with anti-HA antibody (bottom). (D) Nup107–Nup160 complex candidates identified by both Nup43-BioID2 (middle) and Nup43-Linker-BioID2 (right) are labeled gray. Uniquely detected candidates are colored in green, and fusion proteins are indicated with blue. (E) For the entire NPC, Nups identified by both Nup43-BioID2 (left) and Nup43-Linker-BioID2 (right) are labeled gray. Uniquely detected candidates are colored in green, and fusion proteins are indicated with patterned blue.

    Techniques Used: Expressing, Labeling, Confocal Microscopy

    Application of BioID2 to the human Nup107–Nup160 complex. (A) Expression of Nup43-BioID or Nup43-BioID2 biotinylated endogenous proteins at the NPC. The NPCs were labeled with an anti-Nup153 antibody (red). Biotinylated proteins were detected with streptavidin (green). DNA was labeled with Hoechst dye 33258 (blue). To observe more clearly the NPCs, confocal images were taken at the surface of the NE. Scale bar, 10 μm. (B) Proteins biotinylated by Nup43-BioID and Nup43-BioID2 were detected with HRP-conjugated streptavidin (top). Fusion proteins were detected with anti-HA antibody (middle). BioID- or BioID2-only controls were detected by an anti-myc antibody (bottom). Asterisk indicates predicted migration of Nup96 and Nup107. (C) Model of the Nup107–Nup160 complex based on the previous literature and resolved structures ( Hoelz et al. , 2011 ; Bui et al. , 2013 ). Candidates identified by both Nup43-BioID (middle) and Nup43-BioID2 (right) are labeled gray. Uniquely detected candidates are colored in green, and fusion proteins are indicated with blue. Modified from Kim et al. (2014) . (D) The full range of NPC candidates, with those identified by both Nup43-BioID (left) and Nup43-BioID2 (right) labeled gray. Uniquely detected candidates are colored in green, and fusion proteins are indicated with patterned blue. Modified from Kim et al. (2014) .
    Figure Legend Snippet: Application of BioID2 to the human Nup107–Nup160 complex. (A) Expression of Nup43-BioID or Nup43-BioID2 biotinylated endogenous proteins at the NPC. The NPCs were labeled with an anti-Nup153 antibody (red). Biotinylated proteins were detected with streptavidin (green). DNA was labeled with Hoechst dye 33258 (blue). To observe more clearly the NPCs, confocal images were taken at the surface of the NE. Scale bar, 10 μm. (B) Proteins biotinylated by Nup43-BioID and Nup43-BioID2 were detected with HRP-conjugated streptavidin (top). Fusion proteins were detected with anti-HA antibody (middle). BioID- or BioID2-only controls were detected by an anti-myc antibody (bottom). Asterisk indicates predicted migration of Nup96 and Nup107. (C) Model of the Nup107–Nup160 complex based on the previous literature and resolved structures ( Hoelz et al. , 2011 ; Bui et al. , 2013 ). Candidates identified by both Nup43-BioID (middle) and Nup43-BioID2 (right) are labeled gray. Uniquely detected candidates are colored in green, and fusion proteins are indicated with blue. Modified from Kim et al. (2014) . (D) The full range of NPC candidates, with those identified by both Nup43-BioID (left) and Nup43-BioID2 (right) labeled gray. Uniquely detected candidates are colored in green, and fusion proteins are indicated with patterned blue. Modified from Kim et al. (2014) .

    Techniques Used: Expressing, Labeling, Migration, Modification

    39) Product Images from "Homodimerization of the Lymph Vessel Endothelial Receptor LYVE-1 through a Redox-labile Disulfide Is Critical for Hyaluronan Binding in Lymphatic Endothelium *"

    Article Title: Homodimerization of the Lymph Vessel Endothelial Receptor LYVE-1 through a Redox-labile Disulfide Is Critical for Hyaluronan Binding in Lymphatic Endothelium *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.736926

    The Cys-201 disulfide linkage in the LYVE-1 homodimer is highly labile. The sensitivity to reduction of the Cys-201 intermolecular disulfide was investigated after exposure of soluble hLYVE-1 homodimers to varying concentrations of TCEP and assessment of the consequences for both HA binding and dimer disassembly ( A–C ) A and B , concentration-dependent effects of TCEP treatment on HMW bHA binding to immobilized hLYVE-1 (Δ238 His 10 ) homodimers and hLYVE-1 (Δ238 His 10 ) monomer (non-dimerizing C201A mutant), respectively, as detected with a streptavidin HRP conjugate. Values in each case are the mean ± S.E. n = 3. Statistics p values were obtained using the two-tailed unpaired t test ( n.s. , non-significant). C , non-reducing SDS-PAGE analysis showing concentration-dependent effects of TCEP treatment on soluble homodimer disassembly assessed by Western blotting with LYVE-1 polyclonal antibody and green IRdye® 800 conjugate. D , schematic showing the location and numbering of individual cysteines ( yellow circles ) forming Link domain disulfides in the LYVE-1 homodimer; N - and O -linked glycan chains are colored green and red , respectively. Data shown are from representative experiments that were repeated three times.
    Figure Legend Snippet: The Cys-201 disulfide linkage in the LYVE-1 homodimer is highly labile. The sensitivity to reduction of the Cys-201 intermolecular disulfide was investigated after exposure of soluble hLYVE-1 homodimers to varying concentrations of TCEP and assessment of the consequences for both HA binding and dimer disassembly ( A–C ) A and B , concentration-dependent effects of TCEP treatment on HMW bHA binding to immobilized hLYVE-1 (Δ238 His 10 ) homodimers and hLYVE-1 (Δ238 His 10 ) monomer (non-dimerizing C201A mutant), respectively, as detected with a streptavidin HRP conjugate. Values in each case are the mean ± S.E. n = 3. Statistics p values were obtained using the two-tailed unpaired t test ( n.s. , non-significant). C , non-reducing SDS-PAGE analysis showing concentration-dependent effects of TCEP treatment on soluble homodimer disassembly assessed by Western blotting with LYVE-1 polyclonal antibody and green IRdye® 800 conjugate. D , schematic showing the location and numbering of individual cysteines ( yellow circles ) forming Link domain disulfides in the LYVE-1 homodimer; N - and O -linked glycan chains are colored green and red , respectively. Data shown are from representative experiments that were repeated three times.

    Techniques Used: Binding Assay, Concentration Assay, Mutagenesis, Two Tailed Test, SDS Page, Western Blot

    LYVE-1 homodimerization greatly increase HA binding affinity. Binding affinities of soluble hLYVE-1 Δ238 monomer (C201A non-dimerizing mutant) and homodimer derived from transfected CHO cells ( A and B ) were determined by surface plasmon resonance analysis with HMW HA immobilized on the sensor chip ( C and D ) and by microtiter plate analysis ( E ) with immobilized receptor and bHA. A , SDS-PAGE analysis of the purified hLYVE-1 monomer and dimer under non-reducing conditions, detected by staining with Coomassie Blue. B , Superdex-200 size exclusion chromatography profiles of purified monomer and dimer preparations with elution positions of molecular weight calibration markers shown at top. C , Biacore sensorgram and the associated binding curve with Scatchard plot ( inset ; RU , response units) for hLYVE-1 monomer measured at equilibrium with varying analyte concentrations (0.5–128 μ m ). The K D value was determined from independent replicate analyses (mean ± S.E. n = 3) by fitting the data to a 1:1 Langmuir binding isotherm. The Scatchard plot indicates this binding model fits the data well. D , Biacore sensorgram for hLYVE-1 homodimer measured as a single-cycle kinetic experiment using sequential injections at LYVE-1 concentrations of 12.8, 64, and 320 n m ( purple line ). Evaluation and fitting was performed using Biaevaluation software assuming bivalency for the analyte ( black line ). Further analysis including rationalization of the number of parameters is included in the supplemental information . E , analysis of bHA binding to hLYVE-1 Δ238 monomer (non-dimerizing C201A mutant) or intact LYVE-1 Δ238 homodimer immobilized on a microtiter plate, detected colorimetrically with streptavidin HRP (absorbance 490 nm) as described under “Experimental Procedures.” Values are the mean ± S.E. n = 3. Data shown are from representative experiments that were repeated at least three times.
    Figure Legend Snippet: LYVE-1 homodimerization greatly increase HA binding affinity. Binding affinities of soluble hLYVE-1 Δ238 monomer (C201A non-dimerizing mutant) and homodimer derived from transfected CHO cells ( A and B ) were determined by surface plasmon resonance analysis with HMW HA immobilized on the sensor chip ( C and D ) and by microtiter plate analysis ( E ) with immobilized receptor and bHA. A , SDS-PAGE analysis of the purified hLYVE-1 monomer and dimer under non-reducing conditions, detected by staining with Coomassie Blue. B , Superdex-200 size exclusion chromatography profiles of purified monomer and dimer preparations with elution positions of molecular weight calibration markers shown at top. C , Biacore sensorgram and the associated binding curve with Scatchard plot ( inset ; RU , response units) for hLYVE-1 monomer measured at equilibrium with varying analyte concentrations (0.5–128 μ m ). The K D value was determined from independent replicate analyses (mean ± S.E. n = 3) by fitting the data to a 1:1 Langmuir binding isotherm. The Scatchard plot indicates this binding model fits the data well. D , Biacore sensorgram for hLYVE-1 homodimer measured as a single-cycle kinetic experiment using sequential injections at LYVE-1 concentrations of 12.8, 64, and 320 n m ( purple line ). Evaluation and fitting was performed using Biaevaluation software assuming bivalency for the analyte ( black line ). Further analysis including rationalization of the number of parameters is included in the supplemental information . E , analysis of bHA binding to hLYVE-1 Δ238 monomer (non-dimerizing C201A mutant) or intact LYVE-1 Δ238 homodimer immobilized on a microtiter plate, detected colorimetrically with streptavidin HRP (absorbance 490 nm) as described under “Experimental Procedures.” Values are the mean ± S.E. n = 3. Data shown are from representative experiments that were repeated at least three times.

    Techniques Used: Binding Assay, Mutagenesis, Derivative Assay, Transfection, SPR Assay, Chromatin Immunoprecipitation, SDS Page, Purification, Staining, Size-exclusion Chromatography, Molecular Weight, Software

    40) Product Images from "Discovery of a Low Toxicity O-GlcNAc Transferase (OGT) Inhibitor by Structure-based Virtual Screening of Natural Products"

    Article Title: Discovery of a Low Toxicity O-GlcNAc Transferase (OGT) Inhibitor by Structure-based Virtual Screening of Natural Products

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-12522-0

    L01 acts in cells to inhibit OGT and does not grossly perturb cell-surface glycan structures. ( a ) Western blots of COS7 cell lysates after L01 treatment at different doses (0–150 μM) for 24 h. OSMI-1 (0–100 μM) was used as a positive control. ( b ) Immunocytochemistry of COS7 cells treated with 0, 50 μM or 100 μM L01 for 24 h. Immunoreactivity from antibody to O-GlcNAc CTD110.6 is green and DAPI is blue. ( c ) COS7 cells were metabolic labeled with UDP-GlcNAz (GlcNAz) and then administered with L01 (100 μM) or DMSO for 24 h. COS7 cells which did not metabolic label were used as a negative control. All the cells were harvested and then underwent Click reaction with biotin-alkyne. Blots were probed with streptavidin-HRP. ( d ) Western blots of COS7 cell lysates after L01 treatment at 50 μM for indicated times. OSMI-1 (50 μM) was used as a positive control. ( e ) Western blots of immunoprecipitated Nup62 from cell lysates after DMSO (C) or 100 μM L01 treatment for 24 h. After immunoprecipitation, Nup62 was incubated with UDP-GalNAz in the presence of mutant GalT, and chemoselectively labeled. ( f ) Lectin blots of COS7 cell lysates after L01 or OSMI-1 treatment at 50 μM for 24 h.
    Figure Legend Snippet: L01 acts in cells to inhibit OGT and does not grossly perturb cell-surface glycan structures. ( a ) Western blots of COS7 cell lysates after L01 treatment at different doses (0–150 μM) for 24 h. OSMI-1 (0–100 μM) was used as a positive control. ( b ) Immunocytochemistry of COS7 cells treated with 0, 50 μM or 100 μM L01 for 24 h. Immunoreactivity from antibody to O-GlcNAc CTD110.6 is green and DAPI is blue. ( c ) COS7 cells were metabolic labeled with UDP-GlcNAz (GlcNAz) and then administered with L01 (100 μM) or DMSO for 24 h. COS7 cells which did not metabolic label were used as a negative control. All the cells were harvested and then underwent Click reaction with biotin-alkyne. Blots were probed with streptavidin-HRP. ( d ) Western blots of COS7 cell lysates after L01 treatment at 50 μM for indicated times. OSMI-1 (50 μM) was used as a positive control. ( e ) Western blots of immunoprecipitated Nup62 from cell lysates after DMSO (C) or 100 μM L01 treatment for 24 h. After immunoprecipitation, Nup62 was incubated with UDP-GalNAz in the presence of mutant GalT, and chemoselectively labeled. ( f ) Lectin blots of COS7 cell lysates after L01 or OSMI-1 treatment at 50 μM for 24 h.

    Techniques Used: Western Blot, Positive Control, Immunocytochemistry, Labeling, Negative Control, Immunoprecipitation, Incubation, Mutagenesis

    41) Product Images from "Expression and in vitro Function of ?1-lntegrin Laminin Receptors in the Developing Avian Ciliary Ganglion"

    Article Title: Expression and in vitro Function of ?1-lntegrin Laminin Receptors in the Developing Avian Ciliary Ganglion

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

    doi:

    A, Integrin expression by E7-8 CG and purified CG neurons cultured on laminin-1. Whole CG ( lanes 2, 4, 6, 9, 12, 14 ), CG membranes ( lane 16 ), CG neurons plated on laminin-1 ( lanes 1, 5, 8, 11, 13 ), E11 myotubes ( lane 3 ), E11 myotube membranes ( lane 17 ), thrombocytes ( lanes 7, 10 ) and E6–8 retinae ( lane 15 ) were extracted in sample buffer ( lanes 1, 2, 5–17 ) or 1% Triton X-100 ( lanes 3, 4 ) as described in Materials and Methods. Approximately equal amounts of protein were loaded per lane for each blot. Blots were incubated with polyclonal antibodies against α 1 ( lanes 3, 4 ), α 2 cyto (preimmune, lanes 5, 6, 7 ; immune, lanes 8, 9, 10 ), α 3 (Ex2, lanes 11, 12 ), α 6 (Ex, lanes 13, 14, 15 ), or monoclonal antibodies against β 1 (TASC, lanes 1, 2 ) or α 7 (H1, lanes 16, 17 ). Immunoreactivity was visualized with alkaline phosphatase-conjugated anti-rabbit or anti-mouse antibodies. Arrows indicate bands corresponding to each subunit. The doublets around 97 kDa in lanes 3 and 4 are probably breakdown products of α 1 ). The band at 190 kDa in lanes 5 (preimmune) and 8 (immune) is nonspecific. The lower M r band in lane 17 is a common breakdown product of α 7 ). Numbers denote positions of M r marker proteins in kilodaltons. pi , Preimmune. B , α 6 and α 3 heterodimerize with β 1 in CG neurons. β 1 -containing integrin heterodimers were immunoprecipitated from surface-biotinylated CG cells with anti-β 1 (W1B10) mAb coupled to protein A–Sepharose. Immunoprecipitates were visualized directly by chemiluminescent detection of HRP–streptavidin ( lane 1 , protein A–Sepharose control; lane 2 , antibody-coupled Sepharose), then probed with anti-α 6 Ex ( lanes 3, 4 ). Similar immunoprecipitates were probed with anti-α 3 Ex2 ( ip, lane 5 ). Triton X-100 extracts of chick breast fibroblasts ( CEF, lane 6 ) and whole ciliary ganglia ( CG, lane 7 ) were also blotted as positive controls. Arrows indicate bands corresponding to α 6 or α 3 . Numbers indicate M r in kilodaltons.
    Figure Legend Snippet: A, Integrin expression by E7-8 CG and purified CG neurons cultured on laminin-1. Whole CG ( lanes 2, 4, 6, 9, 12, 14 ), CG membranes ( lane 16 ), CG neurons plated on laminin-1 ( lanes 1, 5, 8, 11, 13 ), E11 myotubes ( lane 3 ), E11 myotube membranes ( lane 17 ), thrombocytes ( lanes 7, 10 ) and E6–8 retinae ( lane 15 ) were extracted in sample buffer ( lanes 1, 2, 5–17 ) or 1% Triton X-100 ( lanes 3, 4 ) as described in Materials and Methods. Approximately equal amounts of protein were loaded per lane for each blot. Blots were incubated with polyclonal antibodies against α 1 ( lanes 3, 4 ), α 2 cyto (preimmune, lanes 5, 6, 7 ; immune, lanes 8, 9, 10 ), α 3 (Ex2, lanes 11, 12 ), α 6 (Ex, lanes 13, 14, 15 ), or monoclonal antibodies against β 1 (TASC, lanes 1, 2 ) or α 7 (H1, lanes 16, 17 ). Immunoreactivity was visualized with alkaline phosphatase-conjugated anti-rabbit or anti-mouse antibodies. Arrows indicate bands corresponding to each subunit. The doublets around 97 kDa in lanes 3 and 4 are probably breakdown products of α 1 ). The band at 190 kDa in lanes 5 (preimmune) and 8 (immune) is nonspecific. The lower M r band in lane 17 is a common breakdown product of α 7 ). Numbers denote positions of M r marker proteins in kilodaltons. pi , Preimmune. B , α 6 and α 3 heterodimerize with β 1 in CG neurons. β 1 -containing integrin heterodimers were immunoprecipitated from surface-biotinylated CG cells with anti-β 1 (W1B10) mAb coupled to protein A–Sepharose. Immunoprecipitates were visualized directly by chemiluminescent detection of HRP–streptavidin ( lane 1 , protein A–Sepharose control; lane 2 , antibody-coupled Sepharose), then probed with anti-α 6 Ex ( lanes 3, 4 ). Similar immunoprecipitates were probed with anti-α 3 Ex2 ( ip, lane 5 ). Triton X-100 extracts of chick breast fibroblasts ( CEF, lane 6 ) and whole ciliary ganglia ( CG, lane 7 ) were also blotted as positive controls. Arrows indicate bands corresponding to α 6 or α 3 . Numbers indicate M r in kilodaltons.

    Techniques Used: Expressing, Purification, Cell Culture, Incubation, Marker, Immunoprecipitation

    42) Product Images from "Surface Cathepsin B Protects Cytotoxic Lymphocytes from Self-destruction after Degranulation"

    Article Title: Surface Cathepsin B Protects Cytotoxic Lymphocytes from Self-destruction after Degranulation

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20011836

    Surface cathepsin B on TCR-activated T cells is active and the target of NS-196. (A) Detection of biotinylated NS-196 on the CTL surface after degranulation. Flow cytometry of CD8 + cloned human CTL RS-56 after culture on wells coated with anti-CD3 or isotope control for 2 h, followed by treatment with or without 1 μM NS-196, which was detected by FITC-streptavidin. (B) Identification of cathepsin B as the molecular target of NS-196. CTL clone RS-56 was incubated for 2 h on anti-CD3– or IgG-coated wells, followed by incubation with or without 0.1 μM NS-196. Cells were lysed with Triton X-100 and immunodepleted with beads containing anticathepsin B antibody or control rabbit IgG. The remaining lysate was run on a 12% nonreduced SDS gel, blotted onto nitrocellulose, probed with Streptavidin-HRP, and developed using ECL. The right lane shows the biotinylation pattern when the whole CTL lysate was labeled with 0.1 μM NS-196 and run directly (1/10 of the cell-equivalent input of other lanes).
    Figure Legend Snippet: Surface cathepsin B on TCR-activated T cells is active and the target of NS-196. (A) Detection of biotinylated NS-196 on the CTL surface after degranulation. Flow cytometry of CD8 + cloned human CTL RS-56 after culture on wells coated with anti-CD3 or isotope control for 2 h, followed by treatment with or without 1 μM NS-196, which was detected by FITC-streptavidin. (B) Identification of cathepsin B as the molecular target of NS-196. CTL clone RS-56 was incubated for 2 h on anti-CD3– or IgG-coated wells, followed by incubation with or without 0.1 μM NS-196. Cells were lysed with Triton X-100 and immunodepleted with beads containing anticathepsin B antibody or control rabbit IgG. The remaining lysate was run on a 12% nonreduced SDS gel, blotted onto nitrocellulose, probed with Streptavidin-HRP, and developed using ECL. The right lane shows the biotinylation pattern when the whole CTL lysate was labeled with 0.1 μM NS-196 and run directly (1/10 of the cell-equivalent input of other lanes).

    Techniques Used: CTL Assay, Flow Cytometry, Cytometry, Clone Assay, Incubation, SDS-Gel, Labeling

    43) Product Images from "Selenium-Based S-Adenosylmethionine Analog Reveals the Mammalian Seven-Beta-Strand Methyltransferase METTL10 to Be an EF1A1 Lysine Methyltransferase"

    Article Title: Selenium-Based S-Adenosylmethionine Analog Reveals the Mammalian Seven-Beta-Strand Methyltransferase METTL10 to Be an EF1A1 Lysine Methyltransferase

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0105394

    ProSeAM, a synthetic SAM analog, has a wide spectrum of reactivity for histones and non-histone substrates. A, Schematic overview for analyzing lysine methylation. A synthetic cofactor was used to transfer an alkyne moiety to the ε-amino group of lysine by KMTs (1). The modified proteins were tagged with biotin via CuAAC reaction (2). Tagged-proteins in the crude lysates were pulled down with affinity beads (3), and the precipitants were further analyzed with a LC-MS apparatus (4). B, Chemical structure of SAM (1), propargylated SAM (2) and ProSeAM (3). C, H3 peptide (1-21 a.a.) and ProSeAM was incubated with or without GST-G9a at 20°C for 2 h, then the peptide was analyzed by MALDI-TOF MS. D, full-length Histone H3 (1 µg) and ProSeAM (500 µM) were incubated with indicated KMTs (0.5 µg) for 2 h at 20°C. The histones were separated by SDS-PAGE, transferred to a nitrocellulose membrane and probed with streptavidin-HRP (top) or anti-Histone H3 antibody (bottom). E, The non-histone substrates His-HSP90 and His-HSP70 (1 µg) were incubated with His-SMYD2 and His-METTL21A (1 µg), respectively. After the reaction, proteins were separated by SDS-PAGE (right). Their modifications were detected by western blotting with streptavidin-HRP as in Fig. 1D. *and ** showed automodification of SMYD2 and METTL21A, respectively (left). F, His-HSP70 (WT and K561R) were incubated with or without His-METTL21A in the presence of ProSeAM for 2 h at 20°C. Modified proteins were biotinylated and detected with streptavidin-HRP (top) or anti-HSP70 antibody for the loading control (bottom).
    Figure Legend Snippet: ProSeAM, a synthetic SAM analog, has a wide spectrum of reactivity for histones and non-histone substrates. A, Schematic overview for analyzing lysine methylation. A synthetic cofactor was used to transfer an alkyne moiety to the ε-amino group of lysine by KMTs (1). The modified proteins were tagged with biotin via CuAAC reaction (2). Tagged-proteins in the crude lysates were pulled down with affinity beads (3), and the precipitants were further analyzed with a LC-MS apparatus (4). B, Chemical structure of SAM (1), propargylated SAM (2) and ProSeAM (3). C, H3 peptide (1-21 a.a.) and ProSeAM was incubated with or without GST-G9a at 20°C for 2 h, then the peptide was analyzed by MALDI-TOF MS. D, full-length Histone H3 (1 µg) and ProSeAM (500 µM) were incubated with indicated KMTs (0.5 µg) for 2 h at 20°C. The histones were separated by SDS-PAGE, transferred to a nitrocellulose membrane and probed with streptavidin-HRP (top) or anti-Histone H3 antibody (bottom). E, The non-histone substrates His-HSP90 and His-HSP70 (1 µg) were incubated with His-SMYD2 and His-METTL21A (1 µg), respectively. After the reaction, proteins were separated by SDS-PAGE (right). Their modifications were detected by western blotting with streptavidin-HRP as in Fig. 1D. *and ** showed automodification of SMYD2 and METTL21A, respectively (left). F, His-HSP70 (WT and K561R) were incubated with or without His-METTL21A in the presence of ProSeAM for 2 h at 20°C. Modified proteins were biotinylated and detected with streptavidin-HRP (top) or anti-HSP70 antibody for the loading control (bottom).

    Techniques Used: Methylation, Modification, Liquid Chromatography with Mass Spectroscopy, Incubation, Mass Spectrometry, SDS Page, Western Blot

    Proteomic identification of substrates for seven-beta-strand MTases. A, Schematic protocol for proteomic identification. HEK293T cell lysates were added to either propargylic Se-adenosyl- l -selenomethionine (ProSeAM) alone (1) or ProSeAM plus recombinant KMT (lysate:enzyme ratio was 10∶1) (2). After the in vitro reaction, labeled proteins were tagged with biotin and then precipitated with streptavidin beads. The precipitants were then digested with trypsin, and the trypsinized protein fragments were analyzed by LC-MS/MS. B, ProSeAM competes with SAM in the labeling reaction. HEK293T cell lysates were incubated with ProSeAM (250 µM) in the presence or absence of the indicated amount of SAM (0 to 2.5 mM). Modified proteins were biotinylated and detected with streptavidin-HRP (top). Equal protein loading was confirmed by western blotting with anti-α-tubulin antibody (bottom). C, western blot of labeled proteins. A 5% input of precipitated proteins without ProSeAM (1), with ProSeAM alone (2), with ProSeAM plus GST-G9a (3), with ProSeAM plus His-METTL21A (4) or with ProSeAM plus His-METTL10 was separately analyzed with western blotting with streptavidin-HRP (top) prior to the MS analysis, to compare the labeled proteins. Equal protein loading was confirmed by western blotting with anti-α-tubulin antibody (bottom). D, Doughnut chart of the subcellular distribution of proteins labeled with ProSeAM. HEK293T lysates alone (lane 1 in Fig. 2C) and HEK293T lysates with ProSeAM (lane 2 in Fig. 2C) were analyzed as described in A and Experimental procedures (n = 3). In total, 318 proteins were identified as ProSeAM-labeled proteins. E, List of METTL21A substrates. HEK293T cell lysates and ProSeAM were incubated with or without METTL21A (lane 2 and lane 4 in Fig. 2C), and analyzed as above. Molecular weight, peptide area (reflecting the quantity of detected protein), and fold enrichment of the peptide area are listed: ND, not determined because the substrate was detected only in the condition for lane 4 of B. The total numbers of identified proteins, 2-fold increase (compared to control in each experiment), and overlapped identified numbers of 3 independent experiments are listed in Table S2 .
    Figure Legend Snippet: Proteomic identification of substrates for seven-beta-strand MTases. A, Schematic protocol for proteomic identification. HEK293T cell lysates were added to either propargylic Se-adenosyl- l -selenomethionine (ProSeAM) alone (1) or ProSeAM plus recombinant KMT (lysate:enzyme ratio was 10∶1) (2). After the in vitro reaction, labeled proteins were tagged with biotin and then precipitated with streptavidin beads. The precipitants were then digested with trypsin, and the trypsinized protein fragments were analyzed by LC-MS/MS. B, ProSeAM competes with SAM in the labeling reaction. HEK293T cell lysates were incubated with ProSeAM (250 µM) in the presence or absence of the indicated amount of SAM (0 to 2.5 mM). Modified proteins were biotinylated and detected with streptavidin-HRP (top). Equal protein loading was confirmed by western blotting with anti-α-tubulin antibody (bottom). C, western blot of labeled proteins. A 5% input of precipitated proteins without ProSeAM (1), with ProSeAM alone (2), with ProSeAM plus GST-G9a (3), with ProSeAM plus His-METTL21A (4) or with ProSeAM plus His-METTL10 was separately analyzed with western blotting with streptavidin-HRP (top) prior to the MS analysis, to compare the labeled proteins. Equal protein loading was confirmed by western blotting with anti-α-tubulin antibody (bottom). D, Doughnut chart of the subcellular distribution of proteins labeled with ProSeAM. HEK293T lysates alone (lane 1 in Fig. 2C) and HEK293T lysates with ProSeAM (lane 2 in Fig. 2C) were analyzed as described in A and Experimental procedures (n = 3). In total, 318 proteins were identified as ProSeAM-labeled proteins. E, List of METTL21A substrates. HEK293T cell lysates and ProSeAM were incubated with or without METTL21A (lane 2 and lane 4 in Fig. 2C), and analyzed as above. Molecular weight, peptide area (reflecting the quantity of detected protein), and fold enrichment of the peptide area are listed: ND, not determined because the substrate was detected only in the condition for lane 4 of B. The total numbers of identified proteins, 2-fold increase (compared to control in each experiment), and overlapped identified numbers of 3 independent experiments are listed in Table S2 .

    Techniques Used: Recombinant, In Vitro, Labeling, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Incubation, Modification, Western Blot, Molecular Weight

    44) Product Images from "Chemotactic Chemokines Are Important in the Pathogenesis of Irritable Bowel Syndrome"

    Article Title: Chemotactic Chemokines Are Important in the Pathogenesis of Irritable Bowel Syndrome

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0093144

    A representative image of the various biomarkers detected in the sera of IBS patients and healthy volunteers. Each pair of horizontal blots (bands) represent a different biomarker present in the serum, whereas the intensities of the blots characterize the amount of the respective biomarker. Sera (250 μL) from 60 IBS patients (44 idiopathic IBS and 16 post-infectious IBS) and 40 healthy volunteers were evaluated for the presence of 36 different biomarkers using the Proteome Profiler Human Cytokine Array Panel A Kit (R D Systems, Minneapolis, MN). Samples were incubated with a membrane embedded with biotinylated antibodies that are specific for each of the 36 biomarkers analyzed. For detection, the membranes were probed with the Pierce ECL Western Blotting Substrate (Thermo Scientific, Rockford, IL) and exposed to an X-ray film, following incubation with a secondary antibody labelled with streptavidin-horse radish peroxidase. The exposed film was processed using SRX-101A Medical Film Processor (Konica Minolta). Intensities of the blots were determined and expressed as pixel densities using ImageJ (National Institutes of Health, Bethesda, MD).
    Figure Legend Snippet: A representative image of the various biomarkers detected in the sera of IBS patients and healthy volunteers. Each pair of horizontal blots (bands) represent a different biomarker present in the serum, whereas the intensities of the blots characterize the amount of the respective biomarker. Sera (250 μL) from 60 IBS patients (44 idiopathic IBS and 16 post-infectious IBS) and 40 healthy volunteers were evaluated for the presence of 36 different biomarkers using the Proteome Profiler Human Cytokine Array Panel A Kit (R D Systems, Minneapolis, MN). Samples were incubated with a membrane embedded with biotinylated antibodies that are specific for each of the 36 biomarkers analyzed. For detection, the membranes were probed with the Pierce ECL Western Blotting Substrate (Thermo Scientific, Rockford, IL) and exposed to an X-ray film, following incubation with a secondary antibody labelled with streptavidin-horse radish peroxidase. The exposed film was processed using SRX-101A Medical Film Processor (Konica Minolta). Intensities of the blots were determined and expressed as pixel densities using ImageJ (National Institutes of Health, Bethesda, MD).

    Techniques Used: Biomarker Assay, Incubation, Western Blot

    45) Product Images from "Triptolide Directly Inhibits dCTP Pyrophosphatase"

    Article Title: Triptolide Directly Inhibits dCTP Pyrophosphatase

    Journal: Chembiochem : a European journal of chemical biology

    doi: 10.1002/cbic.201100007

    Identification of DCTPP1 as a triptolide target. (A) silver stained gel and (B) streptavidin-HRP blot of soluble protein lysates of HeLa S3 cells exposed to the reagents indicated: triptolide affinity reagent 2 , affinity reagent 2 + 100-fold excess of
    Figure Legend Snippet: Identification of DCTPP1 as a triptolide target. (A) silver stained gel and (B) streptavidin-HRP blot of soluble protein lysates of HeLa S3 cells exposed to the reagents indicated: triptolide affinity reagent 2 , affinity reagent 2 + 100-fold excess of

    Techniques Used: Staining

    46) Product Images from "Capturing in vivo RNA transcriptional dynamics from the malaria parasite Plasmodium falciparum"

    Article Title: Capturing in vivo RNA transcriptional dynamics from the malaria parasite Plasmodium falciparum

    Journal: Genome Research

    doi: 10.1101/gr.217356.116

    Stage-specific pyrimidine salvage for detection of early gametocyte transcription. To measure mRNA from a subpopulation of cells, we expressed FCU-GFP under the control of the gametocyte-specific promoter pfs16 in the 3D7 parasite line (3D7 pfs16 ). ( A ) Schematic representation of the experimental design including the timing of 4-TU incubation (black arrows) and RNA extraction (dashed black arrows), plasmids transfected into P. falciparum strains, and a depiction of the highly synchronous cell populations that express cam- and pfs16-fcu-gfp throughout the 48-h IDC. All parasites express FCU-GFP from the constitutive cam promoter (green) regardless of developmental stage ( left , 100% GFP + ). Only a small proportion (∼1% GFP + ) of parasites becoming committed (dashed highlighted arrows) during the IDC, and those that have entered gametocytogenesis in the previous cycle and are sexually committed rings express FCU-GFP from the pfs16 promoter (green), whereas asexual parasites do not ( right , ∼99% GFP − ). ( B ) FCU-GFP protein from uninduced asexual cultures of 3D7 cam and 3D7 pfs16 was detected by Western blot probed with anti-yeast cytosine deaminase. ( C ) Detection of the subpopulation of FCU-mediated thiol-tagged RNA was carried out by incubating highly synchronized P.f. 3D7, 3D7 cam , and 3D7 pfs16 in the presence or absence ( top ) of 40 µM 4-TU for 12 h. Total RNA was extracted, biotinylated, and assayed by Northern blot probed with Streptavidin-HRP ( bottom ), demonstrating that thiol tagging occurs at a much lower level in 3D7 pfs16 (*) than in 3D7 cam , representing the minor sexual-stage parasite subpopulation.
    Figure Legend Snippet: Stage-specific pyrimidine salvage for detection of early gametocyte transcription. To measure mRNA from a subpopulation of cells, we expressed FCU-GFP under the control of the gametocyte-specific promoter pfs16 in the 3D7 parasite line (3D7 pfs16 ). ( A ) Schematic representation of the experimental design including the timing of 4-TU incubation (black arrows) and RNA extraction (dashed black arrows), plasmids transfected into P. falciparum strains, and a depiction of the highly synchronous cell populations that express cam- and pfs16-fcu-gfp throughout the 48-h IDC. All parasites express FCU-GFP from the constitutive cam promoter (green) regardless of developmental stage ( left , 100% GFP + ). Only a small proportion (∼1% GFP + ) of parasites becoming committed (dashed highlighted arrows) during the IDC, and those that have entered gametocytogenesis in the previous cycle and are sexually committed rings express FCU-GFP from the pfs16 promoter (green), whereas asexual parasites do not ( right , ∼99% GFP − ). ( B ) FCU-GFP protein from uninduced asexual cultures of 3D7 cam and 3D7 pfs16 was detected by Western blot probed with anti-yeast cytosine deaminase. ( C ) Detection of the subpopulation of FCU-mediated thiol-tagged RNA was carried out by incubating highly synchronized P.f. 3D7, 3D7 cam , and 3D7 pfs16 in the presence or absence ( top ) of 40 µM 4-TU for 12 h. Total RNA was extracted, biotinylated, and assayed by Northern blot probed with Streptavidin-HRP ( bottom ), demonstrating that thiol tagging occurs at a much lower level in 3D7 pfs16 (*) than in 3D7 cam , representing the minor sexual-stage parasite subpopulation.

    Techniques Used: Incubation, RNA Extraction, Transfection, Chick Chorioallantoic Membrane Assay, Western Blot, Northern Blot

    Engineering P. falciparum to salvage pyrimidines and generate thiol-modified RNAs. ( A ) Schematic of 4-TU biosynthetic mRNA capture method. Transgenic P. falciparum expressing a fusion gene containing cytosine deaminase/uracil phosphoribosyltransferase tagged with GFP ( FCU-GFP ) under the control of the calmodulin ( cam ) promoter (3D7 cam ) enables 4-TU salvage and incorporation into RNA. Thiolated-RNA can be biotinylated and detected by Northern blot or affinity purified by streptavidin magnetic beads for analysis by DNA microarray (or RNA-seq). ( B ) Expression of FCU-GFP in 3D7 cam parasites was verified by Western blot when probed with anti-yeast cytosine deaminase and by live fluorescence microscopy: (green) GFP; (blue) nuclear DNA stained with DAPI. ( C ) Both wild-type and 3D7 cam parasites were grown for 12 h in the presence of increasing 4-TU concentrations. The specificity of RNA thiol-incorporation and biotinylation was assessed by running 2 µg of each RNA sample with and without EZ-link Biotin-HPDP incubation ( top ). Total RNA was transferred to a nylon membrane and probed with streptavidin-HRP to detect biotinylated RNAs ( bottom ).
    Figure Legend Snippet: Engineering P. falciparum to salvage pyrimidines and generate thiol-modified RNAs. ( A ) Schematic of 4-TU biosynthetic mRNA capture method. Transgenic P. falciparum expressing a fusion gene containing cytosine deaminase/uracil phosphoribosyltransferase tagged with GFP ( FCU-GFP ) under the control of the calmodulin ( cam ) promoter (3D7 cam ) enables 4-TU salvage and incorporation into RNA. Thiolated-RNA can be biotinylated and detected by Northern blot or affinity purified by streptavidin magnetic beads for analysis by DNA microarray (or RNA-seq). ( B ) Expression of FCU-GFP in 3D7 cam parasites was verified by Western blot when probed with anti-yeast cytosine deaminase and by live fluorescence microscopy: (green) GFP; (blue) nuclear DNA stained with DAPI. ( C ) Both wild-type and 3D7 cam parasites were grown for 12 h in the presence of increasing 4-TU concentrations. The specificity of RNA thiol-incorporation and biotinylation was assessed by running 2 µg of each RNA sample with and without EZ-link Biotin-HPDP incubation ( top ). Total RNA was transferred to a nylon membrane and probed with streptavidin-HRP to detect biotinylated RNAs ( bottom ).

    Techniques Used: Modification, Transgenic Assay, Expressing, Chick Chorioallantoic Membrane Assay, Northern Blot, Affinity Purification, Magnetic Beads, Microarray, RNA Sequencing Assay, Western Blot, Fluorescence, Microscopy, Staining, Incubation

    47) Product Images from "Selenium-Based S-Adenosylmethionine Analog Reveals the Mammalian Seven-Beta-Strand Methyltransferase METTL10 to Be an EF1A1 Lysine Methyltransferase"

    Article Title: Selenium-Based S-Adenosylmethionine Analog Reveals the Mammalian Seven-Beta-Strand Methyltransferase METTL10 to Be an EF1A1 Lysine Methyltransferase

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0105394

    ProSeAM, a synthetic SAM analog, has a wide spectrum of reactivity for histones and non-histone substrates. A, Schematic overview for analyzing lysine methylation. A synthetic cofactor was used to transfer an alkyne moiety to the ε-amino group of lysine by KMTs (1). The modified proteins were tagged with biotin via CuAAC reaction (2). Tagged-proteins in the crude lysates were pulled down with affinity beads (3), and the precipitants were further analyzed with a LC-MS apparatus (4). B, Chemical structure of SAM (1), propargylated SAM (2) and ProSeAM (3). C, H3 peptide (1-21 a.a.) and ProSeAM was incubated with or without GST-G9a at 20°C for 2 h, then the peptide was analyzed by MALDI-TOF MS. D, full-length Histone H3 (1 µg) and ProSeAM (500 µM) were incubated with indicated KMTs (0.5 µg) for 2 h at 20°C. The histones were separated by SDS-PAGE, transferred to a nitrocellulose membrane and probed with streptavidin-HRP (top) or anti-Histone H3 antibody (bottom). E, The non-histone substrates His-HSP90 and His-HSP70 (1 µg) were incubated with His-SMYD2 and His-METTL21A (1 µg), respectively. After the reaction, proteins were separated by SDS-PAGE (right). Their modifications were detected by western blotting with streptavidin-HRP as in Fig. 1D. *and ** showed automodification of SMYD2 and METTL21A, respectively (left). F, His-HSP70 (WT and K561R) were incubated with or without His-METTL21A in the presence of ProSeAM for 2 h at 20°C. Modified proteins were biotinylated and detected with streptavidin-HRP (top) or anti-HSP70 antibody for the loading control (bottom).
    Figure Legend Snippet: ProSeAM, a synthetic SAM analog, has a wide spectrum of reactivity for histones and non-histone substrates. A, Schematic overview for analyzing lysine methylation. A synthetic cofactor was used to transfer an alkyne moiety to the ε-amino group of lysine by KMTs (1). The modified proteins were tagged with biotin via CuAAC reaction (2). Tagged-proteins in the crude lysates were pulled down with affinity beads (3), and the precipitants were further analyzed with a LC-MS apparatus (4). B, Chemical structure of SAM (1), propargylated SAM (2) and ProSeAM (3). C, H3 peptide (1-21 a.a.) and ProSeAM was incubated with or without GST-G9a at 20°C for 2 h, then the peptide was analyzed by MALDI-TOF MS. D, full-length Histone H3 (1 µg) and ProSeAM (500 µM) were incubated with indicated KMTs (0.5 µg) for 2 h at 20°C. The histones were separated by SDS-PAGE, transferred to a nitrocellulose membrane and probed with streptavidin-HRP (top) or anti-Histone H3 antibody (bottom). E, The non-histone substrates His-HSP90 and His-HSP70 (1 µg) were incubated with His-SMYD2 and His-METTL21A (1 µg), respectively. After the reaction, proteins were separated by SDS-PAGE (right). Their modifications were detected by western blotting with streptavidin-HRP as in Fig. 1D. *and ** showed automodification of SMYD2 and METTL21A, respectively (left). F, His-HSP70 (WT and K561R) were incubated with or without His-METTL21A in the presence of ProSeAM for 2 h at 20°C. Modified proteins were biotinylated and detected with streptavidin-HRP (top) or anti-HSP70 antibody for the loading control (bottom).

    Techniques Used: Methylation, Modification, Liquid Chromatography with Mass Spectroscopy, Incubation, Mass Spectrometry, SDS Page, Western Blot

    Proteomic identification of substrates for seven-beta-strand MTases. A, Schematic protocol for proteomic identification. HEK293T cell lysates were added to either propargylic Se-adenosyl- l -selenomethionine (ProSeAM) alone (1) or ProSeAM plus recombinant KMT (lysate:enzyme ratio was 10∶1) (2). After the in vitro reaction, labeled proteins were tagged with biotin and then precipitated with streptavidin beads. The precipitants were then digested with trypsin, and the trypsinized protein fragments were analyzed by LC-MS/MS. B, ProSeAM competes with SAM in the labeling reaction. HEK293T cell lysates were incubated with ProSeAM (250 µM) in the presence or absence of the indicated amount of SAM (0 to 2.5 mM). Modified proteins were biotinylated and detected with streptavidin-HRP (top). Equal protein loading was confirmed by western blotting with anti-α-tubulin antibody (bottom). C, western blot of labeled proteins. A 5% input of precipitated proteins without ProSeAM (1), with ProSeAM alone (2), with ProSeAM plus GST-G9a (3), with ProSeAM plus His-METTL21A (4) or with ProSeAM plus His-METTL10 was separately analyzed with western blotting with streptavidin-HRP (top) prior to the MS analysis, to compare the labeled proteins. Equal protein loading was confirmed by western blotting with anti-α-tubulin antibody (bottom). D, Doughnut chart of the subcellular distribution of proteins labeled with ProSeAM. HEK293T lysates alone (lane 1 in Fig. 2C) and HEK293T lysates with ProSeAM (lane 2 in Fig. 2C) were analyzed as described in A and Experimental procedures (n = 3). In total, 318 proteins were identified as ProSeAM-labeled proteins. E, List of METTL21A substrates. HEK293T cell lysates and ProSeAM were incubated with or without METTL21A (lane 2 and lane 4 in Fig. 2C), and analyzed as above. Molecular weight, peptide area (reflecting the quantity of detected protein), and fold enrichment of the peptide area are listed: ND, not determined because the substrate was detected only in the condition for lane 4 of B. The total numbers of identified proteins, 2-fold increase (compared to control in each experiment), and overlapped identified numbers of 3 independent experiments are listed in Table S2 .
    Figure Legend Snippet: Proteomic identification of substrates for seven-beta-strand MTases. A, Schematic protocol for proteomic identification. HEK293T cell lysates were added to either propargylic Se-adenosyl- l -selenomethionine (ProSeAM) alone (1) or ProSeAM plus recombinant KMT (lysate:enzyme ratio was 10∶1) (2). After the in vitro reaction, labeled proteins were tagged with biotin and then precipitated with streptavidin beads. The precipitants were then digested with trypsin, and the trypsinized protein fragments were analyzed by LC-MS/MS. B, ProSeAM competes with SAM in the labeling reaction. HEK293T cell lysates were incubated with ProSeAM (250 µM) in the presence or absence of the indicated amount of SAM (0 to 2.5 mM). Modified proteins were biotinylated and detected with streptavidin-HRP (top). Equal protein loading was confirmed by western blotting with anti-α-tubulin antibody (bottom). C, western blot of labeled proteins. A 5% input of precipitated proteins without ProSeAM (1), with ProSeAM alone (2), with ProSeAM plus GST-G9a (3), with ProSeAM plus His-METTL21A (4) or with ProSeAM plus His-METTL10 was separately analyzed with western blotting with streptavidin-HRP (top) prior to the MS analysis, to compare the labeled proteins. Equal protein loading was confirmed by western blotting with anti-α-tubulin antibody (bottom). D, Doughnut chart of the subcellular distribution of proteins labeled with ProSeAM. HEK293T lysates alone (lane 1 in Fig. 2C) and HEK293T lysates with ProSeAM (lane 2 in Fig. 2C) were analyzed as described in A and Experimental procedures (n = 3). In total, 318 proteins were identified as ProSeAM-labeled proteins. E, List of METTL21A substrates. HEK293T cell lysates and ProSeAM were incubated with or without METTL21A (lane 2 and lane 4 in Fig. 2C), and analyzed as above. Molecular weight, peptide area (reflecting the quantity of detected protein), and fold enrichment of the peptide area are listed: ND, not determined because the substrate was detected only in the condition for lane 4 of B. The total numbers of identified proteins, 2-fold increase (compared to control in each experiment), and overlapped identified numbers of 3 independent experiments are listed in Table S2 .

    Techniques Used: Recombinant, In Vitro, Labeling, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Incubation, Modification, Western Blot, Molecular Weight

    48) Product Images from "DC3, the 21-kDa Subunit of the Outer Dynein Arm-Docking Complex (ODA-DC), Is a Novel EF-Hand Protein Important for Assembly of Both the Outer Arm and the ODA-DC"

    Article Title: DC3, the 21-kDa Subunit of the Outer Dynein Arm-Docking Complex (ODA-DC), Is a Novel EF-Hand Protein Important for Assembly of Both the Outer Arm and the ODA-DC

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E03-01-0057

    (A) DC1 and DC2 assemble on the axoneme in the absence of DC3, but not vice versa. Top panel: Axonemes from wild type, the DC3-deletion strain (DC3Δ), oda 1, and oda 3 were isolated and analyzed by western blotting. oda 1 is null for DC2; oda 3 is null for DC1. The blot was probed with antibodies to DC1, DC2, and the inner arm IC, IC140 (used as a loading control). As expected, antibodies to DC1 detected protein in wild-type axonemes, but not in oda 1 or oda 3 axonemes, which lack the ODA-DC. Importantly, the antibody also detected protein in axonemes of the DC3-deletion strain. Essentially identical results were obtained with antibodies to DC2 (our unpublished results). These data indicate that DC1 and DC2 can assemble on the axoneme in the complete absence of DC3. Bottom panel: Axonemes from wild type, the DC3-deletion strain (DC3Δ), oda 1, oda 3, and oda 9 were prepared as above ( oda 9 has a defect in IC1 and lacks outer dynein arms but retains the ODA-DC). The blot was probed with a polyclonal antibody to DC3. The antibody detects a single protein of M r ∼25,000 in both oda 9 and wild-type axonemes but detects no protein in DC3-null axonemes, indicating that it is specific for DC3. Axonemes from oda 1 and oda 3 do not contain DC3, indicating that DC3 assembly is dependent on the presence of DC1 and DC2. (B) Immunoprecipitation of the ODA-DC in the absence of Mg 2 + . The DC1 antibody was used to immunoprecipitate the ODA-DC from biotinylated 0.6 M KCl extracts of DC3-transformant (strain W215) and DC3-null axonemes. The immunoprecipitated proteins were then resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and detected using streptavidin-HRP. The anti-DC1 antibody immuno-precipitated three proteins of M r ∼105,000, ∼70,000, and ∼25,000 (arrowheads) from DC3-transformant axonemal extracts (lane 3). These proteins, corresponding to DC1, DC2, and DC3, respectively, were not immunoprecipitated from DC3-transformant axonemal extracts using normal rabbit IgG (lane 4). In contrast, the anti-DC1 antibody immunoprecipitated only DC1 and DC2 (arrowheads) from the DC3-null axonemal extracts (lane 1). These proteins were not immunoprecipitated from DC3-null axonemal extracts using normal rabbit IgG (lane 2). These data confirm that a “partial” docking complex composed of DC1 and DC2 assembles on the axoneme when DC3 is missing and that transformation of the DC3-deletion strain with the DC3 gene restores DC3 to the ODA-DC. Numbers on left indicate molecular weight markers.
    Figure Legend Snippet: (A) DC1 and DC2 assemble on the axoneme in the absence of DC3, but not vice versa. Top panel: Axonemes from wild type, the DC3-deletion strain (DC3Δ), oda 1, and oda 3 were isolated and analyzed by western blotting. oda 1 is null for DC2; oda 3 is null for DC1. The blot was probed with antibodies to DC1, DC2, and the inner arm IC, IC140 (used as a loading control). As expected, antibodies to DC1 detected protein in wild-type axonemes, but not in oda 1 or oda 3 axonemes, which lack the ODA-DC. Importantly, the antibody also detected protein in axonemes of the DC3-deletion strain. Essentially identical results were obtained with antibodies to DC2 (our unpublished results). These data indicate that DC1 and DC2 can assemble on the axoneme in the complete absence of DC3. Bottom panel: Axonemes from wild type, the DC3-deletion strain (DC3Δ), oda 1, oda 3, and oda 9 were prepared as above ( oda 9 has a defect in IC1 and lacks outer dynein arms but retains the ODA-DC). The blot was probed with a polyclonal antibody to DC3. The antibody detects a single protein of M r ∼25,000 in both oda 9 and wild-type axonemes but detects no protein in DC3-null axonemes, indicating that it is specific for DC3. Axonemes from oda 1 and oda 3 do not contain DC3, indicating that DC3 assembly is dependent on the presence of DC1 and DC2. (B) Immunoprecipitation of the ODA-DC in the absence of Mg 2 + . The DC1 antibody was used to immunoprecipitate the ODA-DC from biotinylated 0.6 M KCl extracts of DC3-transformant (strain W215) and DC3-null axonemes. The immunoprecipitated proteins were then resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and detected using streptavidin-HRP. The anti-DC1 antibody immuno-precipitated three proteins of M r ∼105,000, ∼70,000, and ∼25,000 (arrowheads) from DC3-transformant axonemal extracts (lane 3). These proteins, corresponding to DC1, DC2, and DC3, respectively, were not immunoprecipitated from DC3-transformant axonemal extracts using normal rabbit IgG (lane 4). In contrast, the anti-DC1 antibody immunoprecipitated only DC1 and DC2 (arrowheads) from the DC3-null axonemal extracts (lane 1). These proteins were not immunoprecipitated from DC3-null axonemal extracts using normal rabbit IgG (lane 2). These data confirm that a “partial” docking complex composed of DC1 and DC2 assembles on the axoneme when DC3 is missing and that transformation of the DC3-deletion strain with the DC3 gene restores DC3 to the ODA-DC. Numbers on left indicate molecular weight markers.

    Techniques Used: Isolation, Western Blot, Immunoprecipitation, SDS Page, Transformation Assay, Molecular Weight

    49) Product Images from "Development of Adenosine Deaminase-Specific IgY Antibodies: Diagnostic and Inhibitory Application"

    Article Title: Development of Adenosine Deaminase-Specific IgY Antibodies: Diagnostic and Inhibitory Application

    Journal: Applied Biochemistry and Biotechnology

    doi: 10.1007/s12010-017-2626-x

    Sandwich-type ELISA assay for calf adenosine deaminase detection. The plate was coated with anti-cADA affinity-purified IgY antibodies and control IgY antibodies (2.5 μg/ml) followed by incubation with cADA at concentrations ranging from 500 to 0.05 ng/ml. For detection, affinity-purified anti-cADA IgY biotin-labeled antibodies were used at a concentration of 2.5 μg/ml. The complexes were detected with streptavidin conjugate with HRP (1: 5000 dilution). Symbols represent mean ± SD from two independent experiments performed in duplicate for each point and are expressed as the OD 490 * values obtained after subtraction of the background values
    Figure Legend Snippet: Sandwich-type ELISA assay for calf adenosine deaminase detection. The plate was coated with anti-cADA affinity-purified IgY antibodies and control IgY antibodies (2.5 μg/ml) followed by incubation with cADA at concentrations ranging from 500 to 0.05 ng/ml. For detection, affinity-purified anti-cADA IgY biotin-labeled antibodies were used at a concentration of 2.5 μg/ml. The complexes were detected with streptavidin conjugate with HRP (1: 5000 dilution). Symbols represent mean ± SD from two independent experiments performed in duplicate for each point and are expressed as the OD 490 * values obtained after subtraction of the background values

    Techniques Used: Enzyme-linked Immunosorbent Assay, Affinity Purification, Incubation, Labeling, Concentration Assay

    50) Product Images from "Small RNAs are trafficked from the epididymis to developing mammalian sperm"

    Article Title: Small RNAs are trafficked from the epididymis to developing mammalian sperm

    Journal: Developmental cell

    doi: 10.1016/j.devcel.2018.06.023

    Tissue-specific labeling and detection of small RNAs in intact animals A) Schematic of the TU tracer locus. In the absence of Cre, GFP is expressed from a ubiquitous promoter, with no UPRT expression. Cre drives LoxP recombination, eliminating GFP and juxtaposing the promoter with HA-UPRT. B) Western blot showing GFP and HA-UPRT levels in livers and several reproductive tissues isolated from TU-tracer animals not expressing Cre (top) or expressing liver-specific Albumin for uncropped Western blot images. C) Dot blot for RNA isolated from TU-tracer animals not expressing Cre, injected with either 4-TU or vehicle alone. As shown, only positive control (Biotinylated DNA oligo) is detectable upon incubation with Streptavidin-HRP. D) Dot blot for RNA isolated from Alb -Cre X TU tracer mice injected with two different doses of 4-TU. Upon 4-TU injection RNAs are specifically labeled, in a dose-dependent manner, in liver tissue and not in control tissue (cauda epididymis). E) SLAM-Seq analysis of 4-TU-labeled RNAs. TU tracer X Alb -Cre animals were either injected with 4-TU (400 mg/kg body weight) or with vehicle (solvent only), and were sacrificed 5–6 hours after the last injection for tissue harvest. Small RNAs were isolated from either cauda epididymis or from liver, and subject to SLAM-Seq, and sequencing reads were mapped to microRNAs using an error-tolerant pipeline. Mismatches were identified for all reads mapping to a given microRNA, and boxplots (box: 25th/50th/75th percentile; whiskers: 10th/90th percentile) show the frequency of various mismatches for mock-injected and 4-TU-injected animals, as indicated.
    Figure Legend Snippet: Tissue-specific labeling and detection of small RNAs in intact animals A) Schematic of the TU tracer locus. In the absence of Cre, GFP is expressed from a ubiquitous promoter, with no UPRT expression. Cre drives LoxP recombination, eliminating GFP and juxtaposing the promoter with HA-UPRT. B) Western blot showing GFP and HA-UPRT levels in livers and several reproductive tissues isolated from TU-tracer animals not expressing Cre (top) or expressing liver-specific Albumin for uncropped Western blot images. C) Dot blot for RNA isolated from TU-tracer animals not expressing Cre, injected with either 4-TU or vehicle alone. As shown, only positive control (Biotinylated DNA oligo) is detectable upon incubation with Streptavidin-HRP. D) Dot blot for RNA isolated from Alb -Cre X TU tracer mice injected with two different doses of 4-TU. Upon 4-TU injection RNAs are specifically labeled, in a dose-dependent manner, in liver tissue and not in control tissue (cauda epididymis). E) SLAM-Seq analysis of 4-TU-labeled RNAs. TU tracer X Alb -Cre animals were either injected with 4-TU (400 mg/kg body weight) or with vehicle (solvent only), and were sacrificed 5–6 hours after the last injection for tissue harvest. Small RNAs were isolated from either cauda epididymis or from liver, and subject to SLAM-Seq, and sequencing reads were mapped to microRNAs using an error-tolerant pipeline. Mismatches were identified for all reads mapping to a given microRNA, and boxplots (box: 25th/50th/75th percentile; whiskers: 10th/90th percentile) show the frequency of various mismatches for mock-injected and 4-TU-injected animals, as indicated.

    Techniques Used: Labeling, Expressing, Western Blot, Isolation, Dot Blot, Injection, Positive Control, Incubation, Mouse Assay, Sequencing

    51) Product Images from "A Specific Point Mutant at Position 1 of the Influenza Hemagglutinin Fusion Peptide Displays a Hemifusion Phenotype"

    Article Title: A Specific Point Mutant at Position 1 of the Influenza Hemagglutinin Fusion Peptide Displays a Hemifusion Phenotype

    Journal: Molecular Biology of the Cell

    doi:

    Cell surface expression and proteolytic processing of WT and mutant HAs. Stable NIH 3T3 cells expressing WT and mutant HAs were treated with NaButyrate to enhance HA expression. The cells were then labeled with the membrane impermeant reagent NHS-LC-biotin, treated with either trypsin (to cleave HA0) or chymotrypsin (HA0 control), and lysed. Equal amounts of protein from each cell lysate were precipitated with the Site A mAb, and the immune complexes were separated by reducing SDS-PAGE. The gel was transferred to nitrocellulose, probed with streptavidin-HRP, and developed by enhanced chemiluminescence.
    Figure Legend Snippet: Cell surface expression and proteolytic processing of WT and mutant HAs. Stable NIH 3T3 cells expressing WT and mutant HAs were treated with NaButyrate to enhance HA expression. The cells were then labeled with the membrane impermeant reagent NHS-LC-biotin, treated with either trypsin (to cleave HA0) or chymotrypsin (HA0 control), and lysed. Equal amounts of protein from each cell lysate were precipitated with the Site A mAb, and the immune complexes were separated by reducing SDS-PAGE. The gel was transferred to nitrocellulose, probed with streptavidin-HRP, and developed by enhanced chemiluminescence.

    Techniques Used: Expressing, Mutagenesis, Labeling, SDS Page

    52) Product Images from "A morphologic and semi-quantitative technique to analyze synthesis and release of specific proteins in cells"

    Article Title: A morphologic and semi-quantitative technique to analyze synthesis and release of specific proteins in cells

    Journal: BMC Cell Biology

    doi: 10.1186/s12860-014-0045-1

    Schematic diagram showed non-radioactive metabolic incorporation followed by azide-biotin or azide-Alex555 labeling, and biotin signals of proteins were detected by streptavidin-HRP by western blot. HPG is incorporated into newly synthesized proteins by metabolism and protein synthesis and the triazole conjugation between newly alkyne proteins labeled HPG and azide labeled either biotin or Alex555 via CuSO4 catalysis (A) . (B-a) The detection of biotin signals from extracted total proteins labeled by labeling reaction. Normal culture medium was changed to replace DMEM free of L-methionine supplemented with HPG after pulse 4 hr, and proteins were extracted in each of group at various time points including 0, 4, 24 and 72 hr. (B-b) Biotin signals of total proteins were detected. 1: Normal culture condition group; 2: HPG plus anisomycin group; 3: HPG group. (B-c,d,e) Biotin signals of Bcl-2, MMP-9 and IgG were individually detected in the immunoprecipitate pulled down by primary antibodies via siRNA post-transfection followed by non-radioactive metabolic labeling. (B-f) Radioactive isotope 35 S-methonine incorporated into synthesized IgG purified by immunoprecipitation was detected by autoradiography. 1: 35 S-methonine treated human choriocarcinoma cell line BeWo group and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 2: cycloheximide plus 35 S-methonine treated BeWo group then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 3: 35 S-methonine treated skin fibroblast and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins.
    Figure Legend Snippet: Schematic diagram showed non-radioactive metabolic incorporation followed by azide-biotin or azide-Alex555 labeling, and biotin signals of proteins were detected by streptavidin-HRP by western blot. HPG is incorporated into newly synthesized proteins by metabolism and protein synthesis and the triazole conjugation between newly alkyne proteins labeled HPG and azide labeled either biotin or Alex555 via CuSO4 catalysis (A) . (B-a) The detection of biotin signals from extracted total proteins labeled by labeling reaction. Normal culture medium was changed to replace DMEM free of L-methionine supplemented with HPG after pulse 4 hr, and proteins were extracted in each of group at various time points including 0, 4, 24 and 72 hr. (B-b) Biotin signals of total proteins were detected. 1: Normal culture condition group; 2: HPG plus anisomycin group; 3: HPG group. (B-c,d,e) Biotin signals of Bcl-2, MMP-9 and IgG were individually detected in the immunoprecipitate pulled down by primary antibodies via siRNA post-transfection followed by non-radioactive metabolic labeling. (B-f) Radioactive isotope 35 S-methonine incorporated into synthesized IgG purified by immunoprecipitation was detected by autoradiography. 1: 35 S-methonine treated human choriocarcinoma cell line BeWo group and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 2: cycloheximide plus 35 S-methonine treated BeWo group then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 3: 35 S-methonine treated skin fibroblast and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins.

    Techniques Used: Labeling, Western Blot, Synthesized, Conjugation Assay, Transfection, Purification, Immunoprecipitation, Autoradiography

    53) Product Images from "The overlapping host responses to bacterial cyclic dinucleotides"

    Article Title: The overlapping host responses to bacterial cyclic dinucleotides

    Journal: Microbes and infection / Institut Pasteur

    doi: 10.1016/j.micinf.2011.09.002

    Analysis of macrophage extracts with biotin-c-diGMP ( A ) Raw264.7 WCEs (1.5 µl) were incubated with biotin-c-diGMP (0.25, 0.5, 1 4 µg; 20 min, 4° C), treated UV light, fractionation on SDS-PAGE, transferred to nitrocellulose and probed with Streptavidin-HRP. A molar excess of GTP, c-diGMP (cdG) or c-diAMP (cdA) competitors was added simultaneously with biotin-c-diGMP, as indicated. ( B ) PMA treated U937 cells WCEs were UV crosslinked (UVx) with biotin-c-diGMP (1 µg), with or without competitors, as in panel A. ( C ) Crosslinked samples, with or without competition, were collected from UV treated (UVx) U937 cell extracts (left panel) or Raw264.7 cell extracts (right panel) by streptavidin pulldown (UVx-IP), prior to gel fractionation.
    Figure Legend Snippet: Analysis of macrophage extracts with biotin-c-diGMP ( A ) Raw264.7 WCEs (1.5 µl) were incubated with biotin-c-diGMP (0.25, 0.5, 1 4 µg; 20 min, 4° C), treated UV light, fractionation on SDS-PAGE, transferred to nitrocellulose and probed with Streptavidin-HRP. A molar excess of GTP, c-diGMP (cdG) or c-diAMP (cdA) competitors was added simultaneously with biotin-c-diGMP, as indicated. ( B ) PMA treated U937 cells WCEs were UV crosslinked (UVx) with biotin-c-diGMP (1 µg), with or without competitors, as in panel A. ( C ) Crosslinked samples, with or without competition, were collected from UV treated (UVx) U937 cell extracts (left panel) or Raw264.7 cell extracts (right panel) by streptavidin pulldown (UVx-IP), prior to gel fractionation.

    Techniques Used: Incubation, Fractionation, SDS Page

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

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

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.541250

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

    Techniques Used: Electron Microscopy, Generated, Produced

    55) Product Images from "Heterozygous PINK1 p.G411S increases risk of Parkinson’s disease via a dominant-negative mechanism"

    Article Title: Heterozygous PINK1 p.G411S increases risk of Parkinson’s disease via a dominant-negative mechanism

    Journal: Brain

    doi: 10.1093/brain/aww261

    The PINK1 p.G411S mutation exerts a dominant-negative mechanism. HeLa cells were used to confirm a dominant-negative effect of the p.G411S mutation on kinase activity of PINK1 wild-type that translated into reduced activation of parkin downstream. ( A ) HeLa cells were simultaneously transfected with specific PINK1 siRNA and siRNA-resistant PINK1-V5 wild-type or mutants (p.Q456X or p.G411S). Control cells were transfected with the corresponding empty vector (-) and with scrambled (scr) or PINK1 siRNA. Cells were treated with 15 µM CCCP for the indicated times and levels of phosphorylated ubiquitin were assessed by anti-p-Ser65-Ub. Endogenous and overexpressed PINK1 levels were monitored by anti-PINK1 and anti-V5 antibodies, respectively. Anti-GAPDH served as loading control. ( B ) HeLa cells were co-transfected with the indicated combinations of PINK1-V5 and PINK1-mCherry constructs or respective empty vector controls (-) and treated with 15 µM CCCP for 3 h. PINK1-V5 was immunoprecipitated (IP: V5) and the interaction between wild-type and mutant PINK1 was analysed by western blot. PINK1 wild-type and p.G411S strongly interacted with themselves and with each other to a similar extent. Black and grey triangles indicate full-length (wild-type and p.G411S) and truncated (p.Q456X) PINK1 protein, respectively. ( C ) In vitro ubiquitin phosphorylation assay confirms reduced kinase activity of p.G411S mutant and partial dominant-negative effects on PINK1 wild-type. HeLa cells were transfected with V5-tagged PINK1 wild-type, p.G411S, p.Q456X or a combination of wild-type plus p.G411S or p.Q456X. Cells were then treated with 15 µM CCCP for 3 h and PINK1 was immunoprecipitated with anti-V5. V5 immunoprecipitates were incubated with biotinylated ubiquitin in kinase reaction buffer. Anti-V5 antibody was used to show equal PINK1 levels in the IP. Black and grey triangles indicate full-length (wild-type and p.G411S) and truncated (p.Q456X) PINK1 protein, respectively. Phosphorylation of ubiquitin was determined by anti-p-Ser65-Ub antibody and total ubiquitin was detected by streptavidin-HRP that served as a loading control. Quantification of the p-Ser65-Ub/streptavidin ratio from three independent experiments is provided below. Values represent mean ± SEM, normalized to the average of wild-type values. Statistical significance was assessed by one-way ANOVA with Tukey’s post hoc test (* P
    Figure Legend Snippet: The PINK1 p.G411S mutation exerts a dominant-negative mechanism. HeLa cells were used to confirm a dominant-negative effect of the p.G411S mutation on kinase activity of PINK1 wild-type that translated into reduced activation of parkin downstream. ( A ) HeLa cells were simultaneously transfected with specific PINK1 siRNA and siRNA-resistant PINK1-V5 wild-type or mutants (p.Q456X or p.G411S). Control cells were transfected with the corresponding empty vector (-) and with scrambled (scr) or PINK1 siRNA. Cells were treated with 15 µM CCCP for the indicated times and levels of phosphorylated ubiquitin were assessed by anti-p-Ser65-Ub. Endogenous and overexpressed PINK1 levels were monitored by anti-PINK1 and anti-V5 antibodies, respectively. Anti-GAPDH served as loading control. ( B ) HeLa cells were co-transfected with the indicated combinations of PINK1-V5 and PINK1-mCherry constructs or respective empty vector controls (-) and treated with 15 µM CCCP for 3 h. PINK1-V5 was immunoprecipitated (IP: V5) and the interaction between wild-type and mutant PINK1 was analysed by western blot. PINK1 wild-type and p.G411S strongly interacted with themselves and with each other to a similar extent. Black and grey triangles indicate full-length (wild-type and p.G411S) and truncated (p.Q456X) PINK1 protein, respectively. ( C ) In vitro ubiquitin phosphorylation assay confirms reduced kinase activity of p.G411S mutant and partial dominant-negative effects on PINK1 wild-type. HeLa cells were transfected with V5-tagged PINK1 wild-type, p.G411S, p.Q456X or a combination of wild-type plus p.G411S or p.Q456X. Cells were then treated with 15 µM CCCP for 3 h and PINK1 was immunoprecipitated with anti-V5. V5 immunoprecipitates were incubated with biotinylated ubiquitin in kinase reaction buffer. Anti-V5 antibody was used to show equal PINK1 levels in the IP. Black and grey triangles indicate full-length (wild-type and p.G411S) and truncated (p.Q456X) PINK1 protein, respectively. Phosphorylation of ubiquitin was determined by anti-p-Ser65-Ub antibody and total ubiquitin was detected by streptavidin-HRP that served as a loading control. Quantification of the p-Ser65-Ub/streptavidin ratio from three independent experiments is provided below. Values represent mean ± SEM, normalized to the average of wild-type values. Statistical significance was assessed by one-way ANOVA with Tukey’s post hoc test (* P

    Techniques Used: Mutagenesis, Dominant Negative Mutation, Activity Assay, Activation Assay, Transfection, Plasmid Preparation, Construct, Immunoprecipitation, Western Blot, In Vitro, Phosphorylation Assay, Incubation

    56) Product Images from "Clathrin-independent carriers form a high capacity endocytic sorting system at the leading edge of migrating cells"

    Article Title: Clathrin-independent carriers form a high capacity endocytic sorting system at the leading edge of migrating cells

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201002119

    Quantitation of CLIC endocytosis. (A) Cav1 −/− or wild-type (WT) MEFs were left untreated or were treated with Dyngo4a. CTxB-HRP was internalized for 15 s, 1 min, or 2 min. Examples of CTxB-labeled structures after 15 s of uptake are shown (inset, left panel). Substratum indicated by large arrowhead, grid sizes are 2,000 or 200 nm, examples of intersections shown by arrows. (B) 20–25 cells treated as in A were used to calculate the volume fraction (V(v)). Error bars show SEM. (C) NIH3T3 cells not treated with inhibitor were processed as in A and counted as in B. V(v) was calculated for both tubular and vesicular structures. Error bars show SEM. (D) Cav1 −/− MEFs were incubated with HRP for 15 s, 1 min or 2 min and HRP-laden carriers counted as in B. Error bars show SEM. (E) CTxB was conjugated with NHS-SS-biotin and added to untreated Cav1 −/− MEFs or Cav1 −/− MEFs treated with Dyngo4a for 15 s or 2 min or was bound to untreated cells on ice for 10 min (Surface + MesNa). Cells were placed on ice and residual surface biotin cleaved with MesNa. Western blots of cell lysates were probed with streptavidin-HRP. Chart represents the average intensity of streptavidin-HRP across three independent experiments. Residue luminescence in Surface + MesNa samples indicates level of uncleavable biotin. Error bars show SEM. (F) Absolute volume of CLICs was estimated from the volume fraction, V(v), multiplied by the average volume of a Cav1 −/− MEF, 2,347 µm 2 . Surface density (S(v)) was calculated from high resolution images of labeled structures using a cycloid grid as described in Materials and methods and multiplied by the absolute volume to give absolute surface area. Volume of a single carrier was calculated as described in Materials and methods. Number of CLIC budding events per minute per cell was calculated based on the absolute volume internalized by all CLICs divided by the volume of a single carrier. Volume adjustments for overprojection effects are in brackets (see Materials and methods).
    Figure Legend Snippet: Quantitation of CLIC endocytosis. (A) Cav1 −/− or wild-type (WT) MEFs were left untreated or were treated with Dyngo4a. CTxB-HRP was internalized for 15 s, 1 min, or 2 min. Examples of CTxB-labeled structures after 15 s of uptake are shown (inset, left panel). Substratum indicated by large arrowhead, grid sizes are 2,000 or 200 nm, examples of intersections shown by arrows. (B) 20–25 cells treated as in A were used to calculate the volume fraction (V(v)). Error bars show SEM. (C) NIH3T3 cells not treated with inhibitor were processed as in A and counted as in B. V(v) was calculated for both tubular and vesicular structures. Error bars show SEM. (D) Cav1 −/− MEFs were incubated with HRP for 15 s, 1 min or 2 min and HRP-laden carriers counted as in B. Error bars show SEM. (E) CTxB was conjugated with NHS-SS-biotin and added to untreated Cav1 −/− MEFs or Cav1 −/− MEFs treated with Dyngo4a for 15 s or 2 min or was bound to untreated cells on ice for 10 min (Surface + MesNa). Cells were placed on ice and residual surface biotin cleaved with MesNa. Western blots of cell lysates were probed with streptavidin-HRP. Chart represents the average intensity of streptavidin-HRP across three independent experiments. Residue luminescence in Surface + MesNa samples indicates level of uncleavable biotin. Error bars show SEM. (F) Absolute volume of CLICs was estimated from the volume fraction, V(v), multiplied by the average volume of a Cav1 −/− MEF, 2,347 µm 2 . Surface density (S(v)) was calculated from high resolution images of labeled structures using a cycloid grid as described in Materials and methods and multiplied by the absolute volume to give absolute surface area. Volume of a single carrier was calculated as described in Materials and methods. Number of CLIC budding events per minute per cell was calculated based on the absolute volume internalized by all CLICs divided by the volume of a single carrier. Volume adjustments for overprojection effects are in brackets (see Materials and methods).

    Techniques Used: Quantitation Assay, Labeling, Incubation, Western Blot

    57) Product Images from "Biotin-Streptavidin Competition Mediates Sensitive Detection of Biomolecules in Enzyme Linked Immunosorbent Assay"

    Article Title: Biotin-Streptavidin Competition Mediates Sensitive Detection of Biomolecules in Enzyme Linked Immunosorbent Assay

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0151153

    Limit of detection of ESAT-6. (a) ESAT-6 was titrated from 0 to 120 nM. LOD was compared between experiments with and without free biotin added to streptavidin-HRP. (b) Calculation of mean OD. (c) Statistical calculation on improved and conventional ELISA. R 2 values were calculated and they indicate the significance.
    Figure Legend Snippet: Limit of detection of ESAT-6. (a) ESAT-6 was titrated from 0 to 120 nM. LOD was compared between experiments with and without free biotin added to streptavidin-HRP. (b) Calculation of mean OD. (c) Statistical calculation on improved and conventional ELISA. R 2 values were calculated and they indicate the significance.

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Optimization of ESAT-6 antibody. 100 nM of constant ESAT-6 coated on the ELISA well followed by adding the different dilutions (1:500, 1:1000 and 1:5000) of ESAT-6 antibody. And then detected by biotinylated antibody and streptavidin-HRP.1: 500 dilutions of ESAT-6 antibody showed the optimum dilution.
    Figure Legend Snippet: Optimization of ESAT-6 antibody. 100 nM of constant ESAT-6 coated on the ELISA well followed by adding the different dilutions (1:500, 1:1000 and 1:5000) of ESAT-6 antibody. And then detected by biotinylated antibody and streptavidin-HRP.1: 500 dilutions of ESAT-6 antibody showed the optimum dilution.

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Schematic representation of ESAT-6 detection by the biotin-streptavidin interaction. Complete schematics are displayed. ESAT-6 protein was coated onto the plate followed by anti-ESAT-6 and biotinylated anti-mouse-IgG. Streptavidin-HRP with and without free biotin then was added.
    Figure Legend Snippet: Schematic representation of ESAT-6 detection by the biotin-streptavidin interaction. Complete schematics are displayed. ESAT-6 protein was coated onto the plate followed by anti-ESAT-6 and biotinylated anti-mouse-IgG. Streptavidin-HRP with and without free biotin then was added.

    Techniques Used:

    Addition of free biotin improved detection of ESAT-6. 100 nM of ESAT-6 were coated onto the ELISA plate, followed by ESAT-6 antibody and biotinylated anti-mouse-IgG. (a) Detection by streptavidin mixed with different concentrations of free biotin (0 to 60 fM) showed that 8 fM of free biotin resulted in the highest OD. (b) A model explains the enhanced sensitivity of streptavidin-HRP pretreated with a fixed amount (8 fM) of free biotin.
    Figure Legend Snippet: Addition of free biotin improved detection of ESAT-6. 100 nM of ESAT-6 were coated onto the ELISA plate, followed by ESAT-6 antibody and biotinylated anti-mouse-IgG. (a) Detection by streptavidin mixed with different concentrations of free biotin (0 to 60 fM) showed that 8 fM of free biotin resulted in the highest OD. (b) A model explains the enhanced sensitivity of streptavidin-HRP pretreated with a fixed amount (8 fM) of free biotin.

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Addition of free biotin improved the interaction between biotinylated antibody and streptavidin-HRP. The 1:500 dilution of biotinylated antibody was detected by streptavidin-HRP mixed with different concentrations of free biotin (0 to 60 fM). Concentrations ranging from 2 to 15 nM of free biotin improved the detection, as indicated by the visible color change of the solution.
    Figure Legend Snippet: Addition of free biotin improved the interaction between biotinylated antibody and streptavidin-HRP. The 1:500 dilution of biotinylated antibody was detected by streptavidin-HRP mixed with different concentrations of free biotin (0 to 60 fM). Concentrations ranging from 2 to 15 nM of free biotin improved the detection, as indicated by the visible color change of the solution.

    Techniques Used:

    Optimization of biotinylated antibody and streptavidin-HRP. Two different dilutions of biotinylated antibody (1:500 and 1:1000) were coated onto ELISA plates. Different dilutions of streptavidin-HRP (1:1000, 1:5000, and 1:10000) were added and the interaction between biotinylated antibody and streptavidin-HRP was detected. (a) Color change in the solution after the addition of HRP substrate. (b) Graphical representation of the optimization.
    Figure Legend Snippet: Optimization of biotinylated antibody and streptavidin-HRP. Two different dilutions of biotinylated antibody (1:500 and 1:1000) were coated onto ELISA plates. Different dilutions of streptavidin-HRP (1:1000, 1:5000, and 1:10000) were added and the interaction between biotinylated antibody and streptavidin-HRP was detected. (a) Color change in the solution after the addition of HRP substrate. (b) Graphical representation of the optimization.

    Techniques Used: Enzyme-linked Immunosorbent Assay

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

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

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.541250

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

    Techniques Used: Electron Microscopy, Generated, Produced

    59) Product Images from "Curcumin Prevents Palmitoylation of Integrin β4 in Breast Cancer Cells"

    Article Title: Curcumin Prevents Palmitoylation of Integrin β4 in Breast Cancer Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0125399

    Curcumin does not indiscriminately block global cysteine modifications, but does reduce palmitoylation of proteins beside Integrin β4. (A) Acyl-biotin exchange was performed on protein from MDA-MB-231 cells treated with or without 15 μM curcumin for 18 hours. The presence or absence of hydroxylamine (HAM) during the reaction is used as a control for reaction specificity. Western blot analysis with streptavidin-HRP is shown depicting the banding pattern of S-acylated proteins from 1 or 3 mg of total protein. (B) HCC1806 or MDA-MB-231 cells (upper and lower panels where indicated) were pretreated with 15 μM curcumin for 1 hr prior to and during 5hr 17-ODYA labeling. Labeled proteins were reacted to biotin-azide via click chemistry and biotinylated proteins were isolated using streptavidin-sepharose. Palmitoylated protein was detected by western blot analysis using indicated antibodies. Representative blots of 3 independent experiments are displayed with relative input protein included and densitometry in arbitrary units.
    Figure Legend Snippet: Curcumin does not indiscriminately block global cysteine modifications, but does reduce palmitoylation of proteins beside Integrin β4. (A) Acyl-biotin exchange was performed on protein from MDA-MB-231 cells treated with or without 15 μM curcumin for 18 hours. The presence or absence of hydroxylamine (HAM) during the reaction is used as a control for reaction specificity. Western blot analysis with streptavidin-HRP is shown depicting the banding pattern of S-acylated proteins from 1 or 3 mg of total protein. (B) HCC1806 or MDA-MB-231 cells (upper and lower panels where indicated) were pretreated with 15 μM curcumin for 1 hr prior to and during 5hr 17-ODYA labeling. Labeled proteins were reacted to biotin-azide via click chemistry and biotinylated proteins were isolated using streptavidin-sepharose. Palmitoylated protein was detected by western blot analysis using indicated antibodies. Representative blots of 3 independent experiments are displayed with relative input protein included and densitometry in arbitrary units.

    Techniques Used: Blocking Assay, Multiple Displacement Amplification, Western Blot, Labeling, Isolation

    60) Product Images from "Bisulfite-free, single base-resolution analysis of 5-hydroxymethylcytosine in genomic DNA by chemical-mediated mismatch single base-resolution analysis of 5-hydroxymethylcytosine in genomic DNA by chemical-mediated mismatch †Electronic supplementary information (ESI) available. See DOI: 10.1039/c8sc04272a"

    Article Title: Bisulfite-free, single base-resolution analysis of 5-hydroxymethylcytosine in genomic DNA by chemical-mediated mismatch single base-resolution analysis of 5-hydroxymethylcytosine in genomic DNA by chemical-mediated mismatch †Electronic supplementary information (ESI) available. See DOI: 10.1039/c8sc04272a

    Journal: Chemical Science

    doi: 10.1039/c8sc04272a

    Polyacrylamide gel electrophoresis analysis and dot-blot assay. (a) Polyacrylamide gel electrophoresis analysis of the protected 5fC. Lane 1: ODN-5fC without treatment; lane 2: ODN-5fC protected with hydroxylamine; lane 3: ODN-5fC protected with hydroxylamine and then treated with azi-BP. (b) Dot-blot assay of the streptavidin–HRP detection of oxidized 5hmC labeled with azi-BP and DBCO-PEG4-biotin. Dot 1: 80 bp ds ODN-5hmC without treatment; dot 2: 80 bp ds ODN-5hmC ligated with an adapter and oxidized by KRuO 4 and then labeled with azi-BP and DBCO-PEG4-biotin; dot 3: 80 bp ds ODN-5fC; dot 4: 80 bp ds ODN-5fC protected with hydroxylamine and then incubated with azi-BP and DBCO-PEG4-biotin; dot 5: 80 bp ds ODN-5fC treated with azi-BP and DBCO-PEG4-biotin. Only the biotin labeled DNA can produce a dot. And after methylene blue incubation, we can verify the existence of DNA for every dot.
    Figure Legend Snippet: Polyacrylamide gel electrophoresis analysis and dot-blot assay. (a) Polyacrylamide gel electrophoresis analysis of the protected 5fC. Lane 1: ODN-5fC without treatment; lane 2: ODN-5fC protected with hydroxylamine; lane 3: ODN-5fC protected with hydroxylamine and then treated with azi-BP. (b) Dot-blot assay of the streptavidin–HRP detection of oxidized 5hmC labeled with azi-BP and DBCO-PEG4-biotin. Dot 1: 80 bp ds ODN-5hmC without treatment; dot 2: 80 bp ds ODN-5hmC ligated with an adapter and oxidized by KRuO 4 and then labeled with azi-BP and DBCO-PEG4-biotin; dot 3: 80 bp ds ODN-5fC; dot 4: 80 bp ds ODN-5fC protected with hydroxylamine and then incubated with azi-BP and DBCO-PEG4-biotin; dot 5: 80 bp ds ODN-5fC treated with azi-BP and DBCO-PEG4-biotin. Only the biotin labeled DNA can produce a dot. And after methylene blue incubation, we can verify the existence of DNA for every dot.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Dot Blot, Labeling, Incubation

    61) Product Images from "TRAIL death receptors DR4 and DR5 mediate cerebral microvascular endothelial cell apoptosis induced by oligomeric Alzheimer's Aβ"

    Article Title: TRAIL death receptors DR4 and DR5 mediate cerebral microvascular endothelial cell apoptosis induced by oligomeric Alzheimer's Aβ

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2012.55

    Binding of A β variants to death receptor-Fc chimeras. A β homologs – either freshly solubilized (F) or pre-aggregated for 3 days (3d) – were immunoreacted with paramagnetic beads coupled to DR4/Fc (DR4), DR5/Fc (DR5), and Fas/Fc (Fas) chimeras, as well as to the IgG-Fc fragment employed as negative control for nonspecific binding. Immunoprecipitated material was eluted under non-denaturing conditions and analyzed by WB after native 5–30% gradient gel electrophoresis. In all cases, bound panels represent the immunoprecipitated material specifically retained by the respective immobilized receptor chimeras; input panels illustrate the oligomerization state of the starting material incubated with the respective immobilized chimeras. Results are representative of at least three experiments. ( a ) A β -E22Q (Q22), ( b ) A β -L34V (V34), ( c ) A β –WT, and ( d ) reverse A β 40-1 used as negative control and previously conjugated to biotin to allow WB detection as anti-A β 4G8 and 6E10 antibodies are not immunoreactive with the reverse sequence peptide. This panel also illustrates – for control purposes – binding of biotin-conjugated A β -WT to DR4 and DR5; both biotinylated peptides were pre-aggregated for 3 days before binding to the receptor chimeras. In ( d ), WB was probed with streptavidin-HRP as detector reagent. In all cases, fluorograms were developed by chemiluminiscence
    Figure Legend Snippet: Binding of A β variants to death receptor-Fc chimeras. A β homologs – either freshly solubilized (F) or pre-aggregated for 3 days (3d) – were immunoreacted with paramagnetic beads coupled to DR4/Fc (DR4), DR5/Fc (DR5), and Fas/Fc (Fas) chimeras, as well as to the IgG-Fc fragment employed as negative control for nonspecific binding. Immunoprecipitated material was eluted under non-denaturing conditions and analyzed by WB after native 5–30% gradient gel electrophoresis. In all cases, bound panels represent the immunoprecipitated material specifically retained by the respective immobilized receptor chimeras; input panels illustrate the oligomerization state of the starting material incubated with the respective immobilized chimeras. Results are representative of at least three experiments. ( a ) A β -E22Q (Q22), ( b ) A β -L34V (V34), ( c ) A β –WT, and ( d ) reverse A β 40-1 used as negative control and previously conjugated to biotin to allow WB detection as anti-A β 4G8 and 6E10 antibodies are not immunoreactive with the reverse sequence peptide. This panel also illustrates – for control purposes – binding of biotin-conjugated A β -WT to DR4 and DR5; both biotinylated peptides were pre-aggregated for 3 days before binding to the receptor chimeras. In ( d ), WB was probed with streptavidin-HRP as detector reagent. In all cases, fluorograms were developed by chemiluminiscence

    Techniques Used: Binding Assay, Negative Control, Immunoprecipitation, Western Blot, Nucleic Acid Electrophoresis, Incubation, Sequencing

    62) Product Images from "Functionally Active T1-T1 Interfaces Revealed by the Accessibility of Intracellular Thiolate Groups in Kv4 Channels"

    Article Title: Functionally Active T1-T1 Interfaces Revealed by the Accessibility of Intracellular Thiolate Groups in Kv4 Channels

    Journal: The Journal of General Physiology

    doi: 10.1085/jgp.200509288

    Biochemical evidence of the chemical modification of Kv4.2 and Kv4.2-T1 by MTSEA-biotin. (A) Membrane fragments containing Kv4.2 and KChIP3 (Kv4.2:KChIP3, 1:3) were reacted with MTSEA-biotin for 20 min at room temperature, and then electrophoresed and blotted with either anti-Kv4.2 or streptavidin-HRP (MATERIALS AND METHODS). (B) Likewise, the purified T1 domain of Kv4.2 was also reacted with the biotinylated MTSEA reagent and screened with streptavidin-HRP. The indicated molecular weights correspond to those of Kv4.2 a-monomer (67 kD) and the monomeric Kv4.2-T1 protein (14 kD). (C) FPLC profile of the Kv4.2-T1 protein before (black) and after (red) treatment with MTSEA-biotin (0.5 mM). AU280, normalized absorbance units at 280 nM. The expected elution times of the T1 tetramer and the T1 monomer are schematically marked above the abscissa.
    Figure Legend Snippet: Biochemical evidence of the chemical modification of Kv4.2 and Kv4.2-T1 by MTSEA-biotin. (A) Membrane fragments containing Kv4.2 and KChIP3 (Kv4.2:KChIP3, 1:3) were reacted with MTSEA-biotin for 20 min at room temperature, and then electrophoresed and blotted with either anti-Kv4.2 or streptavidin-HRP (MATERIALS AND METHODS). (B) Likewise, the purified T1 domain of Kv4.2 was also reacted with the biotinylated MTSEA reagent and screened with streptavidin-HRP. The indicated molecular weights correspond to those of Kv4.2 a-monomer (67 kD) and the monomeric Kv4.2-T1 protein (14 kD). (C) FPLC profile of the Kv4.2-T1 protein before (black) and after (red) treatment with MTSEA-biotin (0.5 mM). AU280, normalized absorbance units at 280 nM. The expected elution times of the T1 tetramer and the T1 monomer are schematically marked above the abscissa.

    Techniques Used: Modification, Purification, Fast Protein Liquid Chromatography

    63) Product Images from "Characterisation of a Novel Anti-CD52 Antibody with Improved Efficacy and Reduced Immunogenicity"

    Article Title: Characterisation of a Novel Anti-CD52 Antibody with Improved Efficacy and Reduced Immunogenicity

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0138123

    Peptide competition ELISA of selected humanized variants. Titrations of humanized variants were competed against a fixed concentration (35 ng/ml) of biotinylated murine 2E8 for binding to CD52 peptide that was coated directly on an ELISA plate. Binding was detected with streptavidin-HRP and TMB substrate. Antibodies are named in the format ANT10X 1 X 2 where X 1 refers to the VH variant (VH1 to VH5) and X 2 refers to the Vκ variant (Vκ1 to Vκ4).
    Figure Legend Snippet: Peptide competition ELISA of selected humanized variants. Titrations of humanized variants were competed against a fixed concentration (35 ng/ml) of biotinylated murine 2E8 for binding to CD52 peptide that was coated directly on an ELISA plate. Binding was detected with streptavidin-HRP and TMB substrate. Antibodies are named in the format ANT10X 1 X 2 where X 1 refers to the VH variant (VH1 to VH5) and X 2 refers to the Vκ variant (Vκ1 to Vκ4).

    Techniques Used: Enzyme-linked Immunosorbent Assay, Concentration Assay, Binding Assay, Variant Assay

    64) Product Images from "Molecular Targeting of Intracellular Compartments Specifically in Cancer Cells"

    Article Title: Molecular Targeting of Intracellular Compartments Specifically in Cancer Cells

    Journal: Genes & Cancer

    doi: 10.1177/1947601910375274

    Biotin-labeled IL-13.E13K-D2-NLS retains binding to IL-13Rα2 on GBM cells. ( A ) Western blot for the biotin-labeled IL-13.E13K-D2-NLS and IL-13.E13K-D2 probed with streptavidin-HRP. ( B ) Standard curve for the bio-fluoreporter assay for quantification
    Figure Legend Snippet: Biotin-labeled IL-13.E13K-D2-NLS retains binding to IL-13Rα2 on GBM cells. ( A ) Western blot for the biotin-labeled IL-13.E13K-D2-NLS and IL-13.E13K-D2 probed with streptavidin-HRP. ( B ) Standard curve for the bio-fluoreporter assay for quantification

    Techniques Used: Labeling, Binding Assay, Western Blot

    65) Product Images from "Transcription Activator Interactions with Multiple SWI/SNF Subunits"

    Article Title: Transcription Activator Interactions with Multiple SWI/SNF Subunits

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.22.6.1615-1625.2002

    Interaction of Snf5, Swi1, and Swi2/Snf2 in the context of intact SWI/SNF with Hap4. (A) Diagram of the photo-cross-linking reagent Sulfo-SBED. (B) Schematic of the photo-cross-linking experiments. Photo-cross-linking experiments were performed with purified SWI/SNF complex and an activator that was previously conjugated with Sulfo-SBED. Where indicated, the reaction mixtures were exposed to UV light (312 nm) at 6 cm for 8 min at room temperature. The activator was separated from interacting SWI/SNF subunits upon the addition of DTT, which reduces the disulfide bond. (C) Snf5, Swi1, and Swi2/Snf2 are cross-linked by GST-Hap4. The photo-cross-linking reaction mixtures were run on an SDS-4 to 15% gradient polyacrylamide gel. The proteins were transferred to PVDF membrane and immunoblotted with streptavidin-HRP or the indicated SWI/SNF-specific antibodies. The asterisks indicate photo-cross-linked SWI/SNF subunits. (D) SWI/SNF subunits are not cross-linked by GST alone. The photo-cross-linking experiment was performed as described for panels B and C.
    Figure Legend Snippet: Interaction of Snf5, Swi1, and Swi2/Snf2 in the context of intact SWI/SNF with Hap4. (A) Diagram of the photo-cross-linking reagent Sulfo-SBED. (B) Schematic of the photo-cross-linking experiments. Photo-cross-linking experiments were performed with purified SWI/SNF complex and an activator that was previously conjugated with Sulfo-SBED. Where indicated, the reaction mixtures were exposed to UV light (312 nm) at 6 cm for 8 min at room temperature. The activator was separated from interacting SWI/SNF subunits upon the addition of DTT, which reduces the disulfide bond. (C) Snf5, Swi1, and Swi2/Snf2 are cross-linked by GST-Hap4. The photo-cross-linking reaction mixtures were run on an SDS-4 to 15% gradient polyacrylamide gel. The proteins were transferred to PVDF membrane and immunoblotted with streptavidin-HRP or the indicated SWI/SNF-specific antibodies. The asterisks indicate photo-cross-linked SWI/SNF subunits. (D) SWI/SNF subunits are not cross-linked by GST alone. The photo-cross-linking experiment was performed as described for panels B and C.

    Techniques Used: Purification

    66) Product Images from "Wheat germ-based protein libraries for the functional characterisation of the Arabidopsis E2 ubiquitin conjugating enzymes and the RING-type E3 ubiquitin ligase enzymes"

    Article Title: Wheat germ-based protein libraries for the functional characterisation of the Arabidopsis E2 ubiquitin conjugating enzymes and the RING-type E3 ubiquitin ligase enzymes

    Journal: BMC Plant Biology

    doi: 10.1186/s12870-015-0660-9

    Construction of an Arabidopsis E2 protein library with an N-terminus biotin tag using a wheat germ cell-free system. a Flow chart of the wheat germ-based procedure for the high-throughput production of an Arabidopsis E2 library with an N-terminus biotin tag. The first step involves the high-throughput preparation of DNA templates for transcription using 2-step “split-primer” PCR, followed by in vitro transcription using phage-coded SP6 RNA polymerase, and finally translation using the wheat germ cell-free system. All the steps were carried out in 96-well microtiter plates. b Immunoblot analysis of N-bio-E2s expressed by the wheat germ cell-free system. For analysis, 2–6 μL crude recombinant E2 proteins with N-terminus biotin tag were loaded onto SDS-PAGE and detected by streptavidin-HRP antibody. A total of 35 out of 37 predicted Arabidopsis E2s were detected. Arrows on the figure show the expected signal for each E2 and asterisks refer to the E2s used later in vitro ubiquitination analysis (Fig. 4 , Fig. 5 , Fig. 6 )
    Figure Legend Snippet: Construction of an Arabidopsis E2 protein library with an N-terminus biotin tag using a wheat germ cell-free system. a Flow chart of the wheat germ-based procedure for the high-throughput production of an Arabidopsis E2 library with an N-terminus biotin tag. The first step involves the high-throughput preparation of DNA templates for transcription using 2-step “split-primer” PCR, followed by in vitro transcription using phage-coded SP6 RNA polymerase, and finally translation using the wheat germ cell-free system. All the steps were carried out in 96-well microtiter plates. b Immunoblot analysis of N-bio-E2s expressed by the wheat germ cell-free system. For analysis, 2–6 μL crude recombinant E2 proteins with N-terminus biotin tag were loaded onto SDS-PAGE and detected by streptavidin-HRP antibody. A total of 35 out of 37 predicted Arabidopsis E2s were detected. Arrows on the figure show the expected signal for each E2 and asterisks refer to the E2s used later in vitro ubiquitination analysis (Fig. 4 , Fig. 5 , Fig. 6 )

    Techniques Used: Flow Cytometry, High Throughput Screening Assay, Polymerase Chain Reaction, In Vitro, Recombinant, SDS Page

    67) Product Images from "Antiproliferative Factor-Induced Changes in Phosphorylation and Palmitoylation of Cytoskeleton-Associated Protein-4 Regulate Its Nuclear Translocation and DNA Binding"

    Article Title: Antiproliferative Factor-Induced Changes in Phosphorylation and Palmitoylation of Cytoskeleton-Associated Protein-4 Regulate Its Nuclear Translocation and DNA Binding

    Journal: International Journal of Cell Biology

    doi: 10.1155/2012/150918

    Surface-labeled CKAP4 translocates from the plasma membrane into the nucleus following APF exposure . (a) HeLa cell-surface proteins were labeled with Sulfo NHS-biotin as described in Section 2 . Following exposure to 20 nM APF for 24 hours (or no treatment), the cells were harvested and the nuclear protein fraction was isolated (Pierce NE-PER), separated by SDS-PAGE, and transferred to nitrocellulose. The membrane was then probed with streptavidin-HRP (1 : 5000; Pierce) to bind biotinylated proteins, and the signal was detected by ECL (Pierce). Following detection of the biotinylated proteins from the nucleus, the (streptavidin) HRP on the membrane was inactivated by incubating the blot in PBS containing 3% H 2 O 2 and 1% sodium azide. The same membrane was then reprobed with antibodies to CKAP4 (“anti-CLIMP-63”, diluted 1 : 1000, Alexis Biochemicals) and fibrillarin (a nuclear marker and loading control; Abcam; diluted 1 : 1000). (b) HeLa cells were treated with APF (20 nM) for 24 hours, which resulted in a significant increase in the abundance of CKAP4 in the nucleus compared to control samples. Treated cells were harvested and the nuclear and cytosolic fractions were isolated and separated by SDS-PAGE as described in Section 2 . Protein expression was analyzed by Western Blotting with antibodies for β -tubulin (diluted 1 : 1000, Abcam; loading control for the nonnuclear fraction), CKAP4 (“anti-CLIMP-63”, diluted 1 : 1000, Alexis Biochemicals), and fibrillarin (diluted 1 : 1000, Abcam; loading control and specific marker for the nuclear fraction), and then with an HRP-conjugated anti-mouse secondary antibody (1 : 20000; ThermoFisher Scientific). The proteins were detected by ECL (Pierce) with multiple exposures to film. The integrated density of the bands on the film was measured using ImageJ. Exposure times were controlled to ensure that the signals on film were not saturated. (c) The nuclear/cytosolic ratio represents the relative distribution of CKAP4 in the nuclear versus cytosolic fractions extracted from cells treated with or without APF. CKAP4 abundance in the APF-treated and control samples were normalized for loading to β -tubulin for the nonnuclear fractions and to fibrillarin for the nuclear fractions. The nuclear/cytosolic ratio for CKAP4 in the APF and control samples was determined from these normalized values. The standard deviation describes the variability among the normalized, nuclear, and cytosolic ratios from three independent experiments. A two tailed, paired t -test of the two data arrays (plus APF and control) indicate that the difference between these ratios is significant ( P = 0.01; n = 3). Cells treated with APF stop dividing, so the 10 cm dishes containing control and APF treated cells contained fewer cells (and protein) at the end of the experiment, normalizing the CKAP4 signals to loading controls corrected for this disparity. Fibrillarin is a well-characterized nuclear marker that is also known to localize to nucleoli. The data shown are representative of four independent experiments.
    Figure Legend Snippet: Surface-labeled CKAP4 translocates from the plasma membrane into the nucleus following APF exposure . (a) HeLa cell-surface proteins were labeled with Sulfo NHS-biotin as described in Section 2 . Following exposure to 20 nM APF for 24 hours (or no treatment), the cells were harvested and the nuclear protein fraction was isolated (Pierce NE-PER), separated by SDS-PAGE, and transferred to nitrocellulose. The membrane was then probed with streptavidin-HRP (1 : 5000; Pierce) to bind biotinylated proteins, and the signal was detected by ECL (Pierce). Following detection of the biotinylated proteins from the nucleus, the (streptavidin) HRP on the membrane was inactivated by incubating the blot in PBS containing 3% H 2 O 2 and 1% sodium azide. The same membrane was then reprobed with antibodies to CKAP4 (“anti-CLIMP-63”, diluted 1 : 1000, Alexis Biochemicals) and fibrillarin (a nuclear marker and loading control; Abcam; diluted 1 : 1000). (b) HeLa cells were treated with APF (20 nM) for 24 hours, which resulted in a significant increase in the abundance of CKAP4 in the nucleus compared to control samples. Treated cells were harvested and the nuclear and cytosolic fractions were isolated and separated by SDS-PAGE as described in Section 2 . Protein expression was analyzed by Western Blotting with antibodies for β -tubulin (diluted 1 : 1000, Abcam; loading control for the nonnuclear fraction), CKAP4 (“anti-CLIMP-63”, diluted 1 : 1000, Alexis Biochemicals), and fibrillarin (diluted 1 : 1000, Abcam; loading control and specific marker for the nuclear fraction), and then with an HRP-conjugated anti-mouse secondary antibody (1 : 20000; ThermoFisher Scientific). The proteins were detected by ECL (Pierce) with multiple exposures to film. The integrated density of the bands on the film was measured using ImageJ. Exposure times were controlled to ensure that the signals on film were not saturated. (c) The nuclear/cytosolic ratio represents the relative distribution of CKAP4 in the nuclear versus cytosolic fractions extracted from cells treated with or without APF. CKAP4 abundance in the APF-treated and control samples were normalized for loading to β -tubulin for the nonnuclear fractions and to fibrillarin for the nuclear fractions. The nuclear/cytosolic ratio for CKAP4 in the APF and control samples was determined from these normalized values. The standard deviation describes the variability among the normalized, nuclear, and cytosolic ratios from three independent experiments. A two tailed, paired t -test of the two data arrays (plus APF and control) indicate that the difference between these ratios is significant ( P = 0.01; n = 3). Cells treated with APF stop dividing, so the 10 cm dishes containing control and APF treated cells contained fewer cells (and protein) at the end of the experiment, normalizing the CKAP4 signals to loading controls corrected for this disparity. Fibrillarin is a well-characterized nuclear marker that is also known to localize to nucleoli. The data shown are representative of four independent experiments.

    Techniques Used: Labeling, Isolation, SDS Page, Marker, Expressing, Western Blot, Standard Deviation, Two Tailed Test

    68) Product Images from "Dual Topology of the Melanocortin-2 Receptor Accessory Protein Is Stable"

    Article Title: Dual Topology of the Melanocortin-2 Receptor Accessory Protein Is Stable

    Journal: Frontiers in Endocrinology

    doi: 10.3389/fendo.2016.00096

    Kinetics of biotin labeling . CHO cells were transfected with BirA cyt and either (A) AP–MRAP or (B) MRAP–AP, both containing Flag epitope. After overnight incubation in medium without added biotin, 2.5 μM biotin was added for the times shown. MRAPs were immunoprecipitated with anti-Flag antibody, resolved on SDS-PAGE and blotted with either anti-Flag antibody, to identify total MRAP, or HRP–streptavidin, to identify biotin-labeled protein.
    Figure Legend Snippet: Kinetics of biotin labeling . CHO cells were transfected with BirA cyt and either (A) AP–MRAP or (B) MRAP–AP, both containing Flag epitope. After overnight incubation in medium without added biotin, 2.5 μM biotin was added for the times shown. MRAPs were immunoprecipitated with anti-Flag antibody, resolved on SDS-PAGE and blotted with either anti-Flag antibody, to identify total MRAP, or HRP–streptavidin, to identify biotin-labeled protein.

    Techniques Used: Labeling, Transfection, FLAG-tag, Incubation, Immunoprecipitation, SDS Page

    Biotin labeling confirms dual topology of MRAP . (A) CHO cells stably expressing BirA cyt or BirA ER were transfected with MRAP, AP–MRAP, or MRAP–AP; these plasmids also contained a Flag epitope. Following detergent solubilization, proteins were resolved on SDS-PAGE and probed with monoclonal anti-Flag antibody and HRP-anti-mouse IgG or HRP–streptavidin. (B) Expected localization of BirA and predicted orientation of newly synthesized MRAPs. Filled and open circles depict glycosylated and non-glycosylated MRAP and red stars represent biotin.
    Figure Legend Snippet: Biotin labeling confirms dual topology of MRAP . (A) CHO cells stably expressing BirA cyt or BirA ER were transfected with MRAP, AP–MRAP, or MRAP–AP; these plasmids also contained a Flag epitope. Following detergent solubilization, proteins were resolved on SDS-PAGE and probed with monoclonal anti-Flag antibody and HRP-anti-mouse IgG or HRP–streptavidin. (B) Expected localization of BirA and predicted orientation of newly synthesized MRAPs. Filled and open circles depict glycosylated and non-glycosylated MRAP and red stars represent biotin.

    Techniques Used: Labeling, Stable Transfection, Expressing, Transfection, FLAG-tag, SDS Page, Synthesized

    MRAP orientation and function in CHO and adrenal cells . (A) CHO and (B) OS3 adrenal cells were transfected with CRE-luciferase without (control) or with MRAP and MC2 receptor. The day after transfection cells were incubated for 5 h with vehicle, 1 μM ACTH or 20 μM forskolin when luciferase activity, expressed as relative light units (RLU), was measured. Note different scales for responses of OS3 and CHO cells. (C) CHO and (D) OS3 adrenal cells were transfected with BirA cyt and AP–MRAP or MRAP–AP containing Flag epitope with or without MC2 receptor. Cultures were incubated overnight with or without 10 nM ACTH. Lysates were run on SDS-PAGE and blots were incubated with anti-Flag antibody to detect total MRAP or HRP–streptavidin to detect biotin-labeled MRAP. In (D) , the arrows point to a non-specific band, the solid arrowhead to non-glycosylated AP–MRAP, and the open arrowhead to glycosylated MRAP–AP. * P
    Figure Legend Snippet: MRAP orientation and function in CHO and adrenal cells . (A) CHO and (B) OS3 adrenal cells were transfected with CRE-luciferase without (control) or with MRAP and MC2 receptor. The day after transfection cells were incubated for 5 h with vehicle, 1 μM ACTH or 20 μM forskolin when luciferase activity, expressed as relative light units (RLU), was measured. Note different scales for responses of OS3 and CHO cells. (C) CHO and (D) OS3 adrenal cells were transfected with BirA cyt and AP–MRAP or MRAP–AP containing Flag epitope with or without MC2 receptor. Cultures were incubated overnight with or without 10 nM ACTH. Lysates were run on SDS-PAGE and blots were incubated with anti-Flag antibody to detect total MRAP or HRP–streptavidin to detect biotin-labeled MRAP. In (D) , the arrows point to a non-specific band, the solid arrowhead to non-glycosylated AP–MRAP, and the open arrowhead to glycosylated MRAP–AP. * P

    Techniques Used: Transfection, Luciferase, Incubation, Activity Assay, FLAG-tag, SDS Page, Labeling

    69) Product Images from "Detection of the Malaria causing Plasmodium Parasite in Saliva from Infected Patients using Topoisomerase I Activity as a Biomarker"

    Article Title: Detection of the Malaria causing Plasmodium Parasite in Saliva from Infected Patients using Topoisomerase I Activity as a Biomarker

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-22378-7

    Detection of pTopI using a colorimetric readout. ( A ) The individual steps of the colorimetric readout are schematically depicted. (I) shows the RCA primer hybridized to the pTopI generated single stranded DNA circles. II) The primer is elongated in a RCA reaction performed in the presence of biotin labeled nucleotides (red asterisks). III) The biotinylated DNA generated is bound to a silica membrane, which is positioned in a column. IV) Silica bound biotinylated DNA is visualized by coupling to streptavidin conjugated HRP (black asterisks) followed by V) incubation with the colorimetric HRP substrate, TMB. ( B ) Representative pictures of the silica membranes after completion of the colorimetric readout using either a sample without DNA circles or spiked with premade DNA circles (F1 and F2 respectively) as well as after testing saliva samples from uninfected individuals (F3 and F4) or Plasmodium infected individuals (F5 and F6) (patients #4 and #20 respectively, see Table 2 ). ( C ) Box plot representing the result of using the above described colorimetric readout for testing 14 saliva samples from uninfected individuals and 16 samples from Plasmodium infected individuals. The results are shown as fold increase above the average signal obtained from the analysis of samples from uninfected individuals.
    Figure Legend Snippet: Detection of pTopI using a colorimetric readout. ( A ) The individual steps of the colorimetric readout are schematically depicted. (I) shows the RCA primer hybridized to the pTopI generated single stranded DNA circles. II) The primer is elongated in a RCA reaction performed in the presence of biotin labeled nucleotides (red asterisks). III) The biotinylated DNA generated is bound to a silica membrane, which is positioned in a column. IV) Silica bound biotinylated DNA is visualized by coupling to streptavidin conjugated HRP (black asterisks) followed by V) incubation with the colorimetric HRP substrate, TMB. ( B ) Representative pictures of the silica membranes after completion of the colorimetric readout using either a sample without DNA circles or spiked with premade DNA circles (F1 and F2 respectively) as well as after testing saliva samples from uninfected individuals (F3 and F4) or Plasmodium infected individuals (F5 and F6) (patients #4 and #20 respectively, see Table 2 ). ( C ) Box plot representing the result of using the above described colorimetric readout for testing 14 saliva samples from uninfected individuals and 16 samples from Plasmodium infected individuals. The results are shown as fold increase above the average signal obtained from the analysis of samples from uninfected individuals.

    Techniques Used: Generated, Labeling, Incubation, Infection

    70) Product Images from "Plasma Membrane Repair Is Regulated Extracellularly by Proteases Released from Lysosomes"

    Article Title: Plasma Membrane Repair Is Regulated Extracellularly by Proteases Released from Lysosomes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0152583

    Extracellular proteolysis releases surface proteins and promotes PM repair. (A) Effect of proteinase K treatment on Ca 2+- dependent PM repair. NRK cells pre-treated with increasing concentrations of proteinase K were permeabilized with SLO (150 ng/ml), incubated at 37°C for 5 min, stained with PI and analyzed by FACS. The inset on the right shows one example of PI quantification for cells untreated (blue) or pre-treated with 50 μg/ml proteinase K (red). The dotted histogram shows the Ca 2+ -free permeabilization control, which determined the gating (dashed line). The data are representative of three independent experiments. (B) Effect of proteinase K treatment on SM-induced Ca 2+ -free PM repair. NRK cells treated (red) or not (blue) with 50 μg/ml proteinase K were permeabilized with SLO (15 ng/ml), incubated at 37°C for 5 min in Ca 2+ -free DMEM containing 10 μU/ml SM, stained with PI and analyzed by FACS. The dotted black histogram shows the Ca 2+ -free permeabilization control in the absence of sphingomyelinase; the data are representative of five independent experiments. (C, D) Biotinylated surface proteins released in soluble form during SLO wounding and repair. NRK cells were biotinylated at 4°C and permeabilized or not with SLO (100 ng/ml) in the presence or not of Ca 2+ and containing or not a cocktail of protease inhibitors (Prot. Inh.), 100 μM E64, pepstatin-A (PEP-A) or AEBSF for 30 s, followed by collection of the supernatant, centrifugation at 100,000 g and analysis by Western blot with streptavidin-HRP. A diluted sample of the total cell extract before SLO wounding is shown on the left. The data are representative of two (C) or three (D) independent experiments.
    Figure Legend Snippet: Extracellular proteolysis releases surface proteins and promotes PM repair. (A) Effect of proteinase K treatment on Ca 2+- dependent PM repair. NRK cells pre-treated with increasing concentrations of proteinase K were permeabilized with SLO (150 ng/ml), incubated at 37°C for 5 min, stained with PI and analyzed by FACS. The inset on the right shows one example of PI quantification for cells untreated (blue) or pre-treated with 50 μg/ml proteinase K (red). The dotted histogram shows the Ca 2+ -free permeabilization control, which determined the gating (dashed line). The data are representative of three independent experiments. (B) Effect of proteinase K treatment on SM-induced Ca 2+ -free PM repair. NRK cells treated (red) or not (blue) with 50 μg/ml proteinase K were permeabilized with SLO (15 ng/ml), incubated at 37°C for 5 min in Ca 2+ -free DMEM containing 10 μU/ml SM, stained with PI and analyzed by FACS. The dotted black histogram shows the Ca 2+ -free permeabilization control in the absence of sphingomyelinase; the data are representative of five independent experiments. (C, D) Biotinylated surface proteins released in soluble form during SLO wounding and repair. NRK cells were biotinylated at 4°C and permeabilized or not with SLO (100 ng/ml) in the presence or not of Ca 2+ and containing or not a cocktail of protease inhibitors (Prot. Inh.), 100 μM E64, pepstatin-A (PEP-A) or AEBSF for 30 s, followed by collection of the supernatant, centrifugation at 100,000 g and analysis by Western blot with streptavidin-HRP. A diluted sample of the total cell extract before SLO wounding is shown on the left. The data are representative of two (C) or three (D) independent experiments.

    Techniques Used: Incubation, Staining, FACS, Centrifugation, Western Blot

    71) Product Images from "Proteasome inhibition and oxidative reactions disrupt cellular homeostasis during heme stress"

    Article Title: Proteasome inhibition and oxidative reactions disrupt cellular homeostasis during heme stress

    Journal: Cell Death and Differentiation

    doi: 10.1038/cdd.2014.154

    Dissection of proteasome inhibitor and oxidative activities of heme. ( a ) Hmox1 (−/−) MEFs were incubated with different metal porphyrins at 10 μ M for 18 h. Expression of Sqstm1 and ubiquitin protein was probed by western blot. Accumulation of Sqstm1 and high-molecular-weight ubiquitin protein aggregates in cells treated with FePP and CoPP (Fe: iron; Sn: tin; Mn: manganese; Co: cobalt; Zn: zinc). ( b ) Accumulation of constitutively expressed SQSTM-myc in RAW264 cells after treatment with CoPP and bortezomib (Bor, 100 pM) for 12 h (western blot against myc-tag; β -actin as a loading control). ( c ) Inhibition of the proteolytic activity of purified 26S proteasome by CoPP and bortezomib was quantified by monitoring the release of fluorescent AMP from the proteasome substrate peptide Suc-LLVT-Amc. All data for CoPP were corrected for fluorescence quenching by the porphyrin. Data represent mean±S.D. of six biologic replicates. ( d ) Linoleic acid oxidation by FePP but not by CoPP. Equal amounts of each porphyrin (final concentration 10 μ M) were injected into stirred, airtight reaction vessels at 37°C containing linoleic acid as the reactant. Oxygen concentration was measured continuously during the reaction with a Clark type electrode. Data represent mean±S.D. (dashed lines) of three independent experiments. ( e ) Formation of cellular protein-lipid adducts in FePP-treated (20 μ M, 5 h) or CoPP-treated (20 μ M, 5 h) Hmox1 (−/−) MEFs detected by click chemistry facilitated biotinylation of cell lysates and subsequent western blot detection by HRP streptavidin. ( f ) Glutathione (GSH) in Hmox1 (−/−) MEF cells after incubation with different concentrations of FePP and CoPP for 12 h. Data indicate mean±S.D. of luminescence from six biologic replicates. ( g , left) Heme (30 μ M)-triggered lipid peroxidation (TBARS) in soybean lecithin micelles and inhibition by Trolox (10 mM). Data represent mean±S.D. of three biologic replicates. ( g , right) Enzymatic activity of purified 26S proteasome as assessed by monitoring the release of fluorescent AMP from the proteasome substrate peptide Suc-LLVT-Amc in the presence or absence of heme (30 μ M), bortezomib (100 pM)±the antioxidant Trolox (10 mM). Data were corrected for fluorescence quenching by heme, and represent mean±S.D. of six independent replicates
    Figure Legend Snippet: Dissection of proteasome inhibitor and oxidative activities of heme. ( a ) Hmox1 (−/−) MEFs were incubated with different metal porphyrins at 10 μ M for 18 h. Expression of Sqstm1 and ubiquitin protein was probed by western blot. Accumulation of Sqstm1 and high-molecular-weight ubiquitin protein aggregates in cells treated with FePP and CoPP (Fe: iron; Sn: tin; Mn: manganese; Co: cobalt; Zn: zinc). ( b ) Accumulation of constitutively expressed SQSTM-myc in RAW264 cells after treatment with CoPP and bortezomib (Bor, 100 pM) for 12 h (western blot against myc-tag; β -actin as a loading control). ( c ) Inhibition of the proteolytic activity of purified 26S proteasome by CoPP and bortezomib was quantified by monitoring the release of fluorescent AMP from the proteasome substrate peptide Suc-LLVT-Amc. All data for CoPP were corrected for fluorescence quenching by the porphyrin. Data represent mean±S.D. of six biologic replicates. ( d ) Linoleic acid oxidation by FePP but not by CoPP. Equal amounts of each porphyrin (final concentration 10 μ M) were injected into stirred, airtight reaction vessels at 37°C containing linoleic acid as the reactant. Oxygen concentration was measured continuously during the reaction with a Clark type electrode. Data represent mean±S.D. (dashed lines) of three independent experiments. ( e ) Formation of cellular protein-lipid adducts in FePP-treated (20 μ M, 5 h) or CoPP-treated (20 μ M, 5 h) Hmox1 (−/−) MEFs detected by click chemistry facilitated biotinylation of cell lysates and subsequent western blot detection by HRP streptavidin. ( f ) Glutathione (GSH) in Hmox1 (−/−) MEF cells after incubation with different concentrations of FePP and CoPP for 12 h. Data indicate mean±S.D. of luminescence from six biologic replicates. ( g , left) Heme (30 μ M)-triggered lipid peroxidation (TBARS) in soybean lecithin micelles and inhibition by Trolox (10 mM). Data represent mean±S.D. of three biologic replicates. ( g , right) Enzymatic activity of purified 26S proteasome as assessed by monitoring the release of fluorescent AMP from the proteasome substrate peptide Suc-LLVT-Amc in the presence or absence of heme (30 μ M), bortezomib (100 pM)±the antioxidant Trolox (10 mM). Data were corrected for fluorescence quenching by heme, and represent mean±S.D. of six independent replicates

    Techniques Used: Dissection, Incubation, Expressing, Western Blot, Molecular Weight, Inhibition, Activity Assay, Purification, Fluorescence, Concentration Assay, Injection

    72) Product Images from "The Carbohydrate-linked Phosphorylcholine of the Parasitic Nematode Product ES-62 Modulates Complement Activation *"

    Article Title: The Carbohydrate-linked Phosphorylcholine of the Parasitic Nematode Product ES-62 Modulates Complement Activation *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.702746

    High-avidity binding of C-reactive protein to ES-62 is calcium-dependent and can be inhibited by PCh. A , dose response of binding of purified CRP to immobilized ES-62 (2.0 μg/ml, ■), PCh-BSA (0.5 μg/ml, ▴), or CWPS (5 μg/ml, □) on microtiter plates. Various concentrations of CRP were offered, and binding of CRP was detected using polyclonal anti-human CRP-HRP. OD , optical density. B , CRP binding from ES-62 is calcium-dependent and can be inhibited by PCh. Various concentrations of ES-62 were coated onto microtiter plates, and normal serum diluted 1 in 50 to give a final CRP concentration of 50 ng/ml was added. Binding of CRP was detected with the anti-native human CRP monoclonal antibody 2C10 and anti-mouse IgG HRP and 1,1,3,3 tetramethylbenzidine substrate (optical density, 450 nm). Serum was diluted in HBS containing 1 m m CaCl 2 (▴), HBS with 10 m m EDTA (▵), or HBS with 1 m m CaCl 2 and 50 m m phosphorylcholine (□). C , SAP provided in serum diluted 1 in 50 binds weakly to ES-62 (▴) but not PCh-BSA-coated plates (■). SAP was determined using monoclonal anti-SAP and anti-mouse IgG HRP. Controls show binding to ES-62 in the presence of EDTA (▵). D , plates were coated with ES-62 or the positive control acetylated BSA ( ACBSA ) at various concentrations, serum was added in the presence or absence of calcium, and binding was detected with biotinylated anti-ficolin 2 and streptavidin HRP. Data are mean ± S.E. of triplicates. E , ligand blotting of ES-62 following SDS-PAGE demonstrates binding of C-reactive protein to PCh attached to N -linked glycan. Left panel , ES-62 or ES-62 deglycosylated with PNGase stained directly with Coomassie Blue ( CB ). Center panel , ES-62 was transferred to PVDF, and CRP binding in TBSC was detected with anti-CRP and anti-mouse-alkaline phosphatase. Right panel , as for the center panel , but PCh was detected with anti-PCh myeloma protein, TEPC15. WB , Western blotting. F , surface plasmon resonance analysis of interaction. ES-62 was immobilized, and CRP was offered at concentrations of 10, 2.5, 1.25, 0.62, 0.3, 0.16, 0.08, and 0.04 μg/ml. Langmuir 1:1 analysis was performed. Residuals from the association analysis are shown below. RU , response unit. G , surface plasmon resonance analysis of ES-62 (12.5, 6.25, 3, 1.6, 0.8, 0.4, and 0.2 μg/ml) binding to biotinylated CRP immobilized on a streptavidin surface. The superimposed lines show modeled fit. H , CRP and ES-62 form large complexes in fluid phase. The size of the complex was determined using light scattering 5 min after mixing for 50 μg/ml CRP and 65 μg/ml ES-62 in HBS in the presence of 1 m m CaCl 2 .
    Figure Legend Snippet: High-avidity binding of C-reactive protein to ES-62 is calcium-dependent and can be inhibited by PCh. A , dose response of binding of purified CRP to immobilized ES-62 (2.0 μg/ml, ■), PCh-BSA (0.5 μg/ml, ▴), or CWPS (5 μg/ml, □) on microtiter plates. Various concentrations of CRP were offered, and binding of CRP was detected using polyclonal anti-human CRP-HRP. OD , optical density. B , CRP binding from ES-62 is calcium-dependent and can be inhibited by PCh. Various concentrations of ES-62 were coated onto microtiter plates, and normal serum diluted 1 in 50 to give a final CRP concentration of 50 ng/ml was added. Binding of CRP was detected with the anti-native human CRP monoclonal antibody 2C10 and anti-mouse IgG HRP and 1,1,3,3 tetramethylbenzidine substrate (optical density, 450 nm). Serum was diluted in HBS containing 1 m m CaCl 2 (▴), HBS with 10 m m EDTA (▵), or HBS with 1 m m CaCl 2 and 50 m m phosphorylcholine (□). C , SAP provided in serum diluted 1 in 50 binds weakly to ES-62 (▴) but not PCh-BSA-coated plates (■). SAP was determined using monoclonal anti-SAP and anti-mouse IgG HRP. Controls show binding to ES-62 in the presence of EDTA (▵). D , plates were coated with ES-62 or the positive control acetylated BSA ( ACBSA ) at various concentrations, serum was added in the presence or absence of calcium, and binding was detected with biotinylated anti-ficolin 2 and streptavidin HRP. Data are mean ± S.E. of triplicates. E , ligand blotting of ES-62 following SDS-PAGE demonstrates binding of C-reactive protein to PCh attached to N -linked glycan. Left panel , ES-62 or ES-62 deglycosylated with PNGase stained directly with Coomassie Blue ( CB ). Center panel , ES-62 was transferred to PVDF, and CRP binding in TBSC was detected with anti-CRP and anti-mouse-alkaline phosphatase. Right panel , as for the center panel , but PCh was detected with anti-PCh myeloma protein, TEPC15. WB , Western blotting. F , surface plasmon resonance analysis of interaction. ES-62 was immobilized, and CRP was offered at concentrations of 10, 2.5, 1.25, 0.62, 0.3, 0.16, 0.08, and 0.04 μg/ml. Langmuir 1:1 analysis was performed. Residuals from the association analysis are shown below. RU , response unit. G , surface plasmon resonance analysis of ES-62 (12.5, 6.25, 3, 1.6, 0.8, 0.4, and 0.2 μg/ml) binding to biotinylated CRP immobilized on a streptavidin surface. The superimposed lines show modeled fit. H , CRP and ES-62 form large complexes in fluid phase. The size of the complex was determined using light scattering 5 min after mixing for 50 μg/ml CRP and 65 μg/ml ES-62 in HBS in the presence of 1 m m CaCl 2 .

    Techniques Used: Binding Assay, Purification, Concentration Assay, Positive Control, SDS Page, Staining, Western Blot, SPR Assay

    ES-62-bound CRP leads to C4 deposition. A , CRP bound to ES-62 leads to C4 product deposition. Plates were coated with ES-62 or other CRP ligand at concentrations that bound similar amounts of CRP. Serum diluted in VBSCaMg from eight to ten normal healthy donors was added with or without CRP at 0.4 μg/ml and incubated at 37 °C for 30 min. Deposited C4c was determined with biotinylated anti-C4c and streptavidin HRP. Statistical analysis was undertaken by paired t test ( n = 9). B , bound CRP correlates with increased C4c deposition for all three ligands. Data were obtained as in A , but different serum and CRP concentrations were used, and bound CRP was measured and plotted against the deposited C4c. PChBSA, ■; ES-62, ●; CWPS, ▴.
    Figure Legend Snippet: ES-62-bound CRP leads to C4 deposition. A , CRP bound to ES-62 leads to C4 product deposition. Plates were coated with ES-62 or other CRP ligand at concentrations that bound similar amounts of CRP. Serum diluted in VBSCaMg from eight to ten normal healthy donors was added with or without CRP at 0.4 μg/ml and incubated at 37 °C for 30 min. Deposited C4c was determined with biotinylated anti-C4c and streptavidin HRP. Statistical analysis was undertaken by paired t test ( n = 9). B , bound CRP correlates with increased C4c deposition for all three ligands. Data were obtained as in A , but different serum and CRP concentrations were used, and bound CRP was measured and plotted against the deposited C4c. PChBSA, ■; ES-62, ●; CWPS, ▴.

    Techniques Used: Incubation

    ES-62, in contrast to PChBSA and CWPS, does not lead to C3d or C3bi deposition. A , CRP increases deposition of C3d onto PCh in PChBSA and CWPS but not ES-62. Individual sera from healthy donors with or without added CRP in VBSCaMg were incubated at 37 °C in ligand-coated plates in VBSCaMg, and C3d deposition was determined. The background level of complement activation for ES-62 seen without added CRP was not diminished in sera depleted of PCh binding activity by passage through an anti-PCh-Sepharose column. Statistical analysis was undertaken by paired t test. B , the same experiment was performed, but the increase in C3d deposition mediated by CRP was plotted against the amount of CRP bound to the plate under each condition. Ligands: PCh-BSA (■), CPWS (●), or ES-62 (▴). Small symbols represent data obtained for individual donor serum. Larger symbols represent data for pooled serum. C , complement activation by ES-62 and other PCh ligands is through C1q. Normal serum or C1q-depleted pooled sera were used to determine C3d deposition against ES-62, PCh-BSA, and CWPS. Data are mean ± S.E. of four replicates. D , CRP addition to serum increases complement C3bi deposition to ligand PCh-BSA but not ES-62. Following incubation as in A at 37 °C for 30 min, C3bi bound to the surface was detected with biotinylated anti-C3bi and streptavidin HRP. In A , B , and D , the data are for between seven and nine different donors measured in three different experiments.
    Figure Legend Snippet: ES-62, in contrast to PChBSA and CWPS, does not lead to C3d or C3bi deposition. A , CRP increases deposition of C3d onto PCh in PChBSA and CWPS but not ES-62. Individual sera from healthy donors with or without added CRP in VBSCaMg were incubated at 37 °C in ligand-coated plates in VBSCaMg, and C3d deposition was determined. The background level of complement activation for ES-62 seen without added CRP was not diminished in sera depleted of PCh binding activity by passage through an anti-PCh-Sepharose column. Statistical analysis was undertaken by paired t test. B , the same experiment was performed, but the increase in C3d deposition mediated by CRP was plotted against the amount of CRP bound to the plate under each condition. Ligands: PCh-BSA (■), CPWS (●), or ES-62 (▴). Small symbols represent data obtained for individual donor serum. Larger symbols represent data for pooled serum. C , complement activation by ES-62 and other PCh ligands is through C1q. Normal serum or C1q-depleted pooled sera were used to determine C3d deposition against ES-62, PCh-BSA, and CWPS. Data are mean ± S.E. of four replicates. D , CRP addition to serum increases complement C3bi deposition to ligand PCh-BSA but not ES-62. Following incubation as in A at 37 °C for 30 min, C3bi bound to the surface was detected with biotinylated anti-C3bi and streptavidin HRP. In A , B , and D , the data are for between seven and nine different donors measured in three different experiments.

    Techniques Used: Incubation, Activation Assay, Binding Assay, Activity Assay

    73) Product Images from "Antiproliferative Factor-Induced Changes in Phosphorylation and Palmitoylation of Cytoskeleton-Associated Protein-4 Regulate Its Nuclear Translocation and DNA Binding"

    Article Title: Antiproliferative Factor-Induced Changes in Phosphorylation and Palmitoylation of Cytoskeleton-Associated Protein-4 Regulate Its Nuclear Translocation and DNA Binding

    Journal: International Journal of Cell Biology

    doi: 10.1155/2012/150918

    Surface-labeled CKAP4 translocates from the plasma membrane into the nucleus following APF exposure . (a) HeLa cell-surface proteins were labeled with Sulfo NHS-biotin as described in Section 2 . Following exposure to 20 nM APF for 24 hours (or no treatment), the cells were harvested and the nuclear protein fraction was isolated (Pierce NE-PER), separated by SDS-PAGE, and transferred to nitrocellulose. The membrane was then probed with streptavidin-HRP (1 : 5000; Pierce) to bind biotinylated proteins, and the signal was detected by ECL (Pierce). Following detection of the biotinylated proteins from the nucleus, the (streptavidin) HRP on the membrane was inactivated by incubating the blot in PBS containing 3% H 2 O 2 and 1% sodium azide. The same membrane was then reprobed with antibodies to CKAP4 (“anti-CLIMP-63”, diluted 1 : 1000, Alexis Biochemicals) and fibrillarin (a nuclear marker and loading control; Abcam; diluted 1 : 1000). (b) HeLa cells were treated with APF (20 nM) for 24 hours, which resulted in a significant increase in the abundance of CKAP4 in the nucleus compared to control samples. Treated cells were harvested and the nuclear and cytosolic fractions were isolated and separated by SDS-PAGE as described in Section 2 . Protein expression was analyzed by Western Blotting with antibodies for β -tubulin (diluted 1 : 1000, Abcam; loading control for the nonnuclear fraction), CKAP4 (“anti-CLIMP-63”, diluted 1 : 1000, Alexis Biochemicals), and fibrillarin (diluted 1 : 1000, Abcam; loading control and specific marker for the nuclear fraction), and then with an HRP-conjugated anti-mouse secondary antibody (1 : 20000; ThermoFisher Scientific). The proteins were detected by ECL (Pierce) with multiple exposures to film. The integrated density of the bands on the film was measured using ImageJ. Exposure times were controlled to ensure that the signals on film were not saturated. (c) The nuclear/cytosolic ratio represents the relative distribution of CKAP4 in the nuclear versus cytosolic fractions extracted from cells treated with or without APF. CKAP4 abundance in the APF-treated and control samples were normalized for loading to β -tubulin for the nonnuclear fractions and to fibrillarin for the nuclear fractions. The nuclear/cytosolic ratio for CKAP4 in the APF and control samples was determined from these normalized values. The standard deviation describes the variability among the normalized, nuclear, and cytosolic ratios from three independent experiments. A two tailed, paired t -test of the two data arrays (plus APF and control) indicate that the difference between these ratios is significant ( P = 0.01; n = 3). Cells treated with APF stop dividing, so the 10 cm dishes containing control and APF treated cells contained fewer cells (and protein) at the end of the experiment, normalizing the CKAP4 signals to loading controls corrected for this disparity. Fibrillarin is a well-characterized nuclear marker that is also known to localize to nucleoli. The data shown are representative of four independent experiments.
    Figure Legend Snippet: Surface-labeled CKAP4 translocates from the plasma membrane into the nucleus following APF exposure . (a) HeLa cell-surface proteins were labeled with Sulfo NHS-biotin as described in Section 2 . Following exposure to 20 nM APF for 24 hours (or no treatment), the cells were harvested and the nuclear protein fraction was isolated (Pierce NE-PER), separated by SDS-PAGE, and transferred to nitrocellulose. The membrane was then probed with streptavidin-HRP (1 : 5000; Pierce) to bind biotinylated proteins, and the signal was detected by ECL (Pierce). Following detection of the biotinylated proteins from the nucleus, the (streptavidin) HRP on the membrane was inactivated by incubating the blot in PBS containing 3% H 2 O 2 and 1% sodium azide. The same membrane was then reprobed with antibodies to CKAP4 (“anti-CLIMP-63”, diluted 1 : 1000, Alexis Biochemicals) and fibrillarin (a nuclear marker and loading control; Abcam; diluted 1 : 1000). (b) HeLa cells were treated with APF (20 nM) for 24 hours, which resulted in a significant increase in the abundance of CKAP4 in the nucleus compared to control samples. Treated cells were harvested and the nuclear and cytosolic fractions were isolated and separated by SDS-PAGE as described in Section 2 . Protein expression was analyzed by Western Blotting with antibodies for β -tubulin (diluted 1 : 1000, Abcam; loading control for the nonnuclear fraction), CKAP4 (“anti-CLIMP-63”, diluted 1 : 1000, Alexis Biochemicals), and fibrillarin (diluted 1 : 1000, Abcam; loading control and specific marker for the nuclear fraction), and then with an HRP-conjugated anti-mouse secondary antibody (1 : 20000; ThermoFisher Scientific). The proteins were detected by ECL (Pierce) with multiple exposures to film. The integrated density of the bands on the film was measured using ImageJ. Exposure times were controlled to ensure that the signals on film were not saturated. (c) The nuclear/cytosolic ratio represents the relative distribution of CKAP4 in the nuclear versus cytosolic fractions extracted from cells treated with or without APF. CKAP4 abundance in the APF-treated and control samples were normalized for loading to β -tubulin for the nonnuclear fractions and to fibrillarin for the nuclear fractions. The nuclear/cytosolic ratio for CKAP4 in the APF and control samples was determined from these normalized values. The standard deviation describes the variability among the normalized, nuclear, and cytosolic ratios from three independent experiments. A two tailed, paired t -test of the two data arrays (plus APF and control) indicate that the difference between these ratios is significant ( P = 0.01; n = 3). Cells treated with APF stop dividing, so the 10 cm dishes containing control and APF treated cells contained fewer cells (and protein) at the end of the experiment, normalizing the CKAP4 signals to loading controls corrected for this disparity. Fibrillarin is a well-characterized nuclear marker that is also known to localize to nucleoli. The data shown are representative of four independent experiments.

    Techniques Used: Labeling, Isolation, SDS Page, Marker, Expressing, Western Blot, Standard Deviation, Two Tailed Test

    74) Product Images from "Overexpression of TGF-?1 Gene Induces Cell Surface Localized Glucose-Regulated Protein 78-Associated Latency-Associated Peptide/TGF-?"

    Article Title: Overexpression of TGF-?1 Gene Induces Cell Surface Localized Glucose-Regulated Protein 78-Associated Latency-Associated Peptide/TGF-?

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

    doi: 10.4049/jimmunol.0904121

    Identification of surface LAP/TGF-β–associated proteins. A , P3U1–TGF-β clone No. 16 cells or parent P3U1 cells were first surface labeled with biotin. The cell lysates were immunoprecipitated with anti-LAP mAb. The elutes further separated with SA magnetic beads, and blotted with SA-HRP. SA-bound fractions ( lanes 1 , 2 ) and -unbound fractions ( lanes 3 , 4 ) from P3U1 cells ( lanes 1 , 3 ) or P3U1–TGF-β cells ( lanes 2 , 4 ). B , Silver staining of the SA-bound fractions from P3U1 cells ( lane 1 ) or from P3U1–TGF-β cells ( lane 2 ). The band at the arrow was cut and subjected to LC/MS analysis. SA, streptavidin.
    Figure Legend Snippet: Identification of surface LAP/TGF-β–associated proteins. A , P3U1–TGF-β clone No. 16 cells or parent P3U1 cells were first surface labeled with biotin. The cell lysates were immunoprecipitated with anti-LAP mAb. The elutes further separated with SA magnetic beads, and blotted with SA-HRP. SA-bound fractions ( lanes 1 , 2 ) and -unbound fractions ( lanes 3 , 4 ) from P3U1 cells ( lanes 1 , 3 ) or P3U1–TGF-β cells ( lanes 2 , 4 ). B , Silver staining of the SA-bound fractions from P3U1 cells ( lane 1 ) or from P3U1–TGF-β cells ( lane 2 ). The band at the arrow was cut and subjected to LC/MS analysis. SA, streptavidin.

    Techniques Used: Labeling, Immunoprecipitation, Magnetic Beads, Silver Staining, Liquid Chromatography with Mass Spectroscopy

    75) Product Images from "Mechanisms of CPT1C-Dependent AMPAR Trafficking Enhancement"

    Article Title: Mechanisms of CPT1C-Dependent AMPAR Trafficking Enhancement

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2018.00275

    CPT1C acts as depalmitoylating enzyme of GluA1. (A) Palmitoylation levels detected with Acyl-Biotin Exchange (ABE) assay of GluA1 alone (GFP), or together with CPT1C-GFP or CPT1C(H470A) in HEK293AD-GluA1 expressing cells transfected with DHHC3/GODZ palmitoylating enzyme. The biotinylated GluA1 immunoprecipitates subsequent to the ABE assay for all conditions were subjected to SDS-PAGE. Palmitoylation of GluA1 subunit is detected only in plus-hydroxylamine (+HAM) samples (three lanes from the left). −HAM samples control non-specific incorporation of biotin (three lanes from the right). GluA1 palmitoylation levels (right top) were detected by Western blotting with streptavidin-HRP (palmitoylation). The total amount of immunoprecipitated GluA1 was detected by Western blotting with anti-GluA1-NT antibody (anti-GluA1, bottom) after stripping the membranes. (B) Quantification of palmitoylation levels for GluA1 alone (GFP), together with CPT1C or CPT1C(H470A) in HEK293AD cells constitutively expressing GluA1. Ratio of palmitoylated GluA1 to total GluA1 is shown as mean and S.E.M. (** p
    Figure Legend Snippet: CPT1C acts as depalmitoylating enzyme of GluA1. (A) Palmitoylation levels detected with Acyl-Biotin Exchange (ABE) assay of GluA1 alone (GFP), or together with CPT1C-GFP or CPT1C(H470A) in HEK293AD-GluA1 expressing cells transfected with DHHC3/GODZ palmitoylating enzyme. The biotinylated GluA1 immunoprecipitates subsequent to the ABE assay for all conditions were subjected to SDS-PAGE. Palmitoylation of GluA1 subunit is detected only in plus-hydroxylamine (+HAM) samples (three lanes from the left). −HAM samples control non-specific incorporation of biotin (three lanes from the right). GluA1 palmitoylation levels (right top) were detected by Western blotting with streptavidin-HRP (palmitoylation). The total amount of immunoprecipitated GluA1 was detected by Western blotting with anti-GluA1-NT antibody (anti-GluA1, bottom) after stripping the membranes. (B) Quantification of palmitoylation levels for GluA1 alone (GFP), together with CPT1C or CPT1C(H470A) in HEK293AD cells constitutively expressing GluA1. Ratio of palmitoylated GluA1 to total GluA1 is shown as mean and S.E.M. (** p

    Techniques Used: Expressing, Transfection, SDS Page, Western Blot, Immunoprecipitation, Stripping Membranes

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    Cytometry:

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    Blocking Assay:

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    Enzyme-linked Immunosorbent Assay:

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    Incubation:

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    Expressing:

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    Acrylamide Gel Assay:

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    Western Blot:

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    Flow Cytometry:

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    Immunoprecipitation:

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    Imaging:

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    Binding Assay:

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    Nucleic Acid Electrophoresis:

    Article Title: Detecting N-myristoylation and S-acylation of host and pathogen proteins in plants using click chemistry
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    Fluorescence:

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    Article Title: Novel Molecular Multilevel Targeted Antitumor Agents
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    Immunodetection:

    Article Title: Protein Complex Interactor Analysis and Differential Activity of KDM3 Subfamily Members Towards H3K9 Methylation
    Article Snippet: .. Immunodetection reagents used were α-V5 (Invitrogen) in conjunction with α-mouse-HRP (GE Healthcare) to detect V5-SCAI, and Streptavidin-HRP (Pierce) to detect Avi-KDM3A or B. .. Protein bands were visualized using ECL+ (GE Healthcare).

    Labeling:

    Article Title: Selenium-Based S-Adenosylmethionine Analog Reveals the Mammalian Seven-Beta-Strand Methyltransferase METTL10 to Be an EF1A1 Lysine Methyltransferase
    Article Snippet: Paragraph title: Labeling of full-length substrates with ProSeAM ... Proteins were separated on a 12.5% or 15% acrylamide gel and transferred to a nitrocellulose membrane (Pall Corporation, Port Washington, NY, USA); the membrane was incubated with streptavidin-HRP (Thermo Fisher Scientific Inc., Waltham, MA, USA) for 1 h at RT.

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    Purification:

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    Dot Blot:

    Article Title: Bisulfite-free, single base-resolution analysis of 5-hydroxymethylcytosine in genomic DNA by chemical-mediated mismatch single base-resolution analysis of 5-hydroxymethylcytosine in genomic DNA by chemical-mediated mismatch †Electronic supplementary information (ESI) available. See DOI: 10.1039/c8sc0427
    Article Snippet: Paragraph title: Dot-blot assay ... After labeling with streptavidin–HRP (1 : 1500) (Thermo Scientific) at 37 °C for 1 h and washing with 1× TBST four times, the results were visualized by enhanced chemiluminescence (SuperSignal™ West Pico Chemiluminescent Substrate, Cat: 34077, Thermo Scientific) using a Molecular Imager® ChemiDocTM XRS+ Imaging System (Bio-Rad).

    Direct ELISA:

    Article Title: Cross-Reactive HIV-1 Neutralizing Monoclonal Antibodies Selected by Screening of an Immune Human Phage Library against an Envelope Glycoprotein (gp140) Isolated from a Patient (R2) with Broadly HIV-1 Neutralizing Antibodies
    Article Snippet: Direct ELISA was carried out by coating gp120/140s on the plates at a concentration of 1 μg/ml. .. Bound biotinylated Fabs were detected by streptavidin-HRP (Pierce, Rockford, IL) and optical densities were measured.

    Chromatin Immunoprecipitation:

    Article Title: The splicing regulator PTBP2 is an AID interacting protein and promotes binding of AID to switch region DNA
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    SDS Page:

    Article Title: CD9 Regulates Major Histocompatibility Complex Class II Trafficking in Monocyte-Derived Dendritic Cells
    Article Snippet: After washing, beads were resuspended in Laemmli buffer and separated by SDS-PAGE. .. For internalization and recycling assays, biotinylated proteins were revealed using a Fujifilm LAS-4000 system after membrane incubation with streptavidin-HRP (Invitrogen).

    Article Title: Protein Complex Interactor Analysis and Differential Activity of KDM3 Subfamily Members Towards H3K9 Methylation
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    Article Title: Novel Molecular Multilevel Targeted Antitumor Agents
    Article Snippet: .. The biotin-labeled proteins were separated on an SDS-PAGE gel and a Western blot carried out using streptavidin-HRP (Pierce, Rockford, IL) to detect for biotin-labeled proteins. .. The number of biotin molecules attached to the proteins was determined by the FluoReporter Biotin Quantitation assay kit (Invitrogen) as per the manufacturer’s guidelines.

    Ubiquitin Assay:

    Article Title: CD9 Regulates Major Histocompatibility Complex Class II Trafficking in Monocyte-Derived Dendritic Cells
    Article Snippet: For internalization and recycling assays, biotinylated proteins were revealed using a Fujifilm LAS-4000 system after membrane incubation with streptavidin-HRP (Invitrogen). .. For ubiquitination assay, membranes were probed with antiubiquitin antibody (P4D1; Enzo Lifescience).

    Recombinant:

    Article Title: Selenium-Based S-Adenosylmethionine Analog Reveals the Mammalian Seven-Beta-Strand Methyltransferase METTL10 to Be an EF1A1 Lysine Methyltransferase
    Article Snippet: Labeling of full-length substrates with ProSeAM One microgram of recombinant full-length histone H3, His-HSP90 or His-HSP70 was incubated in 1× reaction buffer (50 mM Tris-HCl, pH 8.0) with GST-G9a (0.5 µg), FLAG-tagged KMTs (0.5 µg), His-SMYD2 (1 µg) or His-METTL21A (1 µg) with or without ProSeAM (250 µM, unless otherwise noted) for 2 h at 20°C. .. Proteins were separated on a 12.5% or 15% acrylamide gel and transferred to a nitrocellulose membrane (Pall Corporation, Port Washington, NY, USA); the membrane was incubated with streptavidin-HRP (Thermo Fisher Scientific Inc., Waltham, MA, USA) for 1 h at RT.

    Article Title: Cross-Reactive HIV-1 Neutralizing Monoclonal Antibodies Selected by Screening of an Immune Human Phage Library against an Envelope Glycoprotein (gp140) Isolated from a Patient (R2) with Broadly HIV-1 Neutralizing Antibodies
    Article Snippet: ELISA D7324 capture assays with soluble Fabs (m22, m24 and m46) and recombinant HIV-1 gp120s or gp140s from different isolates were performed by using 96-well Nunc-Immuno™ Maxisorp™ surface plates (Nalgen Nunc International, Denmark) as described ( ). .. Bound biotinylated Fabs were detected by streptavidin-HRP (Pierce, Rockford, IL) and optical densities were measured.

    Quantitation Assay:

    Article Title: Novel Molecular Multilevel Targeted Antitumor Agents
    Article Snippet: The biotin-labeled proteins were separated on an SDS-PAGE gel and a Western blot carried out using streptavidin-HRP (Pierce, Rockford, IL) to detect for biotin-labeled proteins. .. The number of biotin molecules attached to the proteins was determined by the FluoReporter Biotin Quantitation assay kit (Invitrogen) as per the manufacturer’s guidelines.

    Concentration Assay:

    Article Title: PA1b Inhibitor Binding to Subunits c and e of the Vacuolar ATPase Reveals Its Insecticidal Mechanism *
    Article Snippet: .. For the second experiment, 4 μl of V-ATPase (4 μg) was mixed with 3 μl of biotin-PA1b (3 μg) and 3 μl of streptavidin-HRP (15 μg), made up to 60 μl using V-ATPase buffer and incubated for 30 min. Mg·ATP was from a stock solution of 100 mm at pH 7.5 to a final concentration of 5 mm , and the mixture was incubated at room temperature for 5 min to allow for complete turnover. ..

    Article Title: Novel Molecular Multilevel Targeted Antitumor Agents
    Article Snippet: The biotin-labeled proteins were separated on an SDS-PAGE gel and a Western blot carried out using streptavidin-HRP (Pierce, Rockford, IL) to detect for biotin-labeled proteins. .. The fluorescent signals were measured using the BMG Optima plate reader (Ortenberg, Germany) and data was plotted as concentration of the standard Biocytin in pmoles versus relative fluorescence units.

    Article Title: Cross-Reactive HIV-1 Neutralizing Monoclonal Antibodies Selected by Screening of an Immune Human Phage Library against an Envelope Glycoprotein (gp140) Isolated from a Patient (R2) with Broadly HIV-1 Neutralizing Antibodies
    Article Snippet: Following the addition of three-fold serially diluted Fabs (m14, m16, m18, X5) IgGs (b12, 17b) and sCD4, equal volume of biotinylated Fabs (m22, m24 and m46) at a concentration, which led to 70% maximum binding, was simultaneously added to each well. .. Bound biotinylated Fabs were detected by streptavidin-HRP (Pierce, Rockford, IL) and optical densities were measured.

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    Thermo Fisher streptavidin conjugated hrp
    Photoaffinity labeling of various PrP species. <t>Streptavidin-HRP-probed</t> blots of samples photoaffinity labeled with PA-PBD peptide. (A) Samples containing PrP Int1 or PrP C were incubated with or without PA-PBD and exposed to UV light for varying time periods, as indicated. (B) Samples containing α -helical PrP or PrP Int1 were incubated with PA-PBD and exposed to UV light for 5 min. (C) Samples of PrP Int1 were incubated with varying concentrations of PA-PBD, as indicated, and exposed to UV light for 0 or 5 min, as indicated. (D) Sample containing 7 μ g of PrP Int1 photoaffinity labeled with PA-PBD (PA-PrP Int1 ) is compared to a standard curve of biotinylated AviTag PrP for reference.
    Streptavidin Conjugated Hrp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher streptavidin hrp
    Expression of recombinant porcine CCL2. (A) CHO cell line stably expressing the porcine CCL2 fused to GFP. The expression of GFP fusion protein was directly analysed by flow cytometry. Non transfected CHO cells were used as negative control (grey histogram). 5 000 cells were acquired. (B) Western blot of CCL2-GFP produced by transfected CHO cells. Different dilutions of supernatant were resolved by 15% SDS-PAGE under reducing conditions and revealed with biotinylated anti-GFP and <t>streptavidin-HRP.</t> Numbers on the left indicate the position of MW markers. (C) Chemotactic activity of CCL2-GFP on porcine blood monocytes. Chemotaxis was assessed with the Transwell cell migration system and subsequent flow cytometry counting of migrated cells by a 45 s acquisition. (1) FSC versus SSC dot plot of migrated cells in response to supernatants from CHO cells expressing CCL2-GFP or the inverted sequence of pCCL2 fused to GFP (InvCCL2-GFP, negative control). (2) Results expressed as migration index, calculated as the ratio of the number of cells migrating to the chemokine and the number of cells in the negative control. Results from one representative experiment out of three performed are shown. (A color version of this figure is available at www.vetres.org. )
    Streptavidin Hrp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 162 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


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

    Journal: Biochemistry

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

    doi: 10.1021/bi4014825

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

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

    Techniques: Labeling, Incubation

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

    Journal: Nature Communications

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

    doi: 10.1038/ncomms7765

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

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

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

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

    Journal: Veterinary Research

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

    doi: 10.1051/vetres/2010048

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

    Article Snippet: The expression of GFP-fused proteins in these clones was confirmed by Western blot using a biotin-conjugated goat anti-GFP polyclonal antibody and streptavidin-HRP.

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

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

    Journal: The Journal of Biological Chemistry

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

    doi: 10.1074/jbc.M113.541250

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

    Article Snippet: For the second experiment, 4 μl of V-ATPase (4 μg) was mixed with 3 μl of biotin-PA1b (3 μg) and 3 μl of streptavidin-HRP (15 μg), made up to 60 μl using V-ATPase buffer and incubated for 30 min. Mg·ATP was from a stock solution of 100 mm at pH 7.5 to a final concentration of 5 mm , and the mixture was incubated at room temperature for 5 min to allow for complete turnover.

    Techniques: Electron Microscopy, Generated, Produced