streptavidin hrp  (Thermo Fisher)


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    Structured Review

    Thermo Fisher streptavidin hrp
    CD9 regulates MHC-II internalization and recycling in mature MoDCs. (A and B) Immature (A) and LPS-matured (B) WT and CD9 KO MoDCs were incubated with biotinylated MHC-II antibodies for 1 h at 4°C, washed, and incubated for different times at 37°C, and then MHC-II surface expression was detected by flow cytometry after <t>streptavidin</t> labeling in CD11c + cells. Data represent mean fold changes ± SEM of results from three independent experiments performed in triplicate and were analyzed by two-way ANOVA with Bonferroni's post hoc multiple-comparison test. (C and D) Measurement of MHC-II internalization (C) and recycling (D) in mature WT and CD9 KO MoDCs by cell biotinylation assay. After incubation with biotin (blue in the schemes) at 4°C, cells were kept at 37°C (internalization), and the remaining surface biotins were removed by MESNA washing. (D) For recycling experiments, cells were further incubated for different times at 37°C (recycling), and surface biotins removed by MESNA washing. Cell lysates were immunoprecipitated with an anti-MHC-II antibody (M5/114), and biotinylated proteins were detected after membrane incubation with <t>streptavidin-HRP</t> (StrepHRP). Membranes were reprobed with MHC-II antibody for loading measurement. The blots shown are from representative experiments. (C) Only a fractional amount from cells incubated at 4°C in the absence of MESNA (0 min) was loaded. The graph shows the biotinylated MHC-II/total MHC-II signal ratio. The graph shows data determined as follows: [1 − (biotinylated MHC-II/total MHC-II signal ratio)]. Data represent mean fold changes ± SEM of results from four (C) and three (D) independent experiments analyzed by two-way ANOVA with Bonferroni's post hoc multiple-comparison test. **, P
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    Images

    1) 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

    2) 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

    3) 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

    4) 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

    5) 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

    6) 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

    7) 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

    8) 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

    9) 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

    10) 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

    11) 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

    12) Product Images from "TRPC3 Activation by Erythropoietin Is Modulated by TRPC6"

    Article Title: TRPC3 Activation by Erythropoietin Is Modulated by TRPC6

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M804734200

    Modulation of membrane insertion of TRPC3 by Epo detected by cell surface biotinylation. HEK 293T cells transfected ( Tx'd ) with Epo-R and V5-TRPC3 without ( A and B ) or with FLAG-TRPC6 ( C and D ) were stimulated with 40 units/ml Epo. Biotinylation of cell surface proteins was performed, and V5-TRPC3 immunoprecipitated ( IP ) from lysates with anti-V5 antibody. Western blots ( WB ) were probed with streptavidin-HRP to detect biotinylated TRPC3 and anti-V5-HRP to detect total V5-TRPC3. Representative results of Western blots from four experiments are shown in A and C . Biotinylated and total TRPC3 bands were quantitated with densitometry, and the ratio was normalized to time 0. The mean ± S.E. of the biotinylated/total TRPC3 ratios at 0, 1, 5, 10, and 20 min from four experiments are shown ( B and D ). * indicates a significant difference in the ratio compared with time 0 ( p ≤ 0.02). E, Epo-stimulated cell surface expression of endogenous TRPC3 was examined using BFU-E-derived erythroblasts at day 10 of methylcellulose culture (two experiments) or erythroblasts from phase II day 8 of liquid culture (one experiment). Cells were stimulated with 40 units/ml Epo for 0 or 5 min and biotinylated, and TRPC3 was immunoprecipitated from lysates with anti-TRPC3 antibody. Western blots were probed with streptavidin-HRP to detect biotinylated TRPC3 and anti-TRPC3 to detect total TRPC3. A representative result of three Western blots is shown. F, biotinylated and total TRPC3 bands were quantitated with densitometry, and the ratio was normalized to time 0. The mean ± S.E. of the biotinylated/total TRPC3 ratios at 0 and 5 min from the three experiments are shown. No significant difference in the ratio at 5 min compared with time 0 was detected.
    Figure Legend Snippet: Modulation of membrane insertion of TRPC3 by Epo detected by cell surface biotinylation. HEK 293T cells transfected ( Tx'd ) with Epo-R and V5-TRPC3 without ( A and B ) or with FLAG-TRPC6 ( C and D ) were stimulated with 40 units/ml Epo. Biotinylation of cell surface proteins was performed, and V5-TRPC3 immunoprecipitated ( IP ) from lysates with anti-V5 antibody. Western blots ( WB ) were probed with streptavidin-HRP to detect biotinylated TRPC3 and anti-V5-HRP to detect total V5-TRPC3. Representative results of Western blots from four experiments are shown in A and C . Biotinylated and total TRPC3 bands were quantitated with densitometry, and the ratio was normalized to time 0. The mean ± S.E. of the biotinylated/total TRPC3 ratios at 0, 1, 5, 10, and 20 min from four experiments are shown ( B and D ). * indicates a significant difference in the ratio compared with time 0 ( p ≤ 0.02). E, Epo-stimulated cell surface expression of endogenous TRPC3 was examined using BFU-E-derived erythroblasts at day 10 of methylcellulose culture (two experiments) or erythroblasts from phase II day 8 of liquid culture (one experiment). Cells were stimulated with 40 units/ml Epo for 0 or 5 min and biotinylated, and TRPC3 was immunoprecipitated from lysates with anti-TRPC3 antibody. Western blots were probed with streptavidin-HRP to detect biotinylated TRPC3 and anti-TRPC3 to detect total TRPC3. A representative result of three Western blots is shown. F, biotinylated and total TRPC3 bands were quantitated with densitometry, and the ratio was normalized to time 0. The mean ± S.E. of the biotinylated/total TRPC3 ratios at 0 and 5 min from the three experiments are shown. No significant difference in the ratio at 5 min compared with time 0 was detected.

    Techniques Used: Transfection, Immunoprecipitation, Western Blot, Expressing, Derivative Assay

    Plasma membrane insertion of TRPC3/TRPC3 chimeras detected with cell surface biotinylation. Cell surface biotinylation was performed with HEK 293T cells expressing V5-TRPC3, V5-TRPC3-C6C, V5-TRPC3-C6C1, V5-TRPC3-C6C2, FLAG-TRPC6, FLAG-TRPC6-C3C, or FLAG-TRPC6-C3N and Epo-R. Lysates were prepared, and immunoprecipitation ( IP ) performed with anti-V5 antibody or anti-FLAG-agarose. Western blotting ( WB ) was performed on immunoprecipitation pellets with streptavidin-HRP to detect biotinylation and either anti-V5-HRP to detect total TRPC3 chimeras or anti-TRPC6 or anti-TRPC3-N antibodies to detect total TRPC6 chimeras. Representative results of two experiments are shown. Tx'd , transfected.
    Figure Legend Snippet: Plasma membrane insertion of TRPC3/TRPC3 chimeras detected with cell surface biotinylation. Cell surface biotinylation was performed with HEK 293T cells expressing V5-TRPC3, V5-TRPC3-C6C, V5-TRPC3-C6C1, V5-TRPC3-C6C2, FLAG-TRPC6, FLAG-TRPC6-C3C, or FLAG-TRPC6-C3N and Epo-R. Lysates were prepared, and immunoprecipitation ( IP ) performed with anti-V5 antibody or anti-FLAG-agarose. Western blotting ( WB ) was performed on immunoprecipitation pellets with streptavidin-HRP to detect biotinylation and either anti-V5-HRP to detect total TRPC3 chimeras or anti-TRPC6 or anti-TRPC3-N antibodies to detect total TRPC6 chimeras. Representative results of two experiments are shown. Tx'd , transfected.

    Techniques Used: Expressing, Immunoprecipitation, Western Blot, Transfection

    13) Product Images from "Ferritin Blocks Inhibitory Effects of Two-Chain High Molecular Weight Kininogen (HKa) on Adhesion and Survival Signaling in Endothelial Cells"

    Article Title: Ferritin Blocks Inhibitory Effects of Two-Chain High Molecular Weight Kininogen (HKa) on Adhesion and Survival Signaling in Endothelial Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0040030

    Recombinant HFt and LFt bind to HKa and and inhibit its anti-proliferative activity. A. Binding of recombinant HFt and LFt to domain 5 of HKa. Purified and biotinylated recombinant HFt (20 µg) or LFt (20 µg) were incubated with 10 µg GST-D5 and the resulting complexes immunoprecipited with anti-GST antibody. Non-biotinylated HFt and LFt were used in the immunopreciptation shown in lane 4. The membranes were probed with streptavidin-HRP to detect biotinylated ferritin (b-ferritin) as well as with anti-GST antibody. B. Cells were treated with 50 nM HKa alone, or co-treated with 100 nM of HFt or LFt in the presence of 20 ng/ml bFGF and 10 µM ZnCl 2 . Cell viability was assessed using an MTT assay 24 hours post-treatment. Shown are means and standard deviation of triplicate experiments with ** p
    Figure Legend Snippet: Recombinant HFt and LFt bind to HKa and and inhibit its anti-proliferative activity. A. Binding of recombinant HFt and LFt to domain 5 of HKa. Purified and biotinylated recombinant HFt (20 µg) or LFt (20 µg) were incubated with 10 µg GST-D5 and the resulting complexes immunoprecipited with anti-GST antibody. Non-biotinylated HFt and LFt were used in the immunopreciptation shown in lane 4. The membranes were probed with streptavidin-HRP to detect biotinylated ferritin (b-ferritin) as well as with anti-GST antibody. B. Cells were treated with 50 nM HKa alone, or co-treated with 100 nM of HFt or LFt in the presence of 20 ng/ml bFGF and 10 µM ZnCl 2 . Cell viability was assessed using an MTT assay 24 hours post-treatment. Shown are means and standard deviation of triplicate experiments with ** p

    Techniques Used: Recombinant, Activity Assay, Binding Assay, Purification, Incubation, MTT Assay, Standard Deviation

    14) 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

    15) Product Images from "A cryptic Tudor domain links BRWD2/PHIP to COMPASS-mediated histone H3K4 methylation"

    Article Title: A cryptic Tudor domain links BRWD2/PHIP to COMPASS-mediated histone H3K4 methylation

    Journal: Genes & Development

    doi: 10.1101/gad.305201.117

    Characterization of the BRWD2/PHIP CryptoTudor domain. ( A ) Diagram depicting the recombinant BRWD2/PHIP CryptoTudor–bromodomain (BRWD2/PHIP–Crypt–bromo) construct used for binding studies. N-terminal 10X-Histidine (red box) and C-terminal 1XFlag (green box) tags were included for protein purification. ( B ) Coomassie stained SDS-PAGE gel of in vitro chromatin capture experiment using the BRWD2/PHIP–Crypt–bromo module. (Lane 1 ) Input MNase-digested chromatin. (Lane 2 ) Chromatin subjected to anti-Flag immunoprecipitation without BRWD2/PHIP–Crypt–bromo. (Lane 3 ) Chromatin incubated with recombinant BRWD2/PHIP–Crypt–bromo and subjected to anti-Flag immunoprecipitation. ( C ) Quantitative MS analysis of histones captured in B . Bars indicate the fraction of total histone represented by each modification state in the input (gray) or Flag immunoprecipitation material (red). (Un) Unmodified; (me1) monomethylated; (me2) dimethylated; (me3) trimethylated; (Ac) acetylated. ( D ) Coomassie-stained SDS-PAGE gel of histone peptide pull-downs performed with human ( top ) or Drosophila ( bottom ) Crypt–bromo constructs. Recombinant protein was incubated with streptavidin beads alone (SA-beads) or the biotinylated histone peptides indicated. Ten percent of input and 20% of each pull-down sample were loaded. ( Bottom ) Eluted proteins were subjected to dot blotting using streptavidin-HRP (SA-HRP). ( E ) ITC experiments with human BRWD2–Crypt–bromo titrated with H3 unmodified ( left ), H3K4me1 ( middle ), and H3K4me3 ( right ) peptides. ( F ) Coomassie-stained SDS-PAGE gel of pull-downs performed with human BRWD2/PHIP–Crypt–bromo and a panel of histone peptides. Ten percent of input and 20% of each pull-down sample were loaded. Streptavidin-HRP dot blot loading control is shown below the gel image. ( G ) Coomassie-stained SDS-PAGE gel of a panel of histone peptide pull-downs performed with human BRWD2–Crypt–bromo ( top ), the isolated CryptoTudor domain ( middle ), or the isolated tandem bromodomains ( bottom ). Ten percent of input and 20% of each pull-down sample were loaded. Dot blot loading control is shown below each gel image. ( H ) Coomassie-stained gels of histone peptide pull-downs performed with point mutations of the BRWD2 CryptoTudor domain. Dot blot loading controls are shown below the gels. ( I ). Positions of residues predicted to be involved in methyl-lysine binding are highlighted in red.
    Figure Legend Snippet: Characterization of the BRWD2/PHIP CryptoTudor domain. ( A ) Diagram depicting the recombinant BRWD2/PHIP CryptoTudor–bromodomain (BRWD2/PHIP–Crypt–bromo) construct used for binding studies. N-terminal 10X-Histidine (red box) and C-terminal 1XFlag (green box) tags were included for protein purification. ( B ) Coomassie stained SDS-PAGE gel of in vitro chromatin capture experiment using the BRWD2/PHIP–Crypt–bromo module. (Lane 1 ) Input MNase-digested chromatin. (Lane 2 ) Chromatin subjected to anti-Flag immunoprecipitation without BRWD2/PHIP–Crypt–bromo. (Lane 3 ) Chromatin incubated with recombinant BRWD2/PHIP–Crypt–bromo and subjected to anti-Flag immunoprecipitation. ( C ) Quantitative MS analysis of histones captured in B . Bars indicate the fraction of total histone represented by each modification state in the input (gray) or Flag immunoprecipitation material (red). (Un) Unmodified; (me1) monomethylated; (me2) dimethylated; (me3) trimethylated; (Ac) acetylated. ( D ) Coomassie-stained SDS-PAGE gel of histone peptide pull-downs performed with human ( top ) or Drosophila ( bottom ) Crypt–bromo constructs. Recombinant protein was incubated with streptavidin beads alone (SA-beads) or the biotinylated histone peptides indicated. Ten percent of input and 20% of each pull-down sample were loaded. ( Bottom ) Eluted proteins were subjected to dot blotting using streptavidin-HRP (SA-HRP). ( E ) ITC experiments with human BRWD2–Crypt–bromo titrated with H3 unmodified ( left ), H3K4me1 ( middle ), and H3K4me3 ( right ) peptides. ( F ) Coomassie-stained SDS-PAGE gel of pull-downs performed with human BRWD2/PHIP–Crypt–bromo and a panel of histone peptides. Ten percent of input and 20% of each pull-down sample were loaded. Streptavidin-HRP dot blot loading control is shown below the gel image. ( G ) Coomassie-stained SDS-PAGE gel of a panel of histone peptide pull-downs performed with human BRWD2–Crypt–bromo ( top ), the isolated CryptoTudor domain ( middle ), or the isolated tandem bromodomains ( bottom ). Ten percent of input and 20% of each pull-down sample were loaded. Dot blot loading control is shown below each gel image. ( H ) Coomassie-stained gels of histone peptide pull-downs performed with point mutations of the BRWD2 CryptoTudor domain. Dot blot loading controls are shown below the gels. ( I ). Positions of residues predicted to be involved in methyl-lysine binding are highlighted in red.

    Techniques Used: Recombinant, Construct, Binding Assay, Protein Purification, Staining, SDS Page, In Vitro, Immunoprecipitation, Incubation, Mass Spectrometry, Modification, Dot Blot, Isolation

    16) 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

    17) 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

    18) 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

    19) 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

    20) Product Images from "Network analysis of UBE3A/E6AP-associated proteins provides connections to several distinct cellular processes"

    Article Title: Network analysis of UBE3A/E6AP-associated proteins provides connections to several distinct cellular processes

    Journal: Journal of molecular biology

    doi: 10.1016/j.jmb.2018.01.021

    UBE3A promotes the degradation of ASPP2 by the proteasome A.UBE3A coimmunoprecipitates ASPP2. HEK 293T cells were transfected with the indicated vectors. 48 hours after transfection the HA-tagged proteins were immunoprecipitated with anti-HA agarose beads. Protein extracts and immunoprecipitates were analyzed by SDS-PAGE and Western blot using antibodies against HA-tag and Actin. HBH-ASPP2 carrying a biotinylation signal in the HBH tag was detected using streptavidin-HRP. HA-UBE3A: UBE3A isoform 1 C820A, catalytically inactive. B. Coexpression of UBE3A reduces ASPP2 protein levels. HEK 293T cells were transfected with the corresponding vectors. 48 hours post transfection the cells were harvested and the protein extracts were analyzed by SDS-PAGE and Western blot. Proteins were detected using anti HA, V5 and actin antibodies, and streptavidin HRP. Of note, UBE3A runs as a double band while its catalytically inactive form runs as a single band. - indicates that the cells were transfected with the corresponding empty vector. WT: wild type, CA: catalytically inactive form of UBE3A.
    Figure Legend Snippet: UBE3A promotes the degradation of ASPP2 by the proteasome A.UBE3A coimmunoprecipitates ASPP2. HEK 293T cells were transfected with the indicated vectors. 48 hours after transfection the HA-tagged proteins were immunoprecipitated with anti-HA agarose beads. Protein extracts and immunoprecipitates were analyzed by SDS-PAGE and Western blot using antibodies against HA-tag and Actin. HBH-ASPP2 carrying a biotinylation signal in the HBH tag was detected using streptavidin-HRP. HA-UBE3A: UBE3A isoform 1 C820A, catalytically inactive. B. Coexpression of UBE3A reduces ASPP2 protein levels. HEK 293T cells were transfected with the corresponding vectors. 48 hours post transfection the cells were harvested and the protein extracts were analyzed by SDS-PAGE and Western blot. Proteins were detected using anti HA, V5 and actin antibodies, and streptavidin HRP. Of note, UBE3A runs as a double band while its catalytically inactive form runs as a single band. - indicates that the cells were transfected with the corresponding empty vector. WT: wild type, CA: catalytically inactive form of UBE3A.

    Techniques Used: Transfection, Immunoprecipitation, SDS Page, Western Blot, Plasmid Preparation

    21) Product Images from "Liver ubiquitome uncovers nutrient-stress-mediated trafficking and secretion of complement C3"

    Article Title: Liver ubiquitome uncovers nutrient-stress-mediated trafficking and secretion of complement C3

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2016.312

    Pro-C3 is ubiquitylated in the livers of refed mice. ( a and b ) Western blotting after anti-FLAG immunoprecipitation (FLAG IP) ( a ) and histidin-biotin pulldowns (His-Bio pulldown) ( b ) of whole-cell lysates from HEK293T cells ectopically expressing FLAG-tagged C3 and His-Bio-tagged ubiquitin. Cells expressing FLAG alone were used as a control. Cells were treated with DMSO, 10 μ M of proteasome inhibitor MG132 or 50 μ g/ml BFA for 4 h as indicated. Input, FLAG IP or HIS-Bio pulldowns are shown separately. Signals with an antibody against FLAG-tag or revealed by streptavidin conjugated to horseradish peroxidase (Strep-HRP) are shown separately. ( c – e ) Western blotting after FLAG-IP ( c and e ) and a His-Bio pulldown ( d ) of whole-cell lysates from primary murine hepatocytes ectopically expressing FLAG-tagged C3 and His-Bio-tagged ubiquitin. Cells expressing FLAG alone were used as a control. Cells were treated with DMSO, MG132 ( c and d ) or BFA ( e ) as indicated. Input, FLAG IP or HIS-Bio pulldowns are shown separately. Signals with an antibody against FLAG tag or revealed by streptavidin conjugated to horseradish peroxidase (Strep-HRP) are shown separately. ( f ) Western blotting after TUBEs 1 pulldowns of whole-liver lysates from fasted and refed mice as indicated. Input and pulldowns are shown separately. Liver lysates from C3 knockout mice (C3KO) were used as a control. Antibodies against ubiquitin (P4D1) and C3a were used. Arrows indicate endogenous pro-C3 and the alpha chain ( α chain) of mature C3
    Figure Legend Snippet: Pro-C3 is ubiquitylated in the livers of refed mice. ( a and b ) Western blotting after anti-FLAG immunoprecipitation (FLAG IP) ( a ) and histidin-biotin pulldowns (His-Bio pulldown) ( b ) of whole-cell lysates from HEK293T cells ectopically expressing FLAG-tagged C3 and His-Bio-tagged ubiquitin. Cells expressing FLAG alone were used as a control. Cells were treated with DMSO, 10 μ M of proteasome inhibitor MG132 or 50 μ g/ml BFA for 4 h as indicated. Input, FLAG IP or HIS-Bio pulldowns are shown separately. Signals with an antibody against FLAG-tag or revealed by streptavidin conjugated to horseradish peroxidase (Strep-HRP) are shown separately. ( c – e ) Western blotting after FLAG-IP ( c and e ) and a His-Bio pulldown ( d ) of whole-cell lysates from primary murine hepatocytes ectopically expressing FLAG-tagged C3 and His-Bio-tagged ubiquitin. Cells expressing FLAG alone were used as a control. Cells were treated with DMSO, MG132 ( c and d ) or BFA ( e ) as indicated. Input, FLAG IP or HIS-Bio pulldowns are shown separately. Signals with an antibody against FLAG tag or revealed by streptavidin conjugated to horseradish peroxidase (Strep-HRP) are shown separately. ( f ) Western blotting after TUBEs 1 pulldowns of whole-liver lysates from fasted and refed mice as indicated. Input and pulldowns are shown separately. Liver lysates from C3 knockout mice (C3KO) were used as a control. Antibodies against ubiquitin (P4D1) and C3a were used. Arrows indicate endogenous pro-C3 and the alpha chain ( α chain) of mature C3

    Techniques Used: Mouse Assay, Western Blot, Immunoprecipitation, Expressing, FLAG-tag, Knock-Out

    22) 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

    23) 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

    24) 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

    25) 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

    26) 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

    27) 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

    28) 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

    29) 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

    30) 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

    31) 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

    32) 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

    33) 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

    34) 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

    35) 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

    36) 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

    37) 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

    38) Product Images from "Isolation of secreted proteins from Drosophila ovaries and embryos through in vivo BirA-mediated biotinylation"

    Article Title: Isolation of secreted proteins from Drosophila ovaries and embryos through in vivo BirA-mediated biotinylation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0219878

    secBirA expressed in Drosophila ovaries and embryos exhibits biotin ligase activity. Protein extracts from ovaries and embryos expressing (+) or lacking (-) secBirA were incubated with (+) or in the absence of (-) a BirA substrate, Maltose Binding Protein fused in-frame to the Biotin Acceptor Peptide (MBP substrate). After SDS-PAGE and blotting to nitrocellulose, biotinylated MBP was detected with streptavidin-HRP. The lane at far left (labelled C) contains commercially available biotinylated MBP (bio-MBP)(Avidity LLC) as a positive control. When incubated together with substrate, extracts from ovaries and progeny embryos of females expressing secBirA in the germline, and extracts of ovaries from females expressing secBirA in the follicle cell layer, exhibited a strong signal corresponding to biotinylated MBP (see arrows).
    Figure Legend Snippet: secBirA expressed in Drosophila ovaries and embryos exhibits biotin ligase activity. Protein extracts from ovaries and embryos expressing (+) or lacking (-) secBirA were incubated with (+) or in the absence of (-) a BirA substrate, Maltose Binding Protein fused in-frame to the Biotin Acceptor Peptide (MBP substrate). After SDS-PAGE and blotting to nitrocellulose, biotinylated MBP was detected with streptavidin-HRP. The lane at far left (labelled C) contains commercially available biotinylated MBP (bio-MBP)(Avidity LLC) as a positive control. When incubated together with substrate, extracts from ovaries and progeny embryos of females expressing secBirA in the germline, and extracts of ovaries from females expressing secBirA in the follicle cell layer, exhibited a strong signal corresponding to biotinylated MBP (see arrows).

    Techniques Used: Activity Assay, Expressing, Incubation, Binding Assay, SDS Page, Positive Control

    39) Product Images from "Connexin43 recruits PTEN and Csk to inhibit c-Src activity in glioma cells and astrocytes"

    Article Title: Connexin43 recruits PTEN and Csk to inhibit c-Src activity in glioma cells and astrocytes

    Journal: Oncotarget

    doi: 10.18632/oncotarget.10454

    The Cx43 region involved in the recruitment of PTEN and Csk C6 glioma cells were incubated with several cell-penetrating peptides containing the indicated sequences of Cx43 fused to biotin for 30 min. ( A ) The SH3 domain binding motif is shown in grey and the tyrosines phosphorylated by c-Src in red. ( B ) After 30 min, the cells were lysed, and pull-down assays were carried out with avidin-conjugated agarose beads. Western blots before (lysates) and after avidin pull-down for c-Src, Csk and PTEN showing the enrichment of PTEN and Csk in the complex obtained with TAT-Cx43-266-283-Biotin and, to a lesser extent, with TAT-Cx43-245-283-Biotin. Str-HRP, HRP-conjugated streptavidin. ( C ) After 30 min, the cells were fixed, and the uptake of peptides bound to biotin was analyzed by fluorescence microscopy. Scale bars: 15 μm.
    Figure Legend Snippet: The Cx43 region involved in the recruitment of PTEN and Csk C6 glioma cells were incubated with several cell-penetrating peptides containing the indicated sequences of Cx43 fused to biotin for 30 min. ( A ) The SH3 domain binding motif is shown in grey and the tyrosines phosphorylated by c-Src in red. ( B ) After 30 min, the cells were lysed, and pull-down assays were carried out with avidin-conjugated agarose beads. Western blots before (lysates) and after avidin pull-down for c-Src, Csk and PTEN showing the enrichment of PTEN and Csk in the complex obtained with TAT-Cx43-266-283-Biotin and, to a lesser extent, with TAT-Cx43-245-283-Biotin. Str-HRP, HRP-conjugated streptavidin. ( C ) After 30 min, the cells were fixed, and the uptake of peptides bound to biotin was analyzed by fluorescence microscopy. Scale bars: 15 μm.

    Techniques Used: Incubation, Binding Assay, Avidin-Biotin Assay, Western Blot, Fluorescence, Microscopy

    40) 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

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    Article Snippet: .. Anti-phos-PERK (Thr 981) and anti-β -Tubulin antibodies were purchased from Santa-Cruz Biotechnologies (Dallas, TX, USA); anti-eIF2α and anti-phos-eIF2α (Ser 51) antibodies were purchased from Abcam (Cambridge, MA, USA), anti-PARP16 antibody was generated by ourselves; Streptavidin-HRP was obtained from Thermo Fisher (Waltham, MA, USA); BFA, ECG and EGCG were purchased from Sigma-Aldrich (St Louis, MO, USA); TUN was obtained from Cell Signaling Technologies (Beverly, MA, USA); Biotin-labeled NAD+ from Invitrogen (Carlsbad, CA, USA); glutathione Sepharose 4B resin was obtained from GE Healthcare (Pittsburgh, PA, USA). ..

    Cell Culture:

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    Article Snippet: .. Cell culture medium, antibiotics, trypsin-EDTA, sterile PBS, goat anti-rabbit (H-L) and goat anti-mouse (H-L) secondary antibodies, streptavidin Alexa Fluor 647, streptavidin-HRP, and goat anti-mouse HRP were from Invitrogen (Carlsbad, CA). .. Primary antibodies mouse anti-TG2 (CUB 7402 + TG100) and rabbit anti-E-cadherin were from Thermo Fisher Scientific (Waltham, MA) and Cell Signaling Technology (Danvers, MA), respectively.

    Immunoprecipitation:

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

    Article Title: TRPC3 Activation by Erythropoietin Is Modulated by TRPC6
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    other:

    Article Title: Isolation of secreted proteins from Drosophila ovaries and embryos through in vivo BirA-mediated biotinylation
    Article Snippet: Biotinylated proteins were detected using streptavidin-HRP and imaged as described above.

    Sequencing:

    Article Title: TRPC3 Activation by Erythropoietin Is Modulated by TRPC6
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    Western Blot:

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  • 91
    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: 91/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin conjugated hrp/product/Thermo Fisher
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    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: 92/100, based on 152 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin hrp/product/Thermo Fisher
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    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