mbp  (New England Biolabs)


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    Name:
    Anti MBP Monoclonal Antibody
    Description:
    Anti MBP Monoclonal Antibody 0 25 ml
    Catalog Number:
    e8032l
    Price:
    712
    Size:
    0 25 ml
    Category:
    Primary Antibodies
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    Structured Review

    New England Biolabs mbp
    Anti MBP Monoclonal Antibody
    Anti MBP Monoclonal Antibody 0 25 ml
    https://www.bioz.com/result/mbp/product/New England Biolabs
    Average 99 stars, based on 82 article reviews
    Price from $9.99 to $1999.99
    mbp - by Bioz Stars, 2020-07
    99/100 stars

    Images

    1) Product Images from "The Yeast LATS/Ndr Kinase Cbk1 Regulates Growth via Golgi-dependent Glycosylation and Secretion"

    Article Title: The Yeast LATS/Ndr Kinase Cbk1 Regulates Growth via Golgi-dependent Glycosylation and Secretion

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E08-05-0455

    Cbk1 binds and phosphorylates Sec2. (A) Immunoblot showing the results of Sec2 affinity precipitation experiments with MBP fusion proteins. Recombinant MBP-Sec2 1-759 and MBP-Sec2 1-508 precipitate Cbk1-Myc from yeast cell extracts (from FLY2288). MBP-Sec2
    Figure Legend Snippet: Cbk1 binds and phosphorylates Sec2. (A) Immunoblot showing the results of Sec2 affinity precipitation experiments with MBP fusion proteins. Recombinant MBP-Sec2 1-759 and MBP-Sec2 1-508 precipitate Cbk1-Myc from yeast cell extracts (from FLY2288). MBP-Sec2

    Techniques Used: Affinity Precipitation, Recombinant

    2) Product Images from "Crystallogenesis of bacteriophage P22 tail accessory factor gp26 at acidic and neutral pH"

    Article Title: Crystallogenesis of bacteriophage P22 tail accessory factor gp26 at acidic and neutral pH

    Journal:

    doi: 10.1107/S1744309106013856

    Crystallization of gp26 fused to maltose-binding protein at neutral and alkaline pH. ( a ) A chimera of gp26 fused to maltose-binding protein (MBP-gp26) retains full oligomerization on SDS–PAGE. Unboiled MBP-gp26 in lane 1 runs on SDS–PAGE
    Figure Legend Snippet: Crystallization of gp26 fused to maltose-binding protein at neutral and alkaline pH. ( a ) A chimera of gp26 fused to maltose-binding protein (MBP-gp26) retains full oligomerization on SDS–PAGE. Unboiled MBP-gp26 in lane 1 runs on SDS–PAGE

    Techniques Used: Crystallization Assay, Binding Assay, SDS Page

    3) Product Images from "Peptide Mimic of the HIV Envelope gp120-gp41 Interface"

    Article Title: Peptide Mimic of the HIV Envelope gp120-gp41 Interface

    Journal:

    doi: 10.1016/j.jmb.2007.12.001

    Interaction between gp41 fragments and gp120 deletion mutants or MBP-C1/C5
    Figure Legend Snippet: Interaction between gp41 fragments and gp120 deletion mutants or MBP-C1/C5

    Techniques Used:

    4) Product Images from "Legionella effector Lpg1137 shuts down ER-mitochondria communication through cleavage of syntaxin 17"

    Article Title: Legionella effector Lpg1137 shuts down ER-mitochondria communication through cleavage of syntaxin 17

    Journal: Nature Communications

    doi: 10.1038/ncomms15406

    Lpg1137 is a serine protease localized in MAM/mitochondria. ( a ) HeLa-FcγRII cells were transfected with a plasmid encoding GFP (left) or GFP-Lpg1137 (right). At 24 h after transfection, cells were subjected to subcellular fractionation, and equal amounts of fractions were analysed by IB with the indicated antibodies. MS and MT denote microsomes and mitochondria, respectively. ( b ) HeLa-FcγRII cells were transfected with GFP-Lpg1137. At 4 h after transfection, DMSO (Vehicle), PMSF (1 mM) or MG132 (1 μM) was added to cells, and the cells were incubated for 20 h. Equal amounts of cell lysates were analysed by IB with the indicated antibodies. ( c , d ) HeLa-FcγRII cells were transfected with one of the indicated plasmids, and after 24 h equal amounts of lysates were analysed by IB with the ( c ) indicated antibodies. Alternatively, cells were fixed and stained with ( d ) an anti-Stx17 antibody. Scale bar, 5 μm. ( e ) His-Stx17 (0.2 μg) was incubated with MBP, MBP-Lpg1137 wild-type (WT) or MBP-Lpg1137 S68A (each 0.2 μg) for the indicated times at 37 °C. After incubation, samples were subjected to IB with antibodies against Stx17 and MBP. Uncropped images of blots are shown in Supplementary Fig. 7 .
    Figure Legend Snippet: Lpg1137 is a serine protease localized in MAM/mitochondria. ( a ) HeLa-FcγRII cells were transfected with a plasmid encoding GFP (left) or GFP-Lpg1137 (right). At 24 h after transfection, cells were subjected to subcellular fractionation, and equal amounts of fractions were analysed by IB with the indicated antibodies. MS and MT denote microsomes and mitochondria, respectively. ( b ) HeLa-FcγRII cells were transfected with GFP-Lpg1137. At 4 h after transfection, DMSO (Vehicle), PMSF (1 mM) or MG132 (1 μM) was added to cells, and the cells were incubated for 20 h. Equal amounts of cell lysates were analysed by IB with the indicated antibodies. ( c , d ) HeLa-FcγRII cells were transfected with one of the indicated plasmids, and after 24 h equal amounts of lysates were analysed by IB with the ( c ) indicated antibodies. Alternatively, cells were fixed and stained with ( d ) an anti-Stx17 antibody. Scale bar, 5 μm. ( e ) His-Stx17 (0.2 μg) was incubated with MBP, MBP-Lpg1137 wild-type (WT) or MBP-Lpg1137 S68A (each 0.2 μg) for the indicated times at 37 °C. After incubation, samples were subjected to IB with antibodies against Stx17 and MBP. Uncropped images of blots are shown in Supplementary Fig. 7 .

    Techniques Used: Transfection, Plasmid Preparation, Fractionation, Mass Spectrometry, Incubation, Staining

    5) Product Images from "The E3 Ligase AtRDUF1 Positively Regulates Salt Stress Responses in Arabidopsis thaliana"

    Article Title: The E3 Ligase AtRDUF1 Positively Regulates Salt Stress Responses in Arabidopsis thaliana

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0071078

    Analysis of the AtRDUF1 protein. (A) Alignment of the RING finger domains of the AtRDUF1 homologs in Arabidopsis . Black and gray indicate 100% and ≥50% identities, respectively. (B) Subcellular localization of AtRDUF1:GFP fusion protein in Arabidopsis leaf protoplast cells. Bars represent 20 μm. The green and blue fluorescenece are GFP and 4′,6-diamidino-2-phenylindole (DAPI) signals, respectively. (C) Verification of E3 ligase activity of AtRDUF1 by in vitro autoubiquitination assay. CH/Y represents the mutant form of the MBP:AtRDUF1 fusion protein, with substitution of metal ligand positions Cys-3, His-4, and His-5 of the RING motif with Tyr. The numbers at left denote the molecular masses of marker proteins in kilodaltons. Nichel-HRP (Ub), the nickel-horseradish peroxidase used to detect His-tagged ubiquitin. Anti-MBP, the anti-MBP antibody to detect maltose fusion proteins.
    Figure Legend Snippet: Analysis of the AtRDUF1 protein. (A) Alignment of the RING finger domains of the AtRDUF1 homologs in Arabidopsis . Black and gray indicate 100% and ≥50% identities, respectively. (B) Subcellular localization of AtRDUF1:GFP fusion protein in Arabidopsis leaf protoplast cells. Bars represent 20 μm. The green and blue fluorescenece are GFP and 4′,6-diamidino-2-phenylindole (DAPI) signals, respectively. (C) Verification of E3 ligase activity of AtRDUF1 by in vitro autoubiquitination assay. CH/Y represents the mutant form of the MBP:AtRDUF1 fusion protein, with substitution of metal ligand positions Cys-3, His-4, and His-5 of the RING motif with Tyr. The numbers at left denote the molecular masses of marker proteins in kilodaltons. Nichel-HRP (Ub), the nickel-horseradish peroxidase used to detect His-tagged ubiquitin. Anti-MBP, the anti-MBP antibody to detect maltose fusion proteins.

    Techniques Used: Activity Assay, In Vitro, Mutagenesis, Marker

    6) Product Images from "Molecular Basis of Filamin A-FilGAP Interaction and Its Impairment in Congenital Disorders Associated with Filamin A Mutations"

    Article Title: Molecular Basis of Filamin A-FilGAP Interaction and Its Impairment in Congenital Disorders Associated with Filamin A Mutations

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0004928

    Point mutations of FLNa and FilGAP confirms the in silico model of their binding interaction. (A) A point mutation in FLNa (M2474E) abolishes the complexing of FLNa and FilGAP. The upper panel shows amylose beads coated with MBP-FilGAP649-748 pulls down wild-type FLNa but not FLNaM2474E. The lower panel shows wild-type (WT) FLAG-FLNa immobilized on FLAG-specific mAb immobilized on agarose beads, but not FLAG-FLNaM2474E, pull down full-length FilGAP. (B) FLAG-FLNa does not pull point mutants of FilGAP at G730W and V734Y. T728V mutation has no effect on the interaction.
    Figure Legend Snippet: Point mutations of FLNa and FilGAP confirms the in silico model of their binding interaction. (A) A point mutation in FLNa (M2474E) abolishes the complexing of FLNa and FilGAP. The upper panel shows amylose beads coated with MBP-FilGAP649-748 pulls down wild-type FLNa but not FLNaM2474E. The lower panel shows wild-type (WT) FLAG-FLNa immobilized on FLAG-specific mAb immobilized on agarose beads, but not FLAG-FLNaM2474E, pull down full-length FilGAP. (B) FLAG-FLNa does not pull point mutants of FilGAP at G730W and V734Y. T728V mutation has no effect on the interaction.

    Techniques Used: In Silico, Binding Assay, Mutagenesis

    Localization of FLNa-FilGAP binding site. (A) Schematic representation of FilGAP and its truncation series. The pleckstrin-homology (PH), GTPase-activating protein (GAP), and coiled-coil (CC) domains predicted by EMBnet COILS are shown. Right panel shows binding of FLAG-FLNa to GST-FilGAP fragments illustrated in the left panel. Their interactions were analyzed by pull-down using FLAG-specific mAb immobilized on beads. Bound protein was detected by immunoblotting using rabbit pAb to GST. (B) FilGAP fragments were fused to MBP-His-tag and their binding to FLAG-FLNa were analyzed by pull-down using FLAG-specific mAb immobilized on beads. Bound protein was detected by immunoblotting using rabbit pAb to MBP (C) His-tag FilGAP, or FilGAP lacking residues 649–725 (50 nM), were mixed with increasing amounts of FLAG-FLNa and immunoprecipitated with FLAG-specific mAb immobilized on agarose. Bound FilGAP was detected by immunoblotting using anti-His-tag mouse mAb conjugated with horse radish peroxidase (upper panel). The lower panel shows proteins visualized by CBB staining. (D) Molecular weight calibration curve obtained with a Superose 6 10/300 gel filtration column. Molecular size standards (open circle) used were thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), and ovalbumin (43 kDa). Colored circles indicate the sizes of His-FilGAP, His-FilGAP lacking residues 649–725 or FilGAP truncates fused to MBP-His-tag.
    Figure Legend Snippet: Localization of FLNa-FilGAP binding site. (A) Schematic representation of FilGAP and its truncation series. The pleckstrin-homology (PH), GTPase-activating protein (GAP), and coiled-coil (CC) domains predicted by EMBnet COILS are shown. Right panel shows binding of FLAG-FLNa to GST-FilGAP fragments illustrated in the left panel. Their interactions were analyzed by pull-down using FLAG-specific mAb immobilized on beads. Bound protein was detected by immunoblotting using rabbit pAb to GST. (B) FilGAP fragments were fused to MBP-His-tag and their binding to FLAG-FLNa were analyzed by pull-down using FLAG-specific mAb immobilized on beads. Bound protein was detected by immunoblotting using rabbit pAb to MBP (C) His-tag FilGAP, or FilGAP lacking residues 649–725 (50 nM), were mixed with increasing amounts of FLAG-FLNa and immunoprecipitated with FLAG-specific mAb immobilized on agarose. Bound FilGAP was detected by immunoblotting using anti-His-tag mouse mAb conjugated with horse radish peroxidase (upper panel). The lower panel shows proteins visualized by CBB staining. (D) Molecular weight calibration curve obtained with a Superose 6 10/300 gel filtration column. Molecular size standards (open circle) used were thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), and ovalbumin (43 kDa). Colored circles indicate the sizes of His-FilGAP, His-FilGAP lacking residues 649–725 or FilGAP truncates fused to MBP-His-tag.

    Techniques Used: Binding Assay, Immunoprecipitation, Staining, Molecular Weight, Filtration

    FLNa dimerization and hinge-2 are essential for high avidity binding to FilGAP. (A) Full-length FilGAP was pulled down with increasing amounts of wild-type and deletion mutants (Ä23; deletion of IgFLNa23, ÄH2; deletion of FLNa hinge-2, Ä24; deletion of IgFLNa24) of FLNa tagged to FLAG immunoprecipitated with FLAG-specific mAb immobilized on agarose. Bound FilGAP was detected by immunoblotting using rabbit pAbs to FilGAP. (B) Left panel shows purified MBP-FilGAP649-748, IgFLNa23-24, and IgFLNa23-24 ÄH2 separated on SDS-PAGE and stained with CBB. Right panel; IgFLNa23-24 or IgFLNa23-24 ÄH2 were pulled down with amylose beads coated with increasing amounts of the MBP-FilGAP649-748. Proteins were visualized by CBB staining.
    Figure Legend Snippet: FLNa dimerization and hinge-2 are essential for high avidity binding to FilGAP. (A) Full-length FilGAP was pulled down with increasing amounts of wild-type and deletion mutants (Ä23; deletion of IgFLNa23, ÄH2; deletion of FLNa hinge-2, Ä24; deletion of IgFLNa24) of FLNa tagged to FLAG immunoprecipitated with FLAG-specific mAb immobilized on agarose. Bound FilGAP was detected by immunoblotting using rabbit pAbs to FilGAP. (B) Left panel shows purified MBP-FilGAP649-748, IgFLNa23-24, and IgFLNa23-24 ÄH2 separated on SDS-PAGE and stained with CBB. Right panel; IgFLNa23-24 or IgFLNa23-24 ÄH2 were pulled down with amylose beads coated with increasing amounts of the MBP-FilGAP649-748. Proteins were visualized by CBB staining.

    Techniques Used: Binding Assay, Immunoprecipitation, Purification, SDS Page, Staining

    FilGAP specifically interacts with FLNa isoform. (A) Full-length FLNa, but not FLNb, pulls down FilGAP. Increasing amounts of either FLNa or FLNb were incubated with FilGAP and immunoprecipitated with mAbs to FLNa or FLNb. Bound FilGAP was detected by immunoblotting using rabbit pAbs to FilGAP. (B) MBP-FilGAP649-748 specifically binds the C-terminal of FLNa, but not FLNb or FLNc. Equal amounts of repeats 23–24 of FLNa, b, or c (0.2 ìM) were pulled down with increasing amounts of MBP-FilGAP659-748. Proteins were visualized by CBB staining (top and bottom). The top CBB-stained gel was destained and restained with silver (middle). (C) Sequence alignment of the C–E strands of the IgFLN23 isoforms. FLNa A2461T, M2474E and Y2483H point mutants do not interact with FilGAP as shown in Figures 4A , 5A and 6E . (D) Model of the IgFLNa23-FilGAP complex. Residues mutated in this study and some critical residues for their interaction are indicated. The purple dotted line shows the possible stabilizing hydrogen-bond between Tyr2483 and Thr733. (E) A point mutation of FLNa at Ala2461 to Thr or Tyr2483 to His are sufficient to abolish the complexing of FLNa and FilGAP. Full-length FilGAP (input: 10 nM constant) was pulled down with increasing amount of GST-IgFLNa20-24 immobilized on glutathione beads in a dose-dependent fashion. Mutations corresponding to A2461T or Y2483H in FLNa, but not D2467E, disrupt FilGAP binding. Bound FilGAP was detected by immunoblotting using rabbit pAbs to FilGAP. GST-FLNa constructs were detected by CBB staining.
    Figure Legend Snippet: FilGAP specifically interacts with FLNa isoform. (A) Full-length FLNa, but not FLNb, pulls down FilGAP. Increasing amounts of either FLNa or FLNb were incubated with FilGAP and immunoprecipitated with mAbs to FLNa or FLNb. Bound FilGAP was detected by immunoblotting using rabbit pAbs to FilGAP. (B) MBP-FilGAP649-748 specifically binds the C-terminal of FLNa, but not FLNb or FLNc. Equal amounts of repeats 23–24 of FLNa, b, or c (0.2 ìM) were pulled down with increasing amounts of MBP-FilGAP659-748. Proteins were visualized by CBB staining (top and bottom). The top CBB-stained gel was destained and restained with silver (middle). (C) Sequence alignment of the C–E strands of the IgFLN23 isoforms. FLNa A2461T, M2474E and Y2483H point mutants do not interact with FilGAP as shown in Figures 4A , 5A and 6E . (D) Model of the IgFLNa23-FilGAP complex. Residues mutated in this study and some critical residues for their interaction are indicated. The purple dotted line shows the possible stabilizing hydrogen-bond between Tyr2483 and Thr733. (E) A point mutation of FLNa at Ala2461 to Thr or Tyr2483 to His are sufficient to abolish the complexing of FLNa and FilGAP. Full-length FilGAP (input: 10 nM constant) was pulled down with increasing amount of GST-IgFLNa20-24 immobilized on glutathione beads in a dose-dependent fashion. Mutations corresponding to A2461T or Y2483H in FLNa, but not D2467E, disrupt FilGAP binding. Bound FilGAP was detected by immunoblotting using rabbit pAbs to FilGAP. GST-FLNa constructs were detected by CBB staining.

    Techniques Used: Incubation, Immunoprecipitation, Staining, Sequencing, Mutagenesis, Binding Assay, Construct

    7) Product Images from "Cdk1 phosphorylates SPAT-1/Bora to trigger PLK-1 activation and drive mitotic entry in C. elegans embryos"

    Article Title: Cdk1 phosphorylates SPAT-1/Bora to trigger PLK-1 activation and drive mitotic entry in C. elegans embryos

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201408064

    Phospho–SPAT-1 and phospho-Bora promote PLK-1/Plk1 phosphorylation on its activator T loop by the Aurora A kinase. (A) Flow chart of the assay used to test the role of Bora/SPAT-1 phosphorylation by CyclinB/Cdk1 in Plk1 phosphorylation by Aurora A. (B) The T loop of Plk1 is evolutionarily conserved. Sequence alignment of the T-loop region of Plk1 from various species, using PRALINE ( H. s , H. sapiens ; M. m , Mus musculus ; X. l , X. laevis ; D. r , Danio rerio ; C. e , C. elegans ; D. m , D. melanogaster ). Invariant residues are highlighted in red. An arrow marks the activating T210 phosphorylation of human Plk1, which corresponds to T194 in C.e. PLK-1. (C) Western blot of WT and T194A 6×(His)–PLK-1 mutant purified from insect Sf9 cells with the anti-Plk1 pT210 (top) and PLK-1 antibody (bottom). (D) The phospho-specific T210 Plk1 antibody specifically recognizes pT194 in C.e. PLK-1. 6×(His)–PLK-1 WT purified from insect Sf9 cells was incubated with λ phosphatase (λ PPase, +) or heat-inactivated phosphatase (#) and analyzed by SDS-PAGE and Western blotting using the anti-Plk1 phospho-T210 (top) and PLK-1 antibodies (bottom). (E) Western blot showing the result of the kinase assay of 6×(His)-Plk1 by 6×(His)–Aurora A incubated with MBP (lanes 1 and 2) or MBP-Bora (lanes 3 and 4) phosphorylated (+) or not phosphorylated (−) by CyclinB/Cdk1. Blots were probed with MBP (top), anti-pT210 (bottom), and Plk1 (middle) antibodies. (F) Western blot showing the result of the kinase assay of 6×(His)–PLK-1 by 6×(His)–Aurora A incubated with MBP (lanes 1 and 2) or MBP–SPAT-1 (lanes 3 and 4) phosphorylated (+) or not phosphorylated (−) by CyclinB/Cdk1. Blots were probed with MBP (top), anti-pT210 (bottom), and PLK-1 (middle) antibodies. (G) Western blot showing the result of the kinase assay of 6×(His)–PLK-1 by 6×(His)–Aurora A incubated with MBP (lane 1), MBP–SPAT-1 WT (lane 2), or MBP–SPAT-1 13A mutant phosphorylated by CyclinB/Cdk1. Blots were probed with MBP (top), anti-pT210 (bottom), and PLK-1 (middle) antibodies.
    Figure Legend Snippet: Phospho–SPAT-1 and phospho-Bora promote PLK-1/Plk1 phosphorylation on its activator T loop by the Aurora A kinase. (A) Flow chart of the assay used to test the role of Bora/SPAT-1 phosphorylation by CyclinB/Cdk1 in Plk1 phosphorylation by Aurora A. (B) The T loop of Plk1 is evolutionarily conserved. Sequence alignment of the T-loop region of Plk1 from various species, using PRALINE ( H. s , H. sapiens ; M. m , Mus musculus ; X. l , X. laevis ; D. r , Danio rerio ; C. e , C. elegans ; D. m , D. melanogaster ). Invariant residues are highlighted in red. An arrow marks the activating T210 phosphorylation of human Plk1, which corresponds to T194 in C.e. PLK-1. (C) Western blot of WT and T194A 6×(His)–PLK-1 mutant purified from insect Sf9 cells with the anti-Plk1 pT210 (top) and PLK-1 antibody (bottom). (D) The phospho-specific T210 Plk1 antibody specifically recognizes pT194 in C.e. PLK-1. 6×(His)–PLK-1 WT purified from insect Sf9 cells was incubated with λ phosphatase (λ PPase, +) or heat-inactivated phosphatase (#) and analyzed by SDS-PAGE and Western blotting using the anti-Plk1 phospho-T210 (top) and PLK-1 antibodies (bottom). (E) Western blot showing the result of the kinase assay of 6×(His)-Plk1 by 6×(His)–Aurora A incubated with MBP (lanes 1 and 2) or MBP-Bora (lanes 3 and 4) phosphorylated (+) or not phosphorylated (−) by CyclinB/Cdk1. Blots were probed with MBP (top), anti-pT210 (bottom), and Plk1 (middle) antibodies. (F) Western blot showing the result of the kinase assay of 6×(His)–PLK-1 by 6×(His)–Aurora A incubated with MBP (lanes 1 and 2) or MBP–SPAT-1 (lanes 3 and 4) phosphorylated (+) or not phosphorylated (−) by CyclinB/Cdk1. Blots were probed with MBP (top), anti-pT210 (bottom), and PLK-1 (middle) antibodies. (G) Western blot showing the result of the kinase assay of 6×(His)–PLK-1 by 6×(His)–Aurora A incubated with MBP (lane 1), MBP–SPAT-1 WT (lane 2), or MBP–SPAT-1 13A mutant phosphorylated by CyclinB/Cdk1. Blots were probed with MBP (top), anti-pT210 (bottom), and PLK-1 (middle) antibodies.

    Techniques Used: Flow Cytometry, Sequencing, Western Blot, Mutagenesis, Purification, Incubation, SDS Page, Kinase Assay

    SPAT-1 phosphorylation by Cdk1 promotes the interaction between SPAT-1 and PLK-1. (A, top) Embryonic extracts of the indicated genotypes analyzed by Western blotting using SPAT-1 antibodies. (bottom) Tubulin is used as a loading control. 25 µg (lanes 1, 3, 5, and 7) and 50 µg (lanes 2, 4, 6, and 8) of each protein extract were loaded to visualize the modified forms. (B) MBP–SPAT-1 or MBP incubated with CyclinB/Cdk1 kinase in the presence of γ-[ 32 P]ATP. (right) Autoradiograph of the SDS-PAGE gel showing 32 P incorporation in MBP–SPAT-1 but not MBP. (left) Coomassie staining of the same SDS-PAGE gel. (C) Western blot analysis of PLK-1 immunoprecipitates (IP PLK-1) from control (lane 3) or cdk-1(RNAi) (lane 4) embryonic extracts analyzed with SPAT-1 (top) and PLK-1 antibodies (middle). (bottom) Actin was used as a loading control. 10 µg (1:40) of the total extracts (Ext.; lanes 1 and 2) and the flow through (FT) of the immunoprecipitates (lanes 5 and 6) were loaded for comparison. The asterisk marks the phosphorylated SPAT-1 forms that are present in the PLK-1 immunoprecipitation. (D) In vitro assay used to test Cdk1 dependency of the interaction between SPAT-1 and PLK-1. On the left, we show a flow chart describing the assay; on the right, we show the Western blot analysis. Strep–SPAT-1 protein produced in insect Sf9 cells was immobilized on Strep-Tactin Sepharose beads, dephosphorylated with λ phosphatase (λ PPase), and incubated with CylinB/Cdk1 in the presence (+) or absence (−) of ATP. After washing the kinase and ATP, full-length 6×(His)–PLK-1 was added (+) for a typical pull-down experiment. (right) Strep–SPAT-1 was eluted with desthiobiotin, and the elutions were analyzed by SDS-PAGE and Western blotting using PLK-1 and SPAT-1 antibodies.
    Figure Legend Snippet: SPAT-1 phosphorylation by Cdk1 promotes the interaction between SPAT-1 and PLK-1. (A, top) Embryonic extracts of the indicated genotypes analyzed by Western blotting using SPAT-1 antibodies. (bottom) Tubulin is used as a loading control. 25 µg (lanes 1, 3, 5, and 7) and 50 µg (lanes 2, 4, 6, and 8) of each protein extract were loaded to visualize the modified forms. (B) MBP–SPAT-1 or MBP incubated with CyclinB/Cdk1 kinase in the presence of γ-[ 32 P]ATP. (right) Autoradiograph of the SDS-PAGE gel showing 32 P incorporation in MBP–SPAT-1 but not MBP. (left) Coomassie staining of the same SDS-PAGE gel. (C) Western blot analysis of PLK-1 immunoprecipitates (IP PLK-1) from control (lane 3) or cdk-1(RNAi) (lane 4) embryonic extracts analyzed with SPAT-1 (top) and PLK-1 antibodies (middle). (bottom) Actin was used as a loading control. 10 µg (1:40) of the total extracts (Ext.; lanes 1 and 2) and the flow through (FT) of the immunoprecipitates (lanes 5 and 6) were loaded for comparison. The asterisk marks the phosphorylated SPAT-1 forms that are present in the PLK-1 immunoprecipitation. (D) In vitro assay used to test Cdk1 dependency of the interaction between SPAT-1 and PLK-1. On the left, we show a flow chart describing the assay; on the right, we show the Western blot analysis. Strep–SPAT-1 protein produced in insect Sf9 cells was immobilized on Strep-Tactin Sepharose beads, dephosphorylated with λ phosphatase (λ PPase), and incubated with CylinB/Cdk1 in the presence (+) or absence (−) of ATP. After washing the kinase and ATP, full-length 6×(His)–PLK-1 was added (+) for a typical pull-down experiment. (right) Strep–SPAT-1 was eluted with desthiobiotin, and the elutions were analyzed by SDS-PAGE and Western blotting using PLK-1 and SPAT-1 antibodies.

    Techniques Used: Western Blot, Modification, Incubation, Autoradiography, SDS Page, Staining, Flow Cytometry, Immunoprecipitation, In Vitro, Produced

    8) Product Images from "Phosphorylation-Induced Signal Propagation in the Response Regulator NtrC"

    Article Title: Phosphorylation-Induced Signal Propagation in the Response Regulator NtrC

    Journal: Journal of Bacteriology

    doi:

    Summary of cleavage sites and Western analysis. (A) Diagram of cleavage sites, indicated by arrows. The D86C substitution in the N-terminal domain of NtrC, which was derivatized with Fe-BABE, is indicated. For purposes of illustration, all cleavage sites are indicated within a monomer. The site of the phosphorylation-independent cleavage that yielded bands 3 and 2′ lies after position 54 and before position 86. The site of the phosphorylation-dependent cleavage that yielded bands 1 and 5 lies outside the N-terminal domain and is predicted to lie at the end of the linker or the beginning of the central domain. This cleavage occurs between the monomers of a dimer. (B) Partner bands resulting from phosphorylation-dependent (+P) or -independent (+/−P) cleavages are indicated by their mobilities on SDS–8% polyacrylamide gels. For each band, the results of Western analysis using antibodies directed against MBP, the N-terminal domain of NtrC (N), the C-terminal domain (C), or the iron chelate (Fe) are indicated, as is the reaction of streptavidin with biotin (Bio). The mobility of the MBP–N-terminal protein (a protein containing only MBP and the N-terminal domain of NtrC) is indicated by an arrow.
    Figure Legend Snippet: Summary of cleavage sites and Western analysis. (A) Diagram of cleavage sites, indicated by arrows. The D86C substitution in the N-terminal domain of NtrC, which was derivatized with Fe-BABE, is indicated. For purposes of illustration, all cleavage sites are indicated within a monomer. The site of the phosphorylation-independent cleavage that yielded bands 3 and 2′ lies after position 54 and before position 86. The site of the phosphorylation-dependent cleavage that yielded bands 1 and 5 lies outside the N-terminal domain and is predicted to lie at the end of the linker or the beginning of the central domain. This cleavage occurs between the monomers of a dimer. (B) Partner bands resulting from phosphorylation-dependent (+P) or -independent (+/−P) cleavages are indicated by their mobilities on SDS–8% polyacrylamide gels. For each band, the results of Western analysis using antibodies directed against MBP, the N-terminal domain of NtrC (N), the C-terminal domain (C), or the iron chelate (Fe) are indicated, as is the reaction of streptavidin with biotin (Bio). The mobility of the MBP–N-terminal protein (a protein containing only MBP and the N-terminal domain of NtrC) is indicated by an arrow.

    Techniques Used: Western Blot

    9) Product Images from "Cdc42p-Interacting Protein Bem4p Regulates the Filamentous-Growth Mitogen-Activated Protein Kinase Pathway"

    Article Title: Cdc42p-Interacting Protein Bem4p Regulates the Filamentous-Growth Mitogen-Activated Protein Kinase Pathway

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00850-14

    Bem4p binds to and regulates Cdc42p activity. (A) Subcellular fractionation of cells expressing Bem4p-HA and GFP-Cdc42p. Whole-cell extract (WCE), supernatant (S), and pellet (P) fractions from the centrifugation steps (P13, 13,000 × g ; P100, 100,000 × g ; S100, 100,000 × g ) were analyzed by immunoblot analysis. An integral membrane endoplasmic reticulum protein (Dpm1p) and a cytosolic protein (Pgk1p) are shown as controls. (B) (Top) The Gal4p activation domain (GAD)-Bem4p and Gal4p binding domain (GBD)-Cdc42p associate, as determined by two-hybrid analysis. Strains were spotted onto SD-Ura-Leu (-UL) to maintain selection for plasmids and SD-Ura-Leu-His (-ULH) to measure activity of a two-hybrid growth reporter. (Bottom) β-Galactosidase activities (in Miller units) of the two-hybrid reporter GAL7-lacZ . Values are averages from two independent experiments; the standard deviation was less than 10% between trials. (C) MBP-Bem4p and HIS-Cdc42p proteins associate by in vitro pulldown. Fifty percent of the WCE (input) was used for the pulldown. Numbers are band intensities normalized to input protein levels by ImageJ. (D) PAK-binding assay. GFP-Cdc42p was precipitated by beads coated with GST or GST-PAK binding domain. Band intensity was determined by normalizing precipitated GFP-Cdc42p to input levels from wild-type cells or cells lacking BEM4 . Equal amounts of GST and GST-PAK binding domain were loaded onto beads. Values are averages from two separate experiments, which showed less than 10% difference between trials. (E) MBP-Bem4p associates with HIS-Cdc42p in buffer with and without 10 mM EDTA. Numbers represent band intensity normalized to input protein levels by ImageJ. (F) Localization of GFP-Cdc42p in wild-type cells and the bem4 Δ and ste12 Δ mutants. Bar, 2 μm (left and middle) and 0.4 μm (right). Figure S5A in the supplemental material shows additional examples of cells, and quantitation is provided in Fig. S5B and C.
    Figure Legend Snippet: Bem4p binds to and regulates Cdc42p activity. (A) Subcellular fractionation of cells expressing Bem4p-HA and GFP-Cdc42p. Whole-cell extract (WCE), supernatant (S), and pellet (P) fractions from the centrifugation steps (P13, 13,000 × g ; P100, 100,000 × g ; S100, 100,000 × g ) were analyzed by immunoblot analysis. An integral membrane endoplasmic reticulum protein (Dpm1p) and a cytosolic protein (Pgk1p) are shown as controls. (B) (Top) The Gal4p activation domain (GAD)-Bem4p and Gal4p binding domain (GBD)-Cdc42p associate, as determined by two-hybrid analysis. Strains were spotted onto SD-Ura-Leu (-UL) to maintain selection for plasmids and SD-Ura-Leu-His (-ULH) to measure activity of a two-hybrid growth reporter. (Bottom) β-Galactosidase activities (in Miller units) of the two-hybrid reporter GAL7-lacZ . Values are averages from two independent experiments; the standard deviation was less than 10% between trials. (C) MBP-Bem4p and HIS-Cdc42p proteins associate by in vitro pulldown. Fifty percent of the WCE (input) was used for the pulldown. Numbers are band intensities normalized to input protein levels by ImageJ. (D) PAK-binding assay. GFP-Cdc42p was precipitated by beads coated with GST or GST-PAK binding domain. Band intensity was determined by normalizing precipitated GFP-Cdc42p to input levels from wild-type cells or cells lacking BEM4 . Equal amounts of GST and GST-PAK binding domain were loaded onto beads. Values are averages from two separate experiments, which showed less than 10% difference between trials. (E) MBP-Bem4p associates with HIS-Cdc42p in buffer with and without 10 mM EDTA. Numbers represent band intensity normalized to input protein levels by ImageJ. (F) Localization of GFP-Cdc42p in wild-type cells and the bem4 Δ and ste12 Δ mutants. Bar, 2 μm (left and middle) and 0.4 μm (right). Figure S5A in the supplemental material shows additional examples of cells, and quantitation is provided in Fig. S5B and C.

    Techniques Used: Activity Assay, Fractionation, Expressing, Centrifugation, Activation Assay, Binding Assay, Selection, Standard Deviation, In Vitro, Quantitation Assay

    Bem4p interacts with the MAPKKK Ste11p. (A) (Left) Bem4p interacts with Ste11p by two-hybrid analysis but not Ras2p or Pbs2p. (Right) β-Galactosidase activity (in Miller units) of the two-hybrid reporter GAL7-lacZ was measured for the constructs shown. Values are averages from two experiments; the standard deviation was less than 10% between trials. (B) MBP-Bem4p interacts with GST-Ste11p in vitro . Fifty percent of the WCE (input) was used for the pull down. (C) MAPK activity (based on the β-galactosidase activity of the ste4 FUS1-lacZ reporter) in wild-type cells and the bem4 Δ mutant containing the indicated versions of Ste50p and Ste11p. Asterisks denote P values of
    Figure Legend Snippet: Bem4p interacts with the MAPKKK Ste11p. (A) (Left) Bem4p interacts with Ste11p by two-hybrid analysis but not Ras2p or Pbs2p. (Right) β-Galactosidase activity (in Miller units) of the two-hybrid reporter GAL7-lacZ was measured for the constructs shown. Values are averages from two experiments; the standard deviation was less than 10% between trials. (B) MBP-Bem4p interacts with GST-Ste11p in vitro . Fifty percent of the WCE (input) was used for the pull down. (C) MAPK activity (based on the β-galactosidase activity of the ste4 FUS1-lacZ reporter) in wild-type cells and the bem4 Δ mutant containing the indicated versions of Ste50p and Ste11p. Asterisks denote P values of

    Techniques Used: Activity Assay, Construct, Standard Deviation, In Vitro, Mutagenesis

    Bem4p functions with Cdc24p and regulates its localization. (A) P∼Kss1p levels in cells carrying pMyr-Cdc24p-GFP in the wild type, the sho1 Δ mutant, and the bem4 Δ mutant. (B) Bem4p-HA interacts with Cdc24p-GFP by co-IP analysis. Band intensity relative to input levels after background subtraction was determined by ImageJ. Input levels are 10% for WCE. (C) MBP-Bem4p interacts with GST-Cdc24p in vitro . Fifty percent of the WCE (input) was used for the pulldown. (D) Bem4p interacts with the PH domain of Cdc24p. Deletion constructs were made at the indicated residues. Fifty percent of the WCE (input) was used for the pulldowns. (E) Cells in mid-log phase expressing wild-type Cdc24p or Cdc24p-PHΔ. Bar, 25 μm. (F) Localization of Cdc24p-GFP in wild-type cells and the indicated mutants. Cells were grown to mid-log phase in SD or S-Gal complete medium. Bar, 5 μm. Approximately 50 cells were counted for each experiment in separate trials; asterisks denote P values of
    Figure Legend Snippet: Bem4p functions with Cdc24p and regulates its localization. (A) P∼Kss1p levels in cells carrying pMyr-Cdc24p-GFP in the wild type, the sho1 Δ mutant, and the bem4 Δ mutant. (B) Bem4p-HA interacts with Cdc24p-GFP by co-IP analysis. Band intensity relative to input levels after background subtraction was determined by ImageJ. Input levels are 10% for WCE. (C) MBP-Bem4p interacts with GST-Cdc24p in vitro . Fifty percent of the WCE (input) was used for the pulldown. (D) Bem4p interacts with the PH domain of Cdc24p. Deletion constructs were made at the indicated residues. Fifty percent of the WCE (input) was used for the pulldowns. (E) Cells in mid-log phase expressing wild-type Cdc24p or Cdc24p-PHΔ. Bar, 25 μm. (F) Localization of Cdc24p-GFP in wild-type cells and the indicated mutants. Cells were grown to mid-log phase in SD or S-Gal complete medium. Bar, 5 μm. Approximately 50 cells were counted for each experiment in separate trials; asterisks denote P values of

    Techniques Used: Mutagenesis, Co-Immunoprecipitation Assay, In Vitro, Construct, Expressing

    10) Product Images from "Nance-Horan Syndrome-like 1 protein negatively regulates Scar/WAVE-Arp2/3 activity and inhibits lamellipodia stability and cell migration"

    Article Title: Nance-Horan Syndrome-like 1 protein negatively regulates Scar/WAVE-Arp2/3 activity and inhibits lamellipodia stability and cell migration

    Journal: bioRxiv

    doi: 10.1101/2020.05.11.083030

    Abi SH3 domain binds to two fragments of NHSL1 (A) Far western overlay with purified MBP-tagged full-length Abi1 (MBP-Abi1 full length) or an MBP fusion protein with Abi1 in which the SH3 domain had been deleted (MBP-Abi1-delta-SH3) and MBP as control on a blot of different purified GST-NHSL1 fusion proteins covering the entire length of NHSL1. Representative blots from three independent experiments. Fragments 4 and 5 contain three putative SH3 binding si tes. (B) Coomassie gel showing GST fragments covering the entire length of the NHSL1 amino acid sequence (see Fig. 1E for fragment sizes and location within NHSL1) and GST only as control which are used in the Far Western Blot in (A).
    Figure Legend Snippet: Abi SH3 domain binds to two fragments of NHSL1 (A) Far western overlay with purified MBP-tagged full-length Abi1 (MBP-Abi1 full length) or an MBP fusion protein with Abi1 in which the SH3 domain had been deleted (MBP-Abi1-delta-SH3) and MBP as control on a blot of different purified GST-NHSL1 fusion proteins covering the entire length of NHSL1. Representative blots from three independent experiments. Fragments 4 and 5 contain three putative SH3 binding si tes. (B) Coomassie gel showing GST fragments covering the entire length of the NHSL1 amino acid sequence (see Fig. 1E for fragment sizes and location within NHSL1) and GST only as control which are used in the Far Western Blot in (A).

    Techniques Used: Western Blot, Purification, Binding Assay, Sequencing, Far Western Blot

    11) Product Images from "The receptor-like kinase NIK1 targets FLS2/BAK1 immune complex and inversely modulates antiviral and antibacterial immunity"

    Article Title: The receptor-like kinase NIK1 targets FLS2/BAK1 immune complex and inversely modulates antiviral and antibacterial immunity

    Journal: Nature Communications

    doi: 10.1038/s41467-019-12847-6

    Flg22 induces BAK1-mediated NIK1 phosphorylation, which enhances NIK1’s affinity for receptors. a Flg22 perception triggers NIK1 rapid mobility shift. Protoplasts were transfected with NIK1-HA and treated with 100 nM flg22 for the indicated time points. Total input proteins were stained with Coomassie brilliant blue staining (CBB). b Verification of NIK1 in vivo phosphorylation by λPP treatment. Protein extracts from protoplasts transfected with NIK1-HA were treated with λPP following the standard protocol. c The kinase inhibitor K252a blocks flg22-induced NIK1 mobility shift. K252a was applied 1 h before flg22 treatment. Controls were solvent (DMSO) treatment. d Flg22-induced NIK1 phosphorylation requires FLS2 and its kinase activity. Protoplasts isolated from fls2 mutants were transfected with NIK1-HA and empty vector control, FLAG-tagged FLS2 or FLS2 kinase mutant (FLS2km). e Flg22-mediated NIK1 phosphorylation requires BAK1. NIK1-HA was expressed in protoplasts of Col-0 or bak1-4 mutants and flg22 was applied 10 min before samples collection. f Flg22-induced in vivo phosphorylation of NIK1 detected by different phospho-antibodies. Seedlings of NIK1-HA-overexpressing lines were treated with 100 nM flg22 for the indicated time points. NIK1-HA was immunoprecipitated from total protein extracts, fractionated by SDS-PAGE and immunoblotted with α-phosphoserine (α-pSer), α-phosphothreonine (α-Thr), α-phosphotyrosine (α-Tyr) and α-HA antibodies. g BAK1, but not FLS2, directly phosphorylates the NIK1 cytosolic domain. An in vitro kinase assay was performed using MBP-FLS2JK or MBP-BAK1JK as a kinase and GST-NIK1JKKm as the substrate. Phosphorylation was analysed by autoradiography (Upper), and the protein loading was shown by CBB (Lower). h NIK1 Thr474 is phosphorylated by BAK1 in vitro as shown by MS analysis. i Phosphorylation of NIK1 promotes its interaction with FLS2. FLS2-FLAG was co-expressed with NIK1-HA, NIK1Km-HA or NIK1-T474D-HA in protoplasts. Co-IP was performed with α-FLAG Agarose (IP: α-FLAG), and the proteins were immunoblotted with an α-HA antibody (IB: α-HA). j Quantitative data ( n = 30 for h . k A NIK1 phosphomimetic form shows stronger interaction with BAK1. Co-IP assay was performed with the sample co-expressing BAK1-FLAG and T474A-HA or T474D-HA using α-FLAG Agarose. l Quantitative data from three biological replicates for j . Source data are provided as a Source Data file
    Figure Legend Snippet: Flg22 induces BAK1-mediated NIK1 phosphorylation, which enhances NIK1’s affinity for receptors. a Flg22 perception triggers NIK1 rapid mobility shift. Protoplasts were transfected with NIK1-HA and treated with 100 nM flg22 for the indicated time points. Total input proteins were stained with Coomassie brilliant blue staining (CBB). b Verification of NIK1 in vivo phosphorylation by λPP treatment. Protein extracts from protoplasts transfected with NIK1-HA were treated with λPP following the standard protocol. c The kinase inhibitor K252a blocks flg22-induced NIK1 mobility shift. K252a was applied 1 h before flg22 treatment. Controls were solvent (DMSO) treatment. d Flg22-induced NIK1 phosphorylation requires FLS2 and its kinase activity. Protoplasts isolated from fls2 mutants were transfected with NIK1-HA and empty vector control, FLAG-tagged FLS2 or FLS2 kinase mutant (FLS2km). e Flg22-mediated NIK1 phosphorylation requires BAK1. NIK1-HA was expressed in protoplasts of Col-0 or bak1-4 mutants and flg22 was applied 10 min before samples collection. f Flg22-induced in vivo phosphorylation of NIK1 detected by different phospho-antibodies. Seedlings of NIK1-HA-overexpressing lines were treated with 100 nM flg22 for the indicated time points. NIK1-HA was immunoprecipitated from total protein extracts, fractionated by SDS-PAGE and immunoblotted with α-phosphoserine (α-pSer), α-phosphothreonine (α-Thr), α-phosphotyrosine (α-Tyr) and α-HA antibodies. g BAK1, but not FLS2, directly phosphorylates the NIK1 cytosolic domain. An in vitro kinase assay was performed using MBP-FLS2JK or MBP-BAK1JK as a kinase and GST-NIK1JKKm as the substrate. Phosphorylation was analysed by autoradiography (Upper), and the protein loading was shown by CBB (Lower). h NIK1 Thr474 is phosphorylated by BAK1 in vitro as shown by MS analysis. i Phosphorylation of NIK1 promotes its interaction with FLS2. FLS2-FLAG was co-expressed with NIK1-HA, NIK1Km-HA or NIK1-T474D-HA in protoplasts. Co-IP was performed with α-FLAG Agarose (IP: α-FLAG), and the proteins were immunoblotted with an α-HA antibody (IB: α-HA). j Quantitative data ( n = 30 for h . k A NIK1 phosphomimetic form shows stronger interaction with BAK1. Co-IP assay was performed with the sample co-expressing BAK1-FLAG and T474A-HA or T474D-HA using α-FLAG Agarose. l Quantitative data from three biological replicates for j . Source data are provided as a Source Data file

    Techniques Used: Mobility Shift, Transfection, Staining, In Vivo, Activity Assay, Isolation, Plasmid Preparation, Mutagenesis, Immunoprecipitation, SDS Page, In Vitro, Kinase Assay, Autoradiography, Mass Spectrometry, Co-Immunoprecipitation Assay, Expressing

    Interaction between NIK1 and the FLS2/BAK1 receptor complex is enhanced in response to flg22 signalling. a Interaction of NIK1 with BAK1 or FLS2 in Y2H assay. The kinase domains of NIK1, BAK1 or FLS2 were expressed in yeast as GAL4 activation domain (AD) fusions or binding domain (BD) fusions. EV indicates the empty vectors for either pGADT7 or pGBKT7. b In vivo interaction between NIK1 and BAK1 or FLS2 by BiFC analysis. Fluorescence (YFP) and bright field confocal images were acquired of tobacco leaves co-expressing the indicated fusion proteins in the presence of HC-Pro suppressor 48 h after agro-infiltration with the indicated DNA constructs. Scale bars = 20 µm. c NIK1 directly interacts with BAK1 or FLS2 in vitro. GST or GST-NIK1JK immobilized on glutathione Sepharose beads was incubated with MBP, MBP-FLS2JK or MBP-BAK1JK proteins. Beads were washed and pelleted for immunoblot analysis with α-HA antibody. PD, pull-down. d , e NIK1 associates with FLS2 or BAK1 and these interactions are strengthened by flg22 treatment. Arabidopsis protoplasts were co-transfected with NIK1-HA and FLS2-FLAG, BAK1-FLAG or an empty vector control. Protoplasts were treated with (+) or without (−) 100 nM flg22 for 15 min before harvesting. Co-IP was performed with α-FLAG Agarose (IP: α-FLAG), and proteins were analysed using immunoblots with an α-HA antibody (IB: α-HA). f Quantitative data for d, e . Signal intensity was quantified using the ImageJ software, and values represent the mean ± SD ( n = 3). g Flg22-enhanced NIK1 and BAK1 interaction was abolished in a fls2 mutant background. NIK1-HA and BAK1-FLAG were co-expressed in Col-0 or fls2 protoplasts, and flg22 treatments were performed before samples collection. h Quantitative data for g . Images were quantified using the ImageJ software, and values represent the mean ± SD ( n = 3). i Flg22-enhanced NIK1 and FLS2 interaction is largely reduced in a bak1-4 mutant background. NIK1-HA and FLS2-FLAG were co-expressed in Col-0 or bak1-4 protoplasts, and treated with flg22 before sample collection. j Quantitative data for i . Co-IP signals were quantified using the ImageJ software, and values represent the mean ± SD ( n = 3). Source data are provided as a Source Data file
    Figure Legend Snippet: Interaction between NIK1 and the FLS2/BAK1 receptor complex is enhanced in response to flg22 signalling. a Interaction of NIK1 with BAK1 or FLS2 in Y2H assay. The kinase domains of NIK1, BAK1 or FLS2 were expressed in yeast as GAL4 activation domain (AD) fusions or binding domain (BD) fusions. EV indicates the empty vectors for either pGADT7 or pGBKT7. b In vivo interaction between NIK1 and BAK1 or FLS2 by BiFC analysis. Fluorescence (YFP) and bright field confocal images were acquired of tobacco leaves co-expressing the indicated fusion proteins in the presence of HC-Pro suppressor 48 h after agro-infiltration with the indicated DNA constructs. Scale bars = 20 µm. c NIK1 directly interacts with BAK1 or FLS2 in vitro. GST or GST-NIK1JK immobilized on glutathione Sepharose beads was incubated with MBP, MBP-FLS2JK or MBP-BAK1JK proteins. Beads were washed and pelleted for immunoblot analysis with α-HA antibody. PD, pull-down. d , e NIK1 associates with FLS2 or BAK1 and these interactions are strengthened by flg22 treatment. Arabidopsis protoplasts were co-transfected with NIK1-HA and FLS2-FLAG, BAK1-FLAG or an empty vector control. Protoplasts were treated with (+) or without (−) 100 nM flg22 for 15 min before harvesting. Co-IP was performed with α-FLAG Agarose (IP: α-FLAG), and proteins were analysed using immunoblots with an α-HA antibody (IB: α-HA). f Quantitative data for d, e . Signal intensity was quantified using the ImageJ software, and values represent the mean ± SD ( n = 3). g Flg22-enhanced NIK1 and BAK1 interaction was abolished in a fls2 mutant background. NIK1-HA and BAK1-FLAG were co-expressed in Col-0 or fls2 protoplasts, and flg22 treatments were performed before samples collection. h Quantitative data for g . Images were quantified using the ImageJ software, and values represent the mean ± SD ( n = 3). i Flg22-enhanced NIK1 and FLS2 interaction is largely reduced in a bak1-4 mutant background. NIK1-HA and FLS2-FLAG were co-expressed in Col-0 or bak1-4 protoplasts, and treated with flg22 before sample collection. j Quantitative data for i . Co-IP signals were quantified using the ImageJ software, and values represent the mean ± SD ( n = 3). Source data are provided as a Source Data file

    Techniques Used: Y2H Assay, Activation Assay, Binding Assay, In Vivo, Bimolecular Fluorescence Complementation Assay, Fluorescence, Expressing, Construct, In Vitro, Incubation, Transfection, Plasmid Preparation, Co-Immunoprecipitation Assay, Western Blot, Software, Mutagenesis

    12) Product Images from "Lamellipodin and the Scar/WAVE complex cooperate to promote cell migration in vivo"

    Article Title: Lamellipodin and the Scar/WAVE complex cooperate to promote cell migration in vivo

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201304051

    Lpd directly interacts with the SH3 domain of Abi. (A) Pull-down of Lpd from NIH/3T3 cell lysate using the GST-Abi-SH3 domain or GST as control. (B–D) Far Western overlay on different GST-Lpd truncation mutants (B) or GST control using purified (C) MBP-Abi1 full-length or (D) MBP-Abi1 ΔSH3 was detected with anti-MBP antibodies. Three independent experiments were performed. (E) Far-Western overlay with MBP-Abi1 full-length on a peptide array covering the C terminus of Lpd with 12-mer peptides overlapping each other by three amino acids was detected with anti-MBP antibodies. (F) Table shows Abi SH3 domain–binding motifs in the Lpd sequence. The two GST-Lpd fragments highlighted in red correspond to the most strongly interacting Lpd fragments in the Far-Western experiment in C. The amino acid residues highlighted in yellow correspond to the core residues required for class II SH3 domain binding.
    Figure Legend Snippet: Lpd directly interacts with the SH3 domain of Abi. (A) Pull-down of Lpd from NIH/3T3 cell lysate using the GST-Abi-SH3 domain or GST as control. (B–D) Far Western overlay on different GST-Lpd truncation mutants (B) or GST control using purified (C) MBP-Abi1 full-length or (D) MBP-Abi1 ΔSH3 was detected with anti-MBP antibodies. Three independent experiments were performed. (E) Far-Western overlay with MBP-Abi1 full-length on a peptide array covering the C terminus of Lpd with 12-mer peptides overlapping each other by three amino acids was detected with anti-MBP antibodies. (F) Table shows Abi SH3 domain–binding motifs in the Lpd sequence. The two GST-Lpd fragments highlighted in red correspond to the most strongly interacting Lpd fragments in the Far-Western experiment in C. The amino acid residues highlighted in yellow correspond to the core residues required for class II SH3 domain binding.

    Techniques Used: Western Blot, Purification, Peptide Microarray, Binding Assay, Sequencing

    13) Product Images from "Intraflagellar Transport (IFT) Protein IFT25 Is a Phosphoprotein Component of IFT Complex B and Physically Interacts with IFT27 in Chlamydomonas"

    Article Title: Intraflagellar Transport (IFT) Protein IFT25 Is a Phosphoprotein Component of IFT Complex B and Physically Interacts with IFT27 in Chlamydomonas

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0005384

    IFT25 interacts physically with IFT27. Purified GST-tagged IFT27 and MBP-tagged IFT25 were used for in vitro binding assay. The left panel shows that immobilized GST-IFT27 protein on the beads could retain MBP-IFT25 protein but not the control protein, MBP. The right panel shows that immobilized MBP-IFT25 protein on beads could retain GST-IFT27 protein but not the control protein, GST. The molecular marker is labeled on the left of each figure. From top to bottom for both panels, the first figure is the Coomassie blue-stained gel. The second and third figures represent the immunoblots probed with antibodies against IFT25 and IFT27, respectively. The fourth one is the immunoblots probed with antibodies against either MBP (left panel) or GST (right panel). The loading materials for each lane of the gels are shown in the tables at the top of each panel. S stands for supernatant and P for bead pellet.
    Figure Legend Snippet: IFT25 interacts physically with IFT27. Purified GST-tagged IFT27 and MBP-tagged IFT25 were used for in vitro binding assay. The left panel shows that immobilized GST-IFT27 protein on the beads could retain MBP-IFT25 protein but not the control protein, MBP. The right panel shows that immobilized MBP-IFT25 protein on beads could retain GST-IFT27 protein but not the control protein, GST. The molecular marker is labeled on the left of each figure. From top to bottom for both panels, the first figure is the Coomassie blue-stained gel. The second and third figures represent the immunoblots probed with antibodies against IFT25 and IFT27, respectively. The fourth one is the immunoblots probed with antibodies against either MBP (left panel) or GST (right panel). The loading materials for each lane of the gels are shown in the tables at the top of each panel. S stands for supernatant and P for bead pellet.

    Techniques Used: Purification, In Vitro, Binding Assay, Marker, Labeling, Staining, Western Blot

    14) Product Images from "Fibronectin Binding Protein BBK32 of the Lyme Disease Spirochete Promotes Bacterial Attachment to Glycosaminoglycans "

    Article Title: Fibronectin Binding Protein BBK32 of the Lyme Disease Spirochete Promotes Bacterial Attachment to Glycosaminoglycans

    Journal:

    doi: 10.1128/IAI.74.1.435-441.2006

    BBK32 binds to heparin. Recombinant MBP-BBK32 or MBP proteins were added to fibronectin- or GAG-coated wells, and bound protein was quantitated by measuring absorbance at 650 nm after ELISA using MBP antiserum and an anti-rabbit horseradish peroxidase-conjugated
    Figure Legend Snippet: BBK32 binds to heparin. Recombinant MBP-BBK32 or MBP proteins were added to fibronectin- or GAG-coated wells, and bound protein was quantitated by measuring absorbance at 650 nm after ELISA using MBP antiserum and an anti-rabbit horseradish peroxidase-conjugated

    Techniques Used: Recombinant, Enzyme-linked Immunosorbent Assay

    15) Product Images from "Sec16p potentiates the action of COPII proteins to bud transport vesicles"

    Article Title: Sec16p potentiates the action of COPII proteins to bud transport vesicles

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200207053

    Effect of Sec16p on vesicle budding. (A) Microsomal membranes prepared from RSY267 were stripped as described in the legend to Fig. 1 with B88 buffer containing either 0.1 M NaCl or 0.5 M NaCl and centrifuged for 5 min at 10,000 g . Pellets were then resuspended in 100 μl of B88. 10 μl of both pellet (P) and supernatant fractions (S) were separated on 6% SDS-PAGE, transferred to nitrocellulose, and detected with the indicated antibody. (B) Vesicle release (% of total [ 35 S]gpαF released in vesicles) in the presence of saturating amounts of MBP–Sec16p (20 μg/ml) and various amounts of COPII proteins (standard conditions, 1 × COPII: 20 μg/ml Sar1p, 20 μg/ml Sec23/24p, and 50 μg/ml Sec13/31p). Membranes stripped with 0.5 M NaCl were used in budding reactions.
    Figure Legend Snippet: Effect of Sec16p on vesicle budding. (A) Microsomal membranes prepared from RSY267 were stripped as described in the legend to Fig. 1 with B88 buffer containing either 0.1 M NaCl or 0.5 M NaCl and centrifuged for 5 min at 10,000 g . Pellets were then resuspended in 100 μl of B88. 10 μl of both pellet (P) and supernatant fractions (S) were separated on 6% SDS-PAGE, transferred to nitrocellulose, and detected with the indicated antibody. (B) Vesicle release (% of total [ 35 S]gpαF released in vesicles) in the presence of saturating amounts of MBP–Sec16p (20 μg/ml) and various amounts of COPII proteins (standard conditions, 1 × COPII: 20 μg/ml Sar1p, 20 μg/ml Sec23/24p, and 50 μg/ml Sec13/31p). Membranes stripped with 0.5 M NaCl were used in budding reactions.

    Techniques Used: SDS Page

    Effect of MBP–Sec16p and GTP/GMP-PNP on COPII protein recruitment to DOPC/DOPE liposomes. DOPC/DOPE liposomes (corresponding to 25 μg of phospholipids) were incubated with various combinations of COPII proteins, MBP–Sec16p, and nucleotides (48 μg/ml Sar1p, 17 μg/ml Sec23/24p, 20 μg/ml Sec13/31p, 10 μg/ml MBP–Sec16p, and 0.1 mM GDP, GTP, or GMP-PNP) for 15 min at 30°C in a 250-μl reaction. Proteins bound to liposomes were recovered by flotation, resolved on SDS-PAGE, and stained with SYPRO red.
    Figure Legend Snippet: Effect of MBP–Sec16p and GTP/GMP-PNP on COPII protein recruitment to DOPC/DOPE liposomes. DOPC/DOPE liposomes (corresponding to 25 μg of phospholipids) were incubated with various combinations of COPII proteins, MBP–Sec16p, and nucleotides (48 μg/ml Sar1p, 17 μg/ml Sec23/24p, 20 μg/ml Sec13/31p, 10 μg/ml MBP–Sec16p, and 0.1 mM GDP, GTP, or GMP-PNP) for 15 min at 30°C in a 250-μl reaction. Proteins bound to liposomes were recovered by flotation, resolved on SDS-PAGE, and stained with SYPRO red.

    Techniques Used: Incubation, SDS Page, Staining

    Sec16p stimulates COPII vesicle formation from liposomes. (A) Liposomes (corresponding to 12.5 μg phospholipids) were incubated with COPII proteins (80 μg/ml Sar1p, 130 μg/ml Sec23/24p, and 150 μg/ml Sec13/31p), MBP–Sec16p (11 μg/ml, where indicated), and GMP-PNP (100 μM) for 30 min at 27°C, and then sedimented to equilibrium on sucrose density gradients. The fluorescence of Texas red–labeled liposomes in each fraction was measured. (B) Average fluorescence of COPII vesicle peak (fractions 10–12) for three independent gradients (error bars are SEM).
    Figure Legend Snippet: Sec16p stimulates COPII vesicle formation from liposomes. (A) Liposomes (corresponding to 12.5 μg phospholipids) were incubated with COPII proteins (80 μg/ml Sar1p, 130 μg/ml Sec23/24p, and 150 μg/ml Sec13/31p), MBP–Sec16p (11 μg/ml, where indicated), and GMP-PNP (100 μM) for 30 min at 27°C, and then sedimented to equilibrium on sucrose density gradients. The fluorescence of Texas red–labeled liposomes in each fraction was measured. (B) Average fluorescence of COPII vesicle peak (fractions 10–12) for three independent gradients (error bars are SEM).

    Techniques Used: Incubation, Fluorescence, Labeling

    Binding of MBP–Sec16p and COPII proteins to major–minor mix liposomes. (A) Liposomes (corresponding to 25 μg of phospholipids) were incubated with indicated combinations of COPII proteins, MBP–Sec16p, and nucleotides (16 μg/ml Sar1p, 17 μg/ml Sec23/24p, 20 μg/ml Sec13/31p, 10 μg/ml MBP–Sec16p, and 0.1 mM GDP or GMP-PNP) for 15 min at 30°C in 250-μl reactions and then floated on top of a 0.7-M sucrose cushion. Equal amounts of lipids, measured using fluorescent phospholipids ( Matsuoka et al., 1998 ), from floated fractions were applied to 11% SDS-PAGE and stained with SYPRO red. (B) Titration of Sec23/24p. The same amounts of Sar1p, Sec13/31p, MBP–Sec16p, and liposomes as in A were incubated with the indicated amounts of Sec23/24p in the presence of 0.1 mM GMP-PNP, and the binding of proteins was analyzed after liposome flotation. The asterisk indicates a truncated form of Sec31p.
    Figure Legend Snippet: Binding of MBP–Sec16p and COPII proteins to major–minor mix liposomes. (A) Liposomes (corresponding to 25 μg of phospholipids) were incubated with indicated combinations of COPII proteins, MBP–Sec16p, and nucleotides (16 μg/ml Sar1p, 17 μg/ml Sec23/24p, 20 μg/ml Sec13/31p, 10 μg/ml MBP–Sec16p, and 0.1 mM GDP or GMP-PNP) for 15 min at 30°C in 250-μl reactions and then floated on top of a 0.7-M sucrose cushion. Equal amounts of lipids, measured using fluorescent phospholipids ( Matsuoka et al., 1998 ), from floated fractions were applied to 11% SDS-PAGE and stained with SYPRO red. (B) Titration of Sec23/24p. The same amounts of Sar1p, Sec13/31p, MBP–Sec16p, and liposomes as in A were incubated with the indicated amounts of Sec23/24p in the presence of 0.1 mM GMP-PNP, and the binding of proteins was analyzed after liposome flotation. The asterisk indicates a truncated form of Sec31p.

    Techniques Used: Binding Assay, Incubation, SDS Page, Staining, Titration

    Titration of MBP–Sec16p in a liposome binding reaction. (A) Indicated concentrations of MBP–Sec16p were used for supplementation of binding reactions containing major–minor mix liposomes, COPII proteins (16 μg/ml Sar1p, 5 μg/ml Sec23/24p, and 20 μg/ml Sec13/31p), and GMP-PNP (0.1 mM). The asterisk indicates a truncated form of Sec31p. (B) Quantitation of bound proteins shown in A. The amounts of MBP–Sec16p present in 250-μl reactions are listed in the upper right corner of the graph.
    Figure Legend Snippet: Titration of MBP–Sec16p in a liposome binding reaction. (A) Indicated concentrations of MBP–Sec16p were used for supplementation of binding reactions containing major–minor mix liposomes, COPII proteins (16 μg/ml Sar1p, 5 μg/ml Sec23/24p, and 20 μg/ml Sec13/31p), and GMP-PNP (0.1 mM). The asterisk indicates a truncated form of Sec31p. (B) Quantitation of bound proteins shown in A. The amounts of MBP–Sec16p present in 250-μl reactions are listed in the upper right corner of the graph.

    Techniques Used: Titration, Binding Assay, Quantitation Assay

    Overexpression and purification of MBP–Sec16p. (A) Protein composition of salt extracts from ER-enriched microsomes. 100 μg of microsomal membrane proteins from either wild-type FSY3 strain (W) or MBP–Sec16p-overproducing FSY9 strain (O) were incubated on ice in a 100-μl reaction containing 0.5 M NaCl for 15 min. After incubation, mixtures were centrifuged and 10 μl of supernatant fractions were separated on 6% SDS-PAGE and stained with SYPRO red. The left lane contains molecular weight standards (M). (B) Proteins were transferred to nitrocellulose and probed with anti-Sec16p antibody. (C) Salt extract from a 10,000 g membrane pellet was passed through a 6-ml amylose-agarose column and the bound protein was eluted with buffer containing 10 mM maltose. 10 1-ml fractions were collected. 2 μl of salt extract (T), flowthrough (FT), and fractions (E1–10) were separated on 6% SDS-PAGE and stained with SYPRO red stain.
    Figure Legend Snippet: Overexpression and purification of MBP–Sec16p. (A) Protein composition of salt extracts from ER-enriched microsomes. 100 μg of microsomal membrane proteins from either wild-type FSY3 strain (W) or MBP–Sec16p-overproducing FSY9 strain (O) were incubated on ice in a 100-μl reaction containing 0.5 M NaCl for 15 min. After incubation, mixtures were centrifuged and 10 μl of supernatant fractions were separated on 6% SDS-PAGE and stained with SYPRO red. The left lane contains molecular weight standards (M). (B) Proteins were transferred to nitrocellulose and probed with anti-Sec16p antibody. (C) Salt extract from a 10,000 g membrane pellet was passed through a 6-ml amylose-agarose column and the bound protein was eluted with buffer containing 10 mM maltose. 10 1-ml fractions were collected. 2 μl of salt extract (T), flowthrough (FT), and fractions (E1–10) were separated on 6% SDS-PAGE and stained with SYPRO red stain.

    Techniques Used: Over Expression, Purification, Incubation, SDS Page, Staining, Molecular Weight

    Thin-section electron microscopy of major–minor mix liposomes incubated with and without COPII proteins, GMP-PNP, and MBP–Sec16p. (A) No protein addition showing large, uncoated, uni- and multilamellar liposomes. (B) COPII and GMP-PNP promote coating, budding, and coated vesicle formation. (C) COPII, GMP-PNP, and Sec16p produce groups of vesicular profiles in close apposition to larger liposomes. Filamentous material not seen in B tethers liposomes and coated vesicles together. Bars, 0.2 μm.
    Figure Legend Snippet: Thin-section electron microscopy of major–minor mix liposomes incubated with and without COPII proteins, GMP-PNP, and MBP–Sec16p. (A) No protein addition showing large, uncoated, uni- and multilamellar liposomes. (B) COPII and GMP-PNP promote coating, budding, and coated vesicle formation. (C) COPII, GMP-PNP, and Sec16p produce groups of vesicular profiles in close apposition to larger liposomes. Filamentous material not seen in B tethers liposomes and coated vesicles together. Bars, 0.2 μm.

    Techniques Used: Electron Microscopy, Incubation

    Related Articles

    Western Blot:

    Article Title: The Ska complex promotes Aurora B activity to ensure chromosome biorientation
    Article Snippet: .. The following antibodies were used for Western blotting: mouse anti–Aurora B (1:500; AIM-1, 611083, clone 6; BD), mouse anti-His4 (1:2,000; 34670; Qiagen), rabbit anti-INCENP (1:500; ), rabbit anti-MCAK (1:1,000; ), mouse anti-MBP (1:10,000; E8032; New England Biolabs, Inc.), rabbit anti-Ska1 and anti-Ska2 (1:1,000; ), rabbit anti-Ska3 (1:1,000; ), rabbit anti-Survivin (1:1,000; NB500-201; Novus Biologicals), and mouse anti–α-tubulin (1:3,000; T6199, clone DM1A; Sigma-Aldrich). .. Monoclonal antibody production Novel monoclonal antibodies against Ska3 were developed commercially with Moravian Biotechnology.

    Article Title: Fibroblast Growth Factor Binding Protein 3 (FGFBP3) impacts carbohydrate and lipid metabolism
    Article Snippet: .. Bound proteins were detected by western blot analysis with 1 μg/ml of an anti FGFR4 (LD1; a kind gift of Genentech, South San Francisco, CA), anti MBP (New England BioLabs) or anti FGF19 (Abnova, Walnut, CA) mouse monoclonal antibodies. .. Human hepatocellular carcinoma (HepG2) and HEK293 cells were maintained in Dulbecco’s modified Eagle’s medium (Life Technologies) supplemented with 10% (v/v) fetal bovine serum.

    other:

    Article Title: Citrullination of Inhibitor of Growth 4 (ING4) by Peptidylarginine Deminase 4 (PAD4) Disrupts the Interaction between ING4 and p53
    Article Snippet: Anti-modified citrulline (Millipore, Billerica, MA), anti-FLAG (M2, Sigma), anti-MBP (New England Biolabs), anti-PAD4 (Abcam, Cambridge, MA), anti-ING4 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-p53 (GeneTex, Irvine, CA), anti-p53 Clone DO-1 (Sigma), anti-acetyl-p53 (Lys-382) (Millipore), anti-β-tubulin (Sigma), anti-p21WAF1(EMD4 Biosciences, Calbiochem), and HRP-conjugated anti-GST (Millipore, Upstate Biotechnology) antibodies were obtained from commercial sources.

    Labeling:

    Article Title: “DNA Binding Region” of BRCA1 Affects Genetic Stability through modulating the Intra-S-Phase Checkpoint
    Article Snippet: .. Supershift assays were performed by preincubating purified full length BRCA1 protein with MBP antibody (NEB) before adding the labeled oligonucleotide. .. To perform the competition assay to compare the binding affinity of various shaped DNA structures to BRCA1 protein, non-labeled oligonucleotides of the various shapes with various concentrations were added at the start of the incubation reaction with labeled double strand DNA and BRCA1 protein.

    Purification:

    Article Title: “DNA Binding Region” of BRCA1 Affects Genetic Stability through modulating the Intra-S-Phase Checkpoint
    Article Snippet: .. Supershift assays were performed by preincubating purified full length BRCA1 protein with MBP antibody (NEB) before adding the labeled oligonucleotide. .. To perform the competition assay to compare the binding affinity of various shaped DNA structures to BRCA1 protein, non-labeled oligonucleotides of the various shapes with various concentrations were added at the start of the incubation reaction with labeled double strand DNA and BRCA1 protein.

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    New England Biolabs mbp unc 89 ig53 fn2
    Mapping of interaction sites of PPTR-1 and PPTR-2 for <t>UNC-89,</t> and of UNC-89 for PPTR-1 and PPTR-2. (A) When tested by yeast two-hybrid assays with segments that cover all of UNC-89B, PPTR-1 and PPTR-2 only interact with UNC-89 1/3 <t>IK-Ig53-Fn2.</t> Pink rectangles, Ig domains; green rectangles, Fn3 domains; other domains are as indicated. (B) Domain mapping using yeast two-hybrid assays indicates that the minimal region of UNC-89 that interacts with PPTR-1 is 1/3 IK-Ig53-Fn2, but that the minimal region of UNC-89 that interacts with PPTR-2 is Ig53-Fn2.
    Mbp Unc 89 Ig53 Fn2, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    New England Biolabs mbp rangtp
    The predicted <t>RanGTP</t> binding site at HEAT repeats 1–4 of Imp9. ( A ) A zoomed in view (the N-terminal half of Imp9) of the Imp9 • H2A-H2B structure shown in the same orientation as the importins in Figure 4—figure supplement 5D . Imp9 is in blue, H2A in yellow and H2B in red. Imp9 is structurally aligned with the 1 st four HEAT repeats of Kap121 (shown in dark blue) in the Kap121•RanGTP structure (3W3Z). RanGTP from the Kap121•RanGTP structure is not shown but is schematically depicted by the light-green heart-shape drawing to mark the pred icted location of the Ran site on Imp9. ( B ) Top panel, same view of the Imp9•H2A-H2B structure as in A ), with Imp9 residues at the predicted RanGTP binding site colored green. Bottom panel shows the view upon 90° rotation about the vertical axis.( C–E ) Size exclusion chromatography of <t>MBP-Imp9Δ1–144</t> + excess RanGTP ( C ), MBP-Imp9Δ1–144 + H2A-H2B ( D ) and previously purified MBP-Imp9Δ1–144•H2A-H2B + excess RanGTP ( E ). 500 μL protein samples were loaded on to a Superdex S200 Increase 10/300 column and the size exclusion chromatography experiments were performed in buffer containing 20 mM HEPES pH 7.4, 200 mM sodium chloride, 2 mM magnesium acetate, 2 mM DTT and 10% glycerol. The elution volume for each protein peak is shown and proteins in the fractions are visualized by Coomassie-stained SDS-PAGE gels shown above the chromatograms. SEC analysis shows that RanGTP does not interact with the Imp9 mutant. No interaction is seen at micromolar concentrations even when RanGTP is added at a 6-fold molar excess. This is obvious from the SDS-PAGE analysis of SEC fractions, showing that the Imp9 mutant and RanGTP do not co-migrate ( C ). The Imp9 mutant protein is functional as the interaction is maintained with H2A-H2B ( D ). This is consistent with the crystal structure showing that the region spanning HEAT repeats 1–3 of Imp9 (residues 1–144) is only a very small portion of the very large Imp9•H2A-H2B interface. Not surprisingly, like Imp9 mutant alone in C ), the histone-bound Imp9 mutant also does not bind RanGTP when the GTPase is added at a molar excess ( E ).
    Mbp Rangtp, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    New England Biolabs protein expression pet mbp tagrfp
    Biotinylation of <t>TagRFP.</t> ( a ) The C-terminal of TagRFP has 2 lysines (blue) at residue 231 and 235. ( b ) Bacteria displaying the C-terminal peptide from TagRFP were clearly enriched at 3 selection rounds using streptavidin Dynabeads. ( c ) Ten clones isolated after 3 selection rounds were transformed with <t>MBP-TagRFP</t> (expected size 75 kDa). Western blots of the clones showed that 8/10 clones caused the appearance of a streptavidin reacting band at 75 kDa, whereas the 2 negative clones showed a faint band at 22 kDa, which is the expected size of eCPX. An uncropped image of the blot is shown in Supplementary Fig. 12 . ( d ) MBP-TagRFP was not biotinylated by BirA-6xHis, but clone 1 biotinylated TagRFP and TagRFP with K235A mutation. Western blot against MBP showed that TagRFP was present in all lanes. The blots probed with streptavidin and anti-MBP are from separate gels. Uncropped images of the blots probed with streptavidin and anti-MBP, respectively, are shown in Supplementary Figs 14 and 15 .
    Protein Expression Pet Mbp Tagrfp, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    New England Biolabs mbp nod485cc egfp
    Dimerized NOD tip tracks on dynamic MTs and directly interacts with EB1 through a new motif. (A) A mitotic cell coexpressing <t>NOD485CC-EGFP</t> (green) and EB1-mCherry (red). Kymographs of dynamic astral MTs within the white box reveal subpopulations of NOD puncta tip-tracking coincident with EB1, remaining associated with depolymerizing plus ends, and walking toward the MT plus ends. (B) Kymograph of motile and tip-tracking NOD485CC (green) dimers on dynamic MTs (red, plus and minus ends labeled in MT kymograph) incubated in cell lysate and subjected to TIRF imaging. (C) Coomassie-stained SDS-PAGE gels showing purified <t>MBP-GFP</t> and MBP-NOD485CC-EGFP (left) and Drosophila GST-EB1-TagRFP-T (right) used in the pulldown assays. (D) Western blot for TagRFP of pulldown assay showing Drosophila GST-EB1-TagRFP-T specifically interacts with MBP-NOD485CC-EGFP, but not with MBP-EGFP (left blot). Asterisks denote αTagRFP cross-reactivity with MBP-EGFP and MBP-NOD485CC-EGFP, which were detected by Western blot with α-GFP (right blot). (E) Coomassie-stained SDS-PAGE gel showing the purified Drosophila EB1-TagRFP-T (GST cleaved off) used to probe the SPOT peptide arrays. (F) SPOT peptide arrays of NOD 400–485, and including a “perfect” SxIP peptide as a positive control, probed with anti-EB1 serum (control) or purified EB1-TagRFP-T followed by incubation with the anti-EB1 serum. Two peptides with positive EB1 binding are highlighted in red (PT motif-1) and blue (PT motif-2). (G) Alanine scans of the two peptides highlighted in F. (H) Coomassie-stained SDS-PAGE gel showing the purified Drosophila GST-EB1 used in MST. (I) MST was done by titrating GST-EB1 while maintaining a constant concentrations of FITC-labeled “perfect” SxIP aptamer (left) or NOD “PT” motif-1 (right) resulting in measurable changes in the fluorescence signal within a temperature gradient that can be used to calculate dissociation constants (SxIP K d = 807 ± 86 nM and NOD PT motif-1 K d = 725 ± 77 nM). Error bars represent standard deviation of n = 3 (PT motif-1) and 2 (SxIP motif) MST runs. The K d values are reported as mean ± SEM. Horizontal bars: 5 µm (A); 1 µm in all kymographs (A and B). Vertical bars: 20 s.
    Mbp Nod485cc Egfp, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Mapping of interaction sites of PPTR-1 and PPTR-2 for UNC-89, and of UNC-89 for PPTR-1 and PPTR-2. (A) When tested by yeast two-hybrid assays with segments that cover all of UNC-89B, PPTR-1 and PPTR-2 only interact with UNC-89 1/3 IK-Ig53-Fn2. Pink rectangles, Ig domains; green rectangles, Fn3 domains; other domains are as indicated. (B) Domain mapping using yeast two-hybrid assays indicates that the minimal region of UNC-89 that interacts with PPTR-1 is 1/3 IK-Ig53-Fn2, but that the minimal region of UNC-89 that interacts with PPTR-2 is Ig53-Fn2.

    Journal: Molecular Biology of the Cell

    Article Title: Protein phosphatase 2A is crucial for sarcomere organization in Caenorhabditis elegans striated muscle

    doi: 10.1091/mbc.E18-03-0192

    Figure Lengend Snippet: Mapping of interaction sites of PPTR-1 and PPTR-2 for UNC-89, and of UNC-89 for PPTR-1 and PPTR-2. (A) When tested by yeast two-hybrid assays with segments that cover all of UNC-89B, PPTR-1 and PPTR-2 only interact with UNC-89 1/3 IK-Ig53-Fn2. Pink rectangles, Ig domains; green rectangles, Fn3 domains; other domains are as indicated. (B) Domain mapping using yeast two-hybrid assays indicates that the minimal region of UNC-89 that interacts with PPTR-1 is 1/3 IK-Ig53-Fn2, but that the minimal region of UNC-89 that interacts with PPTR-2 is Ig53-Fn2.

    Article Snippet: Blot strips containing His-PPTR-1 and His-PPTR-2 were incubated with either MBP, MBP-UNC-89 Ig53-Fn2, or MBP-UNC-89 1/3 IK-Ig53-Fn2 at 5 μg/ml at room temperature for 1 h, washed multiple times in TBS-T, reacted with anti–MBP-horseradish peroxidase (HRP) (New England BioLabs) at 1:5000 dilution, and washed multiple times, and reactions were visualized by enhanced chemiluminescence (ECL Pierce, Thermo Fisher Scientific).

    Techniques:

    Segments of UNC-89 interact with PPTR-1 and PPTR-2 in vitro. (A) Far-Western assay. His-tagged PPTR-1 and PPTR-2 were separated by SDS–PAGE, transferred to membrane, incubated with MBP, MBP-UNC-89 Ig53-Fn2, or MBP-UNC-89 1/3 IK-Ig53-Fn2, washed, incubated with anti–MBP-HRP, washed, and detected by ECL. Both MBP fusions of these portions of UNC-89, but not MBP itself, bind to either His-PPTR-1 or His-PPTR-2. (B) In-solution pull-down assay. The indicated proteins were incubated together, and then the His-tagged proteins were pelleted using anti-6His beads. Proteins were eluted from the beads and separated by SDS–PAGE and transferred to a membrane, and then a Western blot was performed using anti–MBP-HRP to detect any copelleting MBP or MBP-UNC-89-Ig-Fn. At the bottom is shown the blot after Ponceau S staining, and above it is the Western blot. Both His-PPTR-1 and His-PPTR-2 pull down MBP-UNC-89-Ig-Fn, but not MBP. Positions of proteins are indicated by arrows; * likely degradation products from MBP-UNC-89-Ig-Fn; ** Ig heavy chain; *** Ig light chain. The predicted molecular weights of the bacterially expressed purified proteins are as follows: His-PPTR-1, 51.5 kDa; His-PPTR-2, 60 kDa; MBP, 42.5 kDa; MBP-UNC-89 Ig53-Fn2, 73.5 kDa; and MBP-UNC-89 1/3 IK-Ig53-Fn2, 97.3 kDa.

    Journal: Molecular Biology of the Cell

    Article Title: Protein phosphatase 2A is crucial for sarcomere organization in Caenorhabditis elegans striated muscle

    doi: 10.1091/mbc.E18-03-0192

    Figure Lengend Snippet: Segments of UNC-89 interact with PPTR-1 and PPTR-2 in vitro. (A) Far-Western assay. His-tagged PPTR-1 and PPTR-2 were separated by SDS–PAGE, transferred to membrane, incubated with MBP, MBP-UNC-89 Ig53-Fn2, or MBP-UNC-89 1/3 IK-Ig53-Fn2, washed, incubated with anti–MBP-HRP, washed, and detected by ECL. Both MBP fusions of these portions of UNC-89, but not MBP itself, bind to either His-PPTR-1 or His-PPTR-2. (B) In-solution pull-down assay. The indicated proteins were incubated together, and then the His-tagged proteins were pelleted using anti-6His beads. Proteins were eluted from the beads and separated by SDS–PAGE and transferred to a membrane, and then a Western blot was performed using anti–MBP-HRP to detect any copelleting MBP or MBP-UNC-89-Ig-Fn. At the bottom is shown the blot after Ponceau S staining, and above it is the Western blot. Both His-PPTR-1 and His-PPTR-2 pull down MBP-UNC-89-Ig-Fn, but not MBP. Positions of proteins are indicated by arrows; * likely degradation products from MBP-UNC-89-Ig-Fn; ** Ig heavy chain; *** Ig light chain. The predicted molecular weights of the bacterially expressed purified proteins are as follows: His-PPTR-1, 51.5 kDa; His-PPTR-2, 60 kDa; MBP, 42.5 kDa; MBP-UNC-89 Ig53-Fn2, 73.5 kDa; and MBP-UNC-89 1/3 IK-Ig53-Fn2, 97.3 kDa.

    Article Snippet: Blot strips containing His-PPTR-1 and His-PPTR-2 were incubated with either MBP, MBP-UNC-89 Ig53-Fn2, or MBP-UNC-89 1/3 IK-Ig53-Fn2 at 5 μg/ml at room temperature for 1 h, washed multiple times in TBS-T, reacted with anti–MBP-horseradish peroxidase (HRP) (New England BioLabs) at 1:5000 dilution, and washed multiple times, and reactions were visualized by enhanced chemiluminescence (ECL Pierce, Thermo Fisher Scientific).

    Techniques: In Vitro, Western Blot, SDS Page, Incubation, Pull Down Assay, Staining, Purification

    The predicted RanGTP binding site at HEAT repeats 1–4 of Imp9. ( A ) A zoomed in view (the N-terminal half of Imp9) of the Imp9 • H2A-H2B structure shown in the same orientation as the importins in Figure 4—figure supplement 5D . Imp9 is in blue, H2A in yellow and H2B in red. Imp9 is structurally aligned with the 1 st four HEAT repeats of Kap121 (shown in dark blue) in the Kap121•RanGTP structure (3W3Z). RanGTP from the Kap121•RanGTP structure is not shown but is schematically depicted by the light-green heart-shape drawing to mark the pred icted location of the Ran site on Imp9. ( B ) Top panel, same view of the Imp9•H2A-H2B structure as in A ), with Imp9 residues at the predicted RanGTP binding site colored green. Bottom panel shows the view upon 90° rotation about the vertical axis.( C–E ) Size exclusion chromatography of MBP-Imp9Δ1–144 + excess RanGTP ( C ), MBP-Imp9Δ1–144 + H2A-H2B ( D ) and previously purified MBP-Imp9Δ1–144•H2A-H2B + excess RanGTP ( E ). 500 μL protein samples were loaded on to a Superdex S200 Increase 10/300 column and the size exclusion chromatography experiments were performed in buffer containing 20 mM HEPES pH 7.4, 200 mM sodium chloride, 2 mM magnesium acetate, 2 mM DTT and 10% glycerol. The elution volume for each protein peak is shown and proteins in the fractions are visualized by Coomassie-stained SDS-PAGE gels shown above the chromatograms. SEC analysis shows that RanGTP does not interact with the Imp9 mutant. No interaction is seen at micromolar concentrations even when RanGTP is added at a 6-fold molar excess. This is obvious from the SDS-PAGE analysis of SEC fractions, showing that the Imp9 mutant and RanGTP do not co-migrate ( C ). The Imp9 mutant protein is functional as the interaction is maintained with H2A-H2B ( D ). This is consistent with the crystal structure showing that the region spanning HEAT repeats 1–3 of Imp9 (residues 1–144) is only a very small portion of the very large Imp9•H2A-H2B interface. Not surprisingly, like Imp9 mutant alone in C ), the histone-bound Imp9 mutant also does not bind RanGTP when the GTPase is added at a molar excess ( E ).

    Journal: eLife

    Article Title: Importin-9 wraps around the H2A-H2B core to act as nuclear importer and histone chaperone

    doi: 10.7554/eLife.43630

    Figure Lengend Snippet: The predicted RanGTP binding site at HEAT repeats 1–4 of Imp9. ( A ) A zoomed in view (the N-terminal half of Imp9) of the Imp9 • H2A-H2B structure shown in the same orientation as the importins in Figure 4—figure supplement 5D . Imp9 is in blue, H2A in yellow and H2B in red. Imp9 is structurally aligned with the 1 st four HEAT repeats of Kap121 (shown in dark blue) in the Kap121•RanGTP structure (3W3Z). RanGTP from the Kap121•RanGTP structure is not shown but is schematically depicted by the light-green heart-shape drawing to mark the pred icted location of the Ran site on Imp9. ( B ) Top panel, same view of the Imp9•H2A-H2B structure as in A ), with Imp9 residues at the predicted RanGTP binding site colored green. Bottom panel shows the view upon 90° rotation about the vertical axis.( C–E ) Size exclusion chromatography of MBP-Imp9Δ1–144 + excess RanGTP ( C ), MBP-Imp9Δ1–144 + H2A-H2B ( D ) and previously purified MBP-Imp9Δ1–144•H2A-H2B + excess RanGTP ( E ). 500 μL protein samples were loaded on to a Superdex S200 Increase 10/300 column and the size exclusion chromatography experiments were performed in buffer containing 20 mM HEPES pH 7.4, 200 mM sodium chloride, 2 mM magnesium acetate, 2 mM DTT and 10% glycerol. The elution volume for each protein peak is shown and proteins in the fractions are visualized by Coomassie-stained SDS-PAGE gels shown above the chromatograms. SEC analysis shows that RanGTP does not interact with the Imp9 mutant. No interaction is seen at micromolar concentrations even when RanGTP is added at a 6-fold molar excess. This is obvious from the SDS-PAGE analysis of SEC fractions, showing that the Imp9 mutant and RanGTP do not co-migrate ( C ). The Imp9 mutant protein is functional as the interaction is maintained with H2A-H2B ( D ). This is consistent with the crystal structure showing that the region spanning HEAT repeats 1–3 of Imp9 (residues 1–144) is only a very small portion of the very large Imp9•H2A-H2B interface. Not surprisingly, like Imp9 mutant alone in C ), the histone-bound Imp9 mutant also does not bind RanGTP when the GTPase is added at a molar excess ( E ).

    Article Snippet: Pull-down binding assays Pull-down binding assays were performed by immobilizing purified MBP-Imp9 or MBP-RanGTP (S. cerevisiae Gsp1(1–179/Q71L) on amylose resin (New England BioLabs, Ipswich, MA).

    Techniques: Binding Assay, Size-exclusion Chromatography, Purification, Staining, SDS Page, Mutagenesis, Functional Assay

    Interactions between RanGTP and the Imp9•H2A-H2B complex. ( A ) Pull-down binding assays to probe RanGTP ( S. cerevisiae Gsp1 (1–179/Q71L) interactions with the Imp9•H2A-H2B complex. Increasing concentrations of RanGTP (12.5 μM, 25 μM, 50 μM or 75 μM) was added to 2.5 μM MBP-Imp9•H2A-H2B. After washing, bound proteins were visualized by Coomassie-stained SDS-PAGE. 2% of input RanGTP for the corresponding to binding reactions in lanes 6–9 are shown lanes 15–18. 2% of the flow-through from the corresponding to binding reactions in lanes 6–9 are shown lanes 24–27. ( B ) Pull-down binding assays to probe RanGTP mediated dissociation of MBP-PYNLS from the GST-Kapβ2•MBP-PY-NLS complex. Increasing concentrations of RanGTP (12.5 μM, 25 μM, 50 μM or 75 μM) was added to 2.5 μM GST-Kapβ2•MBP-PY-NLS. After washing, the bound proteins were visualized by Coomassie-stained SDS-PAGE. 2% of input RanGTP for the corresponding binding reactions in lane 6–9 are shown in lanes 21–24. 2% of flow-through from the corresponding binding reactions in lanes 6–9 are shown in lanes 11–14. ( C ) Controls for pull-down binding assays. Imp9 (lanes 3–5), H2A-H2B (lane 7–9) or RanGTP (lane 11–13) was added to immobilized MBP. The flow-through (FT), the last wash and the proteins that remain bound on beads after washing were visualized by Coomassie-stained SDS-PAGE. ( D ) Coomassie-stained SDS-PAGE of the protein inputs for the native gel in Figure 4E .

    Journal: eLife

    Article Title: Importin-9 wraps around the H2A-H2B core to act as nuclear importer and histone chaperone

    doi: 10.7554/eLife.43630

    Figure Lengend Snippet: Interactions between RanGTP and the Imp9•H2A-H2B complex. ( A ) Pull-down binding assays to probe RanGTP ( S. cerevisiae Gsp1 (1–179/Q71L) interactions with the Imp9•H2A-H2B complex. Increasing concentrations of RanGTP (12.5 μM, 25 μM, 50 μM or 75 μM) was added to 2.5 μM MBP-Imp9•H2A-H2B. After washing, bound proteins were visualized by Coomassie-stained SDS-PAGE. 2% of input RanGTP for the corresponding to binding reactions in lanes 6–9 are shown lanes 15–18. 2% of the flow-through from the corresponding to binding reactions in lanes 6–9 are shown lanes 24–27. ( B ) Pull-down binding assays to probe RanGTP mediated dissociation of MBP-PYNLS from the GST-Kapβ2•MBP-PY-NLS complex. Increasing concentrations of RanGTP (12.5 μM, 25 μM, 50 μM or 75 μM) was added to 2.5 μM GST-Kapβ2•MBP-PY-NLS. After washing, the bound proteins were visualized by Coomassie-stained SDS-PAGE. 2% of input RanGTP for the corresponding binding reactions in lane 6–9 are shown in lanes 21–24. 2% of flow-through from the corresponding binding reactions in lanes 6–9 are shown in lanes 11–14. ( C ) Controls for pull-down binding assays. Imp9 (lanes 3–5), H2A-H2B (lane 7–9) or RanGTP (lane 11–13) was added to immobilized MBP. The flow-through (FT), the last wash and the proteins that remain bound on beads after washing were visualized by Coomassie-stained SDS-PAGE. ( D ) Coomassie-stained SDS-PAGE of the protein inputs for the native gel in Figure 4E .

    Article Snippet: Pull-down binding assays Pull-down binding assays were performed by immobilizing purified MBP-Imp9 or MBP-RanGTP (S. cerevisiae Gsp1(1–179/Q71L) on amylose resin (New England BioLabs, Ipswich, MA).

    Techniques: Binding Assay, Staining, SDS Page, Flow Cytometry

    Biotinylation of TagRFP. ( a ) The C-terminal of TagRFP has 2 lysines (blue) at residue 231 and 235. ( b ) Bacteria displaying the C-terminal peptide from TagRFP were clearly enriched at 3 selection rounds using streptavidin Dynabeads. ( c ) Ten clones isolated after 3 selection rounds were transformed with MBP-TagRFP (expected size 75 kDa). Western blots of the clones showed that 8/10 clones caused the appearance of a streptavidin reacting band at 75 kDa, whereas the 2 negative clones showed a faint band at 22 kDa, which is the expected size of eCPX. An uncropped image of the blot is shown in Supplementary Fig. 12 . ( d ) MBP-TagRFP was not biotinylated by BirA-6xHis, but clone 1 biotinylated TagRFP and TagRFP with K235A mutation. Western blot against MBP showed that TagRFP was present in all lanes. The blots probed with streptavidin and anti-MBP are from separate gels. Uncropped images of the blots probed with streptavidin and anti-MBP, respectively, are shown in Supplementary Figs 14 and 15 .

    Journal: Scientific Reports

    Article Title: A bacterial display system for effective selection of protein-biotin ligase BirA variants with novel peptide specificity

    doi: 10.1038/s41598-019-40984-x

    Figure Lengend Snippet: Biotinylation of TagRFP. ( a ) The C-terminal of TagRFP has 2 lysines (blue) at residue 231 and 235. ( b ) Bacteria displaying the C-terminal peptide from TagRFP were clearly enriched at 3 selection rounds using streptavidin Dynabeads. ( c ) Ten clones isolated after 3 selection rounds were transformed with MBP-TagRFP (expected size 75 kDa). Western blots of the clones showed that 8/10 clones caused the appearance of a streptavidin reacting band at 75 kDa, whereas the 2 negative clones showed a faint band at 22 kDa, which is the expected size of eCPX. An uncropped image of the blot is shown in Supplementary Fig. 12 . ( d ) MBP-TagRFP was not biotinylated by BirA-6xHis, but clone 1 biotinylated TagRFP and TagRFP with K235A mutation. Western blot against MBP showed that TagRFP was present in all lanes. The blots probed with streptavidin and anti-MBP are from separate gels. Uncropped images of the blots probed with streptavidin and anti-MBP, respectively, are shown in Supplementary Figs 14 and 15 .

    Article Snippet: Protein expression pET-MBP-TagRFP (and the K231A, K235A and K231A,K235A variants) were transformed into T7 Express lysY/Iq (New England Biolabs) and grown in LB agar plates with kanamycin (Sigma Aldrich) as selection agents.

    Techniques: Selection, Clone Assay, Isolation, Transformation Assay, Western Blot, Mutagenesis

    Dimerized NOD tip tracks on dynamic MTs and directly interacts with EB1 through a new motif. (A) A mitotic cell coexpressing NOD485CC-EGFP (green) and EB1-mCherry (red). Kymographs of dynamic astral MTs within the white box reveal subpopulations of NOD puncta tip-tracking coincident with EB1, remaining associated with depolymerizing plus ends, and walking toward the MT plus ends. (B) Kymograph of motile and tip-tracking NOD485CC (green) dimers on dynamic MTs (red, plus and minus ends labeled in MT kymograph) incubated in cell lysate and subjected to TIRF imaging. (C) Coomassie-stained SDS-PAGE gels showing purified MBP-GFP and MBP-NOD485CC-EGFP (left) and Drosophila GST-EB1-TagRFP-T (right) used in the pulldown assays. (D) Western blot for TagRFP of pulldown assay showing Drosophila GST-EB1-TagRFP-T specifically interacts with MBP-NOD485CC-EGFP, but not with MBP-EGFP (left blot). Asterisks denote αTagRFP cross-reactivity with MBP-EGFP and MBP-NOD485CC-EGFP, which were detected by Western blot with α-GFP (right blot). (E) Coomassie-stained SDS-PAGE gel showing the purified Drosophila EB1-TagRFP-T (GST cleaved off) used to probe the SPOT peptide arrays. (F) SPOT peptide arrays of NOD 400–485, and including a “perfect” SxIP peptide as a positive control, probed with anti-EB1 serum (control) or purified EB1-TagRFP-T followed by incubation with the anti-EB1 serum. Two peptides with positive EB1 binding are highlighted in red (PT motif-1) and blue (PT motif-2). (G) Alanine scans of the two peptides highlighted in F. (H) Coomassie-stained SDS-PAGE gel showing the purified Drosophila GST-EB1 used in MST. (I) MST was done by titrating GST-EB1 while maintaining a constant concentrations of FITC-labeled “perfect” SxIP aptamer (left) or NOD “PT” motif-1 (right) resulting in measurable changes in the fluorescence signal within a temperature gradient that can be used to calculate dissociation constants (SxIP K d = 807 ± 86 nM and NOD PT motif-1 K d = 725 ± 77 nM). Error bars represent standard deviation of n = 3 (PT motif-1) and 2 (SxIP motif) MST runs. The K d values are reported as mean ± SEM. Horizontal bars: 5 µm (A); 1 µm in all kymographs (A and B). Vertical bars: 20 s.

    Journal: The Journal of Cell Biology

    Article Title: NOD is a plus end–directed motor that binds EB1 via a new microtubule tip localization sequence

    doi: 10.1083/jcb.201708109

    Figure Lengend Snippet: Dimerized NOD tip tracks on dynamic MTs and directly interacts with EB1 through a new motif. (A) A mitotic cell coexpressing NOD485CC-EGFP (green) and EB1-mCherry (red). Kymographs of dynamic astral MTs within the white box reveal subpopulations of NOD puncta tip-tracking coincident with EB1, remaining associated with depolymerizing plus ends, and walking toward the MT plus ends. (B) Kymograph of motile and tip-tracking NOD485CC (green) dimers on dynamic MTs (red, plus and minus ends labeled in MT kymograph) incubated in cell lysate and subjected to TIRF imaging. (C) Coomassie-stained SDS-PAGE gels showing purified MBP-GFP and MBP-NOD485CC-EGFP (left) and Drosophila GST-EB1-TagRFP-T (right) used in the pulldown assays. (D) Western blot for TagRFP of pulldown assay showing Drosophila GST-EB1-TagRFP-T specifically interacts with MBP-NOD485CC-EGFP, but not with MBP-EGFP (left blot). Asterisks denote αTagRFP cross-reactivity with MBP-EGFP and MBP-NOD485CC-EGFP, which were detected by Western blot with α-GFP (right blot). (E) Coomassie-stained SDS-PAGE gel showing the purified Drosophila EB1-TagRFP-T (GST cleaved off) used to probe the SPOT peptide arrays. (F) SPOT peptide arrays of NOD 400–485, and including a “perfect” SxIP peptide as a positive control, probed with anti-EB1 serum (control) or purified EB1-TagRFP-T followed by incubation with the anti-EB1 serum. Two peptides with positive EB1 binding are highlighted in red (PT motif-1) and blue (PT motif-2). (G) Alanine scans of the two peptides highlighted in F. (H) Coomassie-stained SDS-PAGE gel showing the purified Drosophila GST-EB1 used in MST. (I) MST was done by titrating GST-EB1 while maintaining a constant concentrations of FITC-labeled “perfect” SxIP aptamer (left) or NOD “PT” motif-1 (right) resulting in measurable changes in the fluorescence signal within a temperature gradient that can be used to calculate dissociation constants (SxIP K d = 807 ± 86 nM and NOD PT motif-1 K d = 725 ± 77 nM). Error bars represent standard deviation of n = 3 (PT motif-1) and 2 (SxIP motif) MST runs. The K d values are reported as mean ± SEM. Horizontal bars: 5 µm (A); 1 µm in all kymographs (A and B). Vertical bars: 20 s.

    Article Snippet: Twofold molar excess of MBP-NOD485CC-EGFP, and MBP-EGFP as control was added to GST-EB1-TagRFP-T in the presence of 5 mg/ml BSA and incubated at 25°C with agitation for 1 h. Protein mixtures were then added to amylose magnetic beads (NEB) and incubated for an additional hour at 25°C.

    Techniques: Labeling, Incubation, Imaging, Staining, SDS Page, Purification, Western Blot, Positive Control, Binding Assay, Microscale Thermophoresis, Fluorescence, Standard Deviation

    Chemically induced NOD dimers exhibit directional motility, and a NOD mutant lacking one of the DNA binding domains is motile and tip tracks. (A) Schematic of the rapamycin-based approach used to dimerize NOD485 molecules. (B) TIRF image of a cell expressing NOD485-FKBP-EGFP and NOD485-FRB treated with rapamycin. (C) Select frames from the region in the white box in B showing a motile NOD puncta (marked by arrows) walking toward the cell periphery (right). (D) Kymograph of dimerized NOD485 walking on a MT toward the cell periphery (right) within the white box in B. (E) TIRF assay of taxol-stabilized MTs (blue) plus lysate containing ATP prepared from cells expressing NOD485-FKBP-EGFP (green) and NOD485-FRB in the absence of rapamycin. (F) TIRF of the same cell lysate in E but in the presence of 100 nM rapamycin. A motile NOD puncta is highlighted in the kymograph. (G) Schematic of rapamycin-induced dimerization of NOD485-FKBP-EGFP and NOD485-FRB-mCherry. (H) Representative images of cells expressing NOD485-FKBP-EGFP (green) and NOD485-FRB-mCherry (red) in the absence (top) and presence (bottom) of rapamycin in which the localization pattern was the same as NOD485 and NOD485CC, respectively. (I) Schematic of NODΔHMG in which one of the chromosome-associating domains is deleted. (J) Kymograph of a NODΔHMG cluster (red) walking along MTs (green) in a cell. (K) Example of NODΔHMG cluster in a cell exhibiting two activities on the same MT (with plus and minus-ends indicated in the tubulin kymograph): tip tracking (closed arrows) and plus end–directed motility (open arrows). (L) Histogram of the velocities of NODΔHMG puncta, with a mean of 7.63 ± 2.03 µm/min; n = 37. Horizontal bars: 10 µm (B); 1 µm (C–F, J, and K); 5 µm (H). Vertical bars: 10 s (B–F, H, and J); 1 min (K). Mean ± SD.

    Journal: The Journal of Cell Biology

    Article Title: NOD is a plus end–directed motor that binds EB1 via a new microtubule tip localization sequence

    doi: 10.1083/jcb.201708109

    Figure Lengend Snippet: Chemically induced NOD dimers exhibit directional motility, and a NOD mutant lacking one of the DNA binding domains is motile and tip tracks. (A) Schematic of the rapamycin-based approach used to dimerize NOD485 molecules. (B) TIRF image of a cell expressing NOD485-FKBP-EGFP and NOD485-FRB treated with rapamycin. (C) Select frames from the region in the white box in B showing a motile NOD puncta (marked by arrows) walking toward the cell periphery (right). (D) Kymograph of dimerized NOD485 walking on a MT toward the cell periphery (right) within the white box in B. (E) TIRF assay of taxol-stabilized MTs (blue) plus lysate containing ATP prepared from cells expressing NOD485-FKBP-EGFP (green) and NOD485-FRB in the absence of rapamycin. (F) TIRF of the same cell lysate in E but in the presence of 100 nM rapamycin. A motile NOD puncta is highlighted in the kymograph. (G) Schematic of rapamycin-induced dimerization of NOD485-FKBP-EGFP and NOD485-FRB-mCherry. (H) Representative images of cells expressing NOD485-FKBP-EGFP (green) and NOD485-FRB-mCherry (red) in the absence (top) and presence (bottom) of rapamycin in which the localization pattern was the same as NOD485 and NOD485CC, respectively. (I) Schematic of NODΔHMG in which one of the chromosome-associating domains is deleted. (J) Kymograph of a NODΔHMG cluster (red) walking along MTs (green) in a cell. (K) Example of NODΔHMG cluster in a cell exhibiting two activities on the same MT (with plus and minus-ends indicated in the tubulin kymograph): tip tracking (closed arrows) and plus end–directed motility (open arrows). (L) Histogram of the velocities of NODΔHMG puncta, with a mean of 7.63 ± 2.03 µm/min; n = 37. Horizontal bars: 10 µm (B); 1 µm (C–F, J, and K); 5 µm (H). Vertical bars: 10 s (B–F, H, and J); 1 min (K). Mean ± SD.

    Article Snippet: Twofold molar excess of MBP-NOD485CC-EGFP, and MBP-EGFP as control was added to GST-EB1-TagRFP-T in the presence of 5 mg/ml BSA and incubated at 25°C with agitation for 1 h. Protein mixtures were then added to amylose magnetic beads (NEB) and incubated for an additional hour at 25°C.

    Techniques: Mutagenesis, Binding Assay, Expressing

    Dimerized NOD485 exhibits ATP-dependent, plus end–directed motility in cells and in vitro. (A) TIRF microscopy of an interphase cell expressing NOD485CC. (B) Select frames from a TIRF time-lapse zoomed on the region in the black box in A showing a motile NOD485CC dimer (marked by arrow). (C) Kymograph of NOD485CC dimer from the black box in A walking on MTs toward the cell periphery (right side of kymograph). (D) Histogram of velocities of motile NOD485CC dimers in cells, with a mean velocity of 8.70 ± 3.61 µm/min; n = 55. (E) TIRF microscopy of NOD dimers (green) on taxol-stabilized MTs (red) using a cell lysate from NOD485CC-mCherry–expressing cells in the presence of ATP (top) or AMP-PNP (bottom). (F) Histogram of the velocities of motile NOD485CC dimers in cell lysates, with a mean velocity of 8.62 ± 2.32 µm/min; n = 47. (G) TIRF microscopy of taxol-stabilized MTs (red) incubated with Strep-tagged NOD485CC-mCherry (green) purified from S2 cells in the presence of ATP (top) or AMP-PNP (bottom). (H) Histogram of the velocities of purified Strep-tagged NOD485CC puncta, with a mean velocity of 5.79 ± 1.56 µm/min; n = 58. (I) Silver staining of SDS-PAGE gel (left) and Western blot (right) showing the purification of NOD485CC-mCherry from Drosophila S2 cells and mock purification from wild-type cell extracts. Arrows indicate the NOD bands. (J) Kymograph from a TIRF time-lapse of NOD485CC lysate (red) plus purified kinesin-1-EGFP (green) on taxol-stabilized MTs (blue). Horizontal bars: 10 µm (A); 1 µm (B, C, E, G, and J). Vertical bars: 10 s (A–C, E, and G); 20 s (J). Means ± SD.

    Journal: The Journal of Cell Biology

    Article Title: NOD is a plus end–directed motor that binds EB1 via a new microtubule tip localization sequence

    doi: 10.1083/jcb.201708109

    Figure Lengend Snippet: Dimerized NOD485 exhibits ATP-dependent, plus end–directed motility in cells and in vitro. (A) TIRF microscopy of an interphase cell expressing NOD485CC. (B) Select frames from a TIRF time-lapse zoomed on the region in the black box in A showing a motile NOD485CC dimer (marked by arrow). (C) Kymograph of NOD485CC dimer from the black box in A walking on MTs toward the cell periphery (right side of kymograph). (D) Histogram of velocities of motile NOD485CC dimers in cells, with a mean velocity of 8.70 ± 3.61 µm/min; n = 55. (E) TIRF microscopy of NOD dimers (green) on taxol-stabilized MTs (red) using a cell lysate from NOD485CC-mCherry–expressing cells in the presence of ATP (top) or AMP-PNP (bottom). (F) Histogram of the velocities of motile NOD485CC dimers in cell lysates, with a mean velocity of 8.62 ± 2.32 µm/min; n = 47. (G) TIRF microscopy of taxol-stabilized MTs (red) incubated with Strep-tagged NOD485CC-mCherry (green) purified from S2 cells in the presence of ATP (top) or AMP-PNP (bottom). (H) Histogram of the velocities of purified Strep-tagged NOD485CC puncta, with a mean velocity of 5.79 ± 1.56 µm/min; n = 58. (I) Silver staining of SDS-PAGE gel (left) and Western blot (right) showing the purification of NOD485CC-mCherry from Drosophila S2 cells and mock purification from wild-type cell extracts. Arrows indicate the NOD bands. (J) Kymograph from a TIRF time-lapse of NOD485CC lysate (red) plus purified kinesin-1-EGFP (green) on taxol-stabilized MTs (blue). Horizontal bars: 10 µm (A); 1 µm (B, C, E, G, and J). Vertical bars: 10 s (A–C, E, and G); 20 s (J). Means ± SD.

    Article Snippet: Twofold molar excess of MBP-NOD485CC-EGFP, and MBP-EGFP as control was added to GST-EB1-TagRFP-T in the presence of 5 mg/ml BSA and incubated at 25°C with agitation for 1 h. Protein mixtures were then added to amylose magnetic beads (NEB) and incubated for an additional hour at 25°C.

    Techniques: In Vitro, Microscopy, Expressing, Incubation, Purification, Silver Staining, SDS Page, Western Blot