atduo1  (New England Biolabs)


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    Structured Review

    New England Biolabs atduo1
    Characterization of potential DUO1 target genes in Marchantia. a RNA in situ hybridization of genes involved in sperm morphogenesis, the ortholog of At DAZ1 and At DAZ2 genes, and RWP-RK transcription factors during spermatogenesis (see Supplementary Figure 2 for sense-probe controls). Bars, 25 μm. Cell shape schematics (top) represent the developmental stages referred to in Fig. 1c . b Expression levels of genes involved in sperm morphogenesis, the ortholog of <t>AtDUO1</t> target genes, and RWP-RK transcription factors in antheridiophores of WT (gray bars) and Mp duo1-1 ko (white bars). The expression of each gene in WT is set to 1. Error bars indicate mean ± SD; n = 3 (* p
    Atduo1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 107 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Transcription factor DUO1 generated by neo-functionalization is associated with evolution of sperm differentiation in plants"

    Article Title: Transcription factor DUO1 generated by neo-functionalization is associated with evolution of sperm differentiation in plants

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07728-3

    Characterization of potential DUO1 target genes in Marchantia. a RNA in situ hybridization of genes involved in sperm morphogenesis, the ortholog of At DAZ1 and At DAZ2 genes, and RWP-RK transcription factors during spermatogenesis (see Supplementary Figure 2 for sense-probe controls). Bars, 25 μm. Cell shape schematics (top) represent the developmental stages referred to in Fig. 1c . b Expression levels of genes involved in sperm morphogenesis, the ortholog of AtDUO1 target genes, and RWP-RK transcription factors in antheridiophores of WT (gray bars) and Mp duo1-1 ko (white bars). The expression of each gene in WT is set to 1. Error bars indicate mean ± SD; n = 3 (* p
    Figure Legend Snippet: Characterization of potential DUO1 target genes in Marchantia. a RNA in situ hybridization of genes involved in sperm morphogenesis, the ortholog of At DAZ1 and At DAZ2 genes, and RWP-RK transcription factors during spermatogenesis (see Supplementary Figure 2 for sense-probe controls). Bars, 25 μm. Cell shape schematics (top) represent the developmental stages referred to in Fig. 1c . b Expression levels of genes involved in sperm morphogenesis, the ortholog of AtDUO1 target genes, and RWP-RK transcription factors in antheridiophores of WT (gray bars) and Mp duo1-1 ko (white bars). The expression of each gene in WT is set to 1. Error bars indicate mean ± SD; n = 3 (* p

    Techniques Used: RNA In Situ Hybridization, Expressing

    Characterization of DUO1 transcription factor activity. a Amino acid sequence alignment of regions A, B, and C among AtDUO1, MpDUO1, and MpR2R3-MYB21 (top). Dots indicate matching residues with AtDUO1. Asterisks indicate putative DNA-interacting residues in region C. Structural modeling of MpDUO1 in complex with DNA using SWISS-MODEL (bottom). The MpDUO1 MYB domain is overlaid onto the structure of the AMV v-MYB-DNA complex (PDB code: 1H8A). b in vivo transcriptional activation potentials of MpDUO1 and chimeras. Schematic diagram of constructs (left) are color-coded light blue (AtDUO1), dark blue (MpDUO1), and orange (MpR2R3-MYB21). DUO1 transcriptional activation potentials were measured by relative luciferase activity (right). n = 4 (upper), n = 8 (lower) (** p
    Figure Legend Snippet: Characterization of DUO1 transcription factor activity. a Amino acid sequence alignment of regions A, B, and C among AtDUO1, MpDUO1, and MpR2R3-MYB21 (top). Dots indicate matching residues with AtDUO1. Asterisks indicate putative DNA-interacting residues in region C. Structural modeling of MpDUO1 in complex with DNA using SWISS-MODEL (bottom). The MpDUO1 MYB domain is overlaid onto the structure of the AMV v-MYB-DNA complex (PDB code: 1H8A). b in vivo transcriptional activation potentials of MpDUO1 and chimeras. Schematic diagram of constructs (left) are color-coded light blue (AtDUO1), dark blue (MpDUO1), and orange (MpR2R3-MYB21). DUO1 transcriptional activation potentials were measured by relative luciferase activity (right). n = 4 (upper), n = 8 (lower) (** p

    Techniques Used: Activity Assay, Sequencing, In Vivo, Activation Assay, Construct, Luciferase

    2) Product Images from "Stimulation of NeuroD activity by huntingtin and huntingtin-associated proteins HAP1 and MLK2"

    Article Title: Stimulation of NeuroD activity by huntingtin and huntingtin-associated proteins HAP1 and MLK2

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1133382100

    MLK2 phosphorylates ND. ( A ) To examine the phosphorylation state of ND in vivo , immunoblotting experiments were performed by transfecting N2A cells with plasmids expressing MT-ND. The cell lysate was treated with alkaline phosphatase (AP) before immunoblotting (lane 2; lane 1 is untreated cell lysate as negative control). MT-ND was detected by immunoblotting with an antibody against the Myc epitope tag (MT, Upper ). To test the phosphorylation of ND by MLK2 in vivo , immunoblotting experiments were performed by transfecting N2A cells with plasmids expressing MT-ND with either wild-type (WT) (lane 3) or kinase-dead (KD) (lane 4) HA-MLK2. MT-ND was detected by immunoblotting with an antibody against the Myc epitope tag (MT, Upper ). To allow the resolution of the ND banding pattern, lane 3 was loaded as 20% of lane 4, in Upper only. The amount of HA-MLK2 in the cell lysate was analyzed by immunoblotting with an antibody against the HA epitope tag ( Lower ). ( B ) To test the phosphorylation of ND by MLK2 in vitro , immunocomplex kinase assay experiments were performed by transfecting N2A cells with empty vector (lane 1) or with the plasmid expressing MT-ND (lanes 2–4). MT-ND was immunoprecipitated with an antibody against the Myc epitope tag and then added to kinase reaction buffer supplemented with radiolabeled ATP and the following proteins: WT purified recombinant MLK2 (MBP-MLK2) (lane 1); MBP2 (lane 2); WT MBP-MLK2 (lane 3); KD MBP-MLK2 (lane 4). MT-ND was resolved by SDS/PAGE electrophoresis, and incorporation of radiolabeled ATP was then analyzed by PhosphorImager autoradiography ( 32 P, Upper ). The amount of MT-ND in the precipitate was analyzed by immunoblotting with an antibody against the Myc epitope tag (MT, Lower ). Results shown are representative of at least three independent experiments.
    Figure Legend Snippet: MLK2 phosphorylates ND. ( A ) To examine the phosphorylation state of ND in vivo , immunoblotting experiments were performed by transfecting N2A cells with plasmids expressing MT-ND. The cell lysate was treated with alkaline phosphatase (AP) before immunoblotting (lane 2; lane 1 is untreated cell lysate as negative control). MT-ND was detected by immunoblotting with an antibody against the Myc epitope tag (MT, Upper ). To test the phosphorylation of ND by MLK2 in vivo , immunoblotting experiments were performed by transfecting N2A cells with plasmids expressing MT-ND with either wild-type (WT) (lane 3) or kinase-dead (KD) (lane 4) HA-MLK2. MT-ND was detected by immunoblotting with an antibody against the Myc epitope tag (MT, Upper ). To allow the resolution of the ND banding pattern, lane 3 was loaded as 20% of lane 4, in Upper only. The amount of HA-MLK2 in the cell lysate was analyzed by immunoblotting with an antibody against the HA epitope tag ( Lower ). ( B ) To test the phosphorylation of ND by MLK2 in vitro , immunocomplex kinase assay experiments were performed by transfecting N2A cells with empty vector (lane 1) or with the plasmid expressing MT-ND (lanes 2–4). MT-ND was immunoprecipitated with an antibody against the Myc epitope tag and then added to kinase reaction buffer supplemented with radiolabeled ATP and the following proteins: WT purified recombinant MLK2 (MBP-MLK2) (lane 1); MBP2 (lane 2); WT MBP-MLK2 (lane 3); KD MBP-MLK2 (lane 4). MT-ND was resolved by SDS/PAGE electrophoresis, and incorporation of radiolabeled ATP was then analyzed by PhosphorImager autoradiography ( 32 P, Upper ). The amount of MT-ND in the precipitate was analyzed by immunoblotting with an antibody against the Myc epitope tag (MT, Lower ). Results shown are representative of at least three independent experiments.

    Techniques Used: In Vivo, Expressing, Negative Control, In Vitro, Kinase Assay, Plasmid Preparation, Immunoprecipitation, Purification, Recombinant, SDS Page, Electrophoresis, Autoradiography

    3) Product Images from "Molecular Cloning and Characterization of Taurocyamine Kinase from Clonorchis sinensis: A Candidate Chemotherapeutic Target"

    Article Title: Molecular Cloning and Characterization of Taurocyamine Kinase from Clonorchis sinensis: A Candidate Chemotherapeutic Target

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0002548

    Reactivity of anti-CsTKD1 mouse immune serum to C. sinensis CsTK D1D2 and recombinant CsTK D1 proteins by immunoblotting.
    Figure Legend Snippet: Reactivity of anti-CsTKD1 mouse immune serum to C. sinensis CsTK D1D2 and recombinant CsTK D1 proteins by immunoblotting.

    Techniques Used: Recombinant

    4) Product Images from "Immunoreactivity of the Mycobacterium avium subsp. paratuberculosis 19-kDa lipoprotein"

    Article Title: Immunoreactivity of the Mycobacterium avium subsp. paratuberculosis 19-kDa lipoprotein

    Journal: BMC Microbiology

    doi: 10.1186/1471-2180-5-3

    Amino acid sequence comparison of the mycobacterial 19 kDa protein . Non-conserved residues are shaded in black and gaps in amino acid sequence are indicated by hyphens (-). The predicted signal peptidase cleavage site for M. tuberculosis is indicated by a single arrow (↓), while the site for M. avium subsp. paratuberculosis is indicated by a double arrow (⇓). The GenBank accession number and abbreviations are M. avium subsp. paratuberculosis (M. paratb; AAS02578 ), M. avium subsp. avium (M. avium; AAB25888 ), M. intracellulare (M. intracel; AAB25885), M. bovis (M. bovis; S11234) and M. tuberculosis (M. tuberc; NP_218280).
    Figure Legend Snippet: Amino acid sequence comparison of the mycobacterial 19 kDa protein . Non-conserved residues are shaded in black and gaps in amino acid sequence are indicated by hyphens (-). The predicted signal peptidase cleavage site for M. tuberculosis is indicated by a single arrow (↓), while the site for M. avium subsp. paratuberculosis is indicated by a double arrow (⇓). The GenBank accession number and abbreviations are M. avium subsp. paratuberculosis (M. paratb; AAS02578 ), M. avium subsp. avium (M. avium; AAB25888 ), M. intracellulare (M. intracel; AAB25885), M. bovis (M. bovis; S11234) and M. tuberculosis (M. tuberc; NP_218280).

    Techniques Used: Sequencing

    5) Product Images from "DER containing two consecutive GTP-binding domains plays an essential role in chloroplast ribosomal RNA processing and ribosome biogenesis in higher plants"

    Article Title: DER containing two consecutive GTP-binding domains plays an essential role in chloroplast ribosomal RNA processing and ribosome biogenesis in higher plants

    Journal: Journal of Experimental Botany

    doi: 10.1093/jxb/ert360

    Binding of recombinant NbDER and its variants to 23S and 16S rRNA. (A, B) MBP:NbDER, MBP:∆CTD, and MBP:CTD fusion proteins (75 pmol) were incubated with 200ng of 23S (A) or 16S rRNA (B) in the absence or presence of GTP (100 μM); bound (asterisks) and unbound (U) RNAs were resolved on an agarose gel and visualized by ethidium bromide staining; filled circles indicate the wells of the agarose gel. (C) Increasing concentrations (25, 50, and 100 pmol) of MBP:NbDER fusion proteins were incubated with radiolabelled 23S and 16S rRNAs; bound (B) and unbound (U) RNAs were resolved on an agarose gel and radioactive RNA bands were visualized by PhosphorImager. (D) Gel-mobility shift assays were performed with or without cold competitors; 25 pmol MBP:NbDER fusion protein were incubated with different ratios of radiolabelled and unlabelled 23S and 16S rRNAs; the ratios of radiolabelled RNA:unlabelled RNA are as follows: 1:5 (lane 3), 1:20 (lane 4), 1:5 (lane 7), and 1:20 (lane 8).
    Figure Legend Snippet: Binding of recombinant NbDER and its variants to 23S and 16S rRNA. (A, B) MBP:NbDER, MBP:∆CTD, and MBP:CTD fusion proteins (75 pmol) were incubated with 200ng of 23S (A) or 16S rRNA (B) in the absence or presence of GTP (100 μM); bound (asterisks) and unbound (U) RNAs were resolved on an agarose gel and visualized by ethidium bromide staining; filled circles indicate the wells of the agarose gel. (C) Increasing concentrations (25, 50, and 100 pmol) of MBP:NbDER fusion proteins were incubated with radiolabelled 23S and 16S rRNAs; bound (B) and unbound (U) RNAs were resolved on an agarose gel and radioactive RNA bands were visualized by PhosphorImager. (D) Gel-mobility shift assays were performed with or without cold competitors; 25 pmol MBP:NbDER fusion protein were incubated with different ratios of radiolabelled and unlabelled 23S and 16S rRNAs; the ratios of radiolabelled RNA:unlabelled RNA are as follows: 1:5 (lane 3), 1:20 (lane 4), 1:5 (lane 7), and 1:20 (lane 8).

    Techniques Used: Binding Assay, Recombinant, Incubation, Agarose Gel Electrophoresis, Staining, Mobility Shift

    6) Product Images from "Chibby forms a homodimer through a heptad repeat of leucine residues in its C-terminal coiled-coil motif"

    Article Title: Chibby forms a homodimer through a heptad repeat of leucine residues in its C-terminal coiled-coil motif

    Journal: BMC Molecular Biology

    doi: 10.1186/1471-2199-10-41

    Cby homodimerization is dispensable for its interaction with β-catenin and for repression of β-catenin signaling activity . (A) Binding of Cby point mutants to β-catenin was evaluated by in vitro pull-down assays. Bacterially produced MBP or individual MBP-Cby protein was incubated with His-tagged β-catenin C-terminal domain (His-βcatR10-C). The protein complexes were then pulled down with amylose resin and subjected to Western blotting using anti-β-catenin antibody (top panel). The input lane was loaded with one-fiftieth of the amount of His-βcatR10-C used in the binding reactions (lane 1). One-thirtieth of each pull-down sample was run on a separate SDS-PAGE and immunoblotted with anti-MBP antibody, showing that similar amounts of MBP-Cby protein were pulled down (bottom panel). (B) The ability of Cby mutants to repress β-catenin signaling was tested by TOPFLASH assays. HEK293T cells were transfected with 60 ng of TOPFLASH or mutant FOPFLASH luciferase reporter, with or without 40 ng of an expression vector for stabilized β-catenin (β-catenin-Myc), and the indicated amounts of a Flag-tagged Cby expression vector. Luciferase activity was measured 24 hr post-transfection, and normalized to Renila luciferase activity used as an internal control. Transfections were carried out in triplicate and the means ± SD are shown. Note that, to compensate protein levels, higher amounts of plasmid DNA for the Cby mutants were used for transfection.
    Figure Legend Snippet: Cby homodimerization is dispensable for its interaction with β-catenin and for repression of β-catenin signaling activity . (A) Binding of Cby point mutants to β-catenin was evaluated by in vitro pull-down assays. Bacterially produced MBP or individual MBP-Cby protein was incubated with His-tagged β-catenin C-terminal domain (His-βcatR10-C). The protein complexes were then pulled down with amylose resin and subjected to Western blotting using anti-β-catenin antibody (top panel). The input lane was loaded with one-fiftieth of the amount of His-βcatR10-C used in the binding reactions (lane 1). One-thirtieth of each pull-down sample was run on a separate SDS-PAGE and immunoblotted with anti-MBP antibody, showing that similar amounts of MBP-Cby protein were pulled down (bottom panel). (B) The ability of Cby mutants to repress β-catenin signaling was tested by TOPFLASH assays. HEK293T cells were transfected with 60 ng of TOPFLASH or mutant FOPFLASH luciferase reporter, with or without 40 ng of an expression vector for stabilized β-catenin (β-catenin-Myc), and the indicated amounts of a Flag-tagged Cby expression vector. Luciferase activity was measured 24 hr post-transfection, and normalized to Renila luciferase activity used as an internal control. Transfections were carried out in triplicate and the means ± SD are shown. Note that, to compensate protein levels, higher amounts of plasmid DNA for the Cby mutants were used for transfection.

    Techniques Used: Activity Assay, Binding Assay, In Vitro, Produced, Incubation, Western Blot, SDS Page, Transfection, Mutagenesis, Luciferase, Expressing, Plasmid Preparation

    7) Product Images from "Regulation of APC/CCdc20 activity by RASSF1A-APC/CCdc20 circuitry"

    Article Title: Regulation of APC/CCdc20 activity by RASSF1A-APC/CCdc20 circuitry

    Journal: Oncogene

    doi: 10.1038/onc.2011.372

    Inhibition of APC/C Cdc20 by RASSF1A depends on the D-box clusters. ( a ) RASSF1A inhibits APC/C Cdc20 in vitro . Recombinant MBP-RASSF1A or MBP was added to the in vitro ubiquitination assay in the presence of Cdc20(WT) or Cdc20(7A). The 35 S-labeled N-terminal fragment of Cyclin B1 was used as the substrate (left panel). The amount of poly-ubiquitinated Cyclin B1 was quantified and the APC/C activities in each reaction relative to the control reaction containing Cdc20(7A) and MBP were calculated (right panel). ( b ) Deletion of the D-box clusters abolishes the inhibitory effect of RASSF1A in vitro . In vitro APC/C inhibitory assay was performed with recombinant RASSF1A DBD mutants (left panel) and the activity of APC/C in the reaction was determined (right panel). The loadings of different RASSF1A mutants were determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and Coomassie Brilliant Blue (CBB) stain.
    Figure Legend Snippet: Inhibition of APC/C Cdc20 by RASSF1A depends on the D-box clusters. ( a ) RASSF1A inhibits APC/C Cdc20 in vitro . Recombinant MBP-RASSF1A or MBP was added to the in vitro ubiquitination assay in the presence of Cdc20(WT) or Cdc20(7A). The 35 S-labeled N-terminal fragment of Cyclin B1 was used as the substrate (left panel). The amount of poly-ubiquitinated Cyclin B1 was quantified and the APC/C activities in each reaction relative to the control reaction containing Cdc20(7A) and MBP were calculated (right panel). ( b ) Deletion of the D-box clusters abolishes the inhibitory effect of RASSF1A in vitro . In vitro APC/C inhibitory assay was performed with recombinant RASSF1A DBD mutants (left panel) and the activity of APC/C in the reaction was determined (right panel). The loadings of different RASSF1A mutants were determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and Coomassie Brilliant Blue (CBB) stain.

    Techniques Used: Inhibition, In Vitro, Recombinant, Ubiquitin Assay, Labeling, Activity Assay, Polyacrylamide Gel Electrophoresis, SDS Page, Staining

    8) Product Images from "A Septin-based Hierarchy of Proteins Required for Localized Deposition of Chitin in the Saccharomyces cerevisiae Cell Wall "

    Article Title: A Septin-based Hierarchy of Proteins Required for Localized Deposition of Chitin in the Saccharomyces cerevisiae Cell Wall

    Journal: The Journal of Cell Biology

    doi:

    Immunoblot analysis of Bni4p-specific antibodies. Extracts of bni4-Δ1 / bni4-Δ1 strain DDY175 (lane 1 ); wild-type strain YEF473 (lane 2 ); and strain YEF473 containing the high-copy BNI4 plasmid p356 (lane 3 ) were analyzed using affinity-purified antibodies to Bni4p (see Materials and Methods). The mobilities of molecular mass markers are indicated.
    Figure Legend Snippet: Immunoblot analysis of Bni4p-specific antibodies. Extracts of bni4-Δ1 / bni4-Δ1 strain DDY175 (lane 1 ); wild-type strain YEF473 (lane 2 ); and strain YEF473 containing the high-copy BNI4 plasmid p356 (lane 3 ) were analyzed using affinity-purified antibodies to Bni4p (see Materials and Methods). The mobilities of molecular mass markers are indicated.

    Techniques Used: Plasmid Preparation, Affinity Purification

    Amino acid sequences of Chs4p and Bni4p. Asterisks represent the termination codons. ( A ) Portions of the predicted amino acid sequence of Chs4p that include the possible calcium-binding domain (amino acids 237–250; underlined ) and the COOH terminus including the CAAX motif (amino acids 693– 696; underlined ). ( B ) Predicted sequence of Bni4p. The predicted coiled-coil domain (amino acids 106–139) is underlined, and the first residue translated from the BNI4 -containing AD fusion clone is overlined.
    Figure Legend Snippet: Amino acid sequences of Chs4p and Bni4p. Asterisks represent the termination codons. ( A ) Portions of the predicted amino acid sequence of Chs4p that include the possible calcium-binding domain (amino acids 237–250; underlined ) and the COOH terminus including the CAAX motif (amino acids 693– 696; underlined ). ( B ) Predicted sequence of Bni4p. The predicted coiled-coil domain (amino acids 106–139) is underlined, and the first residue translated from the BNI4 -containing AD fusion clone is overlined.

    Techniques Used: Sequencing, Binding Assay

    Localization of Bni4p. Cells were grown in rich (YM-P) medium except for those shown in B , which were grown in SC medium. ( A–G ) Exponentially growing cells of strains ( A and B ) YEF473 (wild-type); ( C ) DDY175 ( bni4-Δ1 / bni4-Δ1 ); ( D ) DDY175 containing plasmid p356 (high-copy BNI4 ); ( E ) DDY174 ( chs4-Δ1 / chs4-Δ1 ); ( F ) DDY186 ( chs3-Δ1 / chs3-Δ1 ); and ( G ) DDY185-1A ( cdc10-Δ1 ) were examined by immunofluorescence microscopy using Bni4p-specific antibodies. ( H–K ) Cells of strain JPTA1493-H01 ( cdc12-6 / cdc12-6 ) growing exponentially at 23°C ( H and J ) or collected 5 min after a shift to 37°C ( I and K ) were examined by immunofluorescence microscopy using Bni4p-specific antibodies ( H and I ) or Cdc11p-specific antibodies ( J and K ). Arrows and arrowheads indicate structures discussed in the text.
    Figure Legend Snippet: Localization of Bni4p. Cells were grown in rich (YM-P) medium except for those shown in B , which were grown in SC medium. ( A–G ) Exponentially growing cells of strains ( A and B ) YEF473 (wild-type); ( C ) DDY175 ( bni4-Δ1 / bni4-Δ1 ); ( D ) DDY175 containing plasmid p356 (high-copy BNI4 ); ( E ) DDY174 ( chs4-Δ1 / chs4-Δ1 ); ( F ) DDY186 ( chs3-Δ1 / chs3-Δ1 ); and ( G ) DDY185-1A ( cdc10-Δ1 ) were examined by immunofluorescence microscopy using Bni4p-specific antibodies. ( H–K ) Cells of strain JPTA1493-H01 ( cdc12-6 / cdc12-6 ) growing exponentially at 23°C ( H and J ) or collected 5 min after a shift to 37°C ( I and K ) were examined by immunofluorescence microscopy using Bni4p-specific antibodies ( H and I ) or Cdc11p-specific antibodies ( J and K ). Arrows and arrowheads indicate structures discussed in the text.

    Techniques Used: Plasmid Preparation, Immunofluorescence, Microscopy

    Model for the interactions among the chitin synthase III complex, Bni4p, and the septins. ( A ) The transmembrane protein Chs3p synthesizes chitin, which is deposited in the cell wall ( solid arrows ). Chs4p, anchored to the cytoplasmic face of the plasma membrane by its (presumably) prenylated COOH terminus, interacts both with Chs3p and with Bni4p, which is tethered to the septin complex by its interaction with Cdc10p. The box with the question mark indicates a postulated protein that is sufficient to anchor the septin complex to the cell membrane in the absence of Bni4p, Chs4p, and/or Chs3p. ( B ) In the absence of Bni4p or the septin complex, the chitin synthase III complex diffuses through the lipid bilayer ( dashed arrows ), resulting in delocalized chitin delocalization.
    Figure Legend Snippet: Model for the interactions among the chitin synthase III complex, Bni4p, and the septins. ( A ) The transmembrane protein Chs3p synthesizes chitin, which is deposited in the cell wall ( solid arrows ). Chs4p, anchored to the cytoplasmic face of the plasma membrane by its (presumably) prenylated COOH terminus, interacts both with Chs3p and with Bni4p, which is tethered to the septin complex by its interaction with Cdc10p. The box with the question mark indicates a postulated protein that is sufficient to anchor the septin complex to the cell membrane in the absence of Bni4p, Chs4p, and/or Chs3p. ( B ) In the absence of Bni4p or the septin complex, the chitin synthase III complex diffuses through the lipid bilayer ( dashed arrows ), resulting in delocalized chitin delocalization.

    Techniques Used:

    9) Product Images from "A Class VI Unconventional Myosin Is Associated with a Homologue of a Microtubule-binding Protein, Cytoplasmic Linker Protein-170, in Neurons and at the Posterior Pole of Drosophila Embryos "

    Article Title: A Class VI Unconventional Myosin Is Associated with a Homologue of a Microtubule-binding Protein, Cytoplasmic Linker Protein-170, in Neurons and at the Posterior Pole of Drosophila Embryos

    Journal: The Journal of Cell Biology

    doi:

    Sequence analysis of the 195-kD protein. ( A ) cDNAs encoding the 195-kD protein that were sequenced are depicted in schematic form. The ov 6D1a cDNA was isolated from an ovary expression library, and the partial cDNAs were isolated from an 8–12-h cDNA library. The Em 10C cDNA appears to extend to the 5′ end of the transcript whereas the Em 10A cDNA extends to the poly (A) tail. The sequence data are available from GenBank/ EMBL/DDBJ under the accession number AF041382 . ( B ) An alignment of the entire amino acid sequence of D-CLIP-190 ( uppercase ) and CLIP-170 ( lowercase ) is shown. Identities ( vertical lines ), conservative substitutions ( two dots ), and less conservative substitutions ( one dot ) are determined by the Genetic Computer Group program Bestfit ( Devereux et al., 1984 ). The first 48 amino acids of D-CLIP-190 do not align with CLIP-170. The amino terminus contains two repeats, rep1 and rep2, ( black lines ) that have been demonstrated to mediate binding of CLIP-170 to microtubules. The central region of both proteins is predicted to be an extended region of coiled coil ( arrows indicate start and end points; Lupas et al., 1991 ). The predicted coiled-coil domain of D-CLIP-190 is interrupted after ∼100 amino acids by four prolines ( asterisks ), which are thought to disrupt α helices. The carboxy-terminal domain has two conserved sequences: a metal-binding motif ( shaded ) and a cysteine-rich sequence of 23 amino acids that does not conform to any previously recognized motif ( dotted line ).
    Figure Legend Snippet: Sequence analysis of the 195-kD protein. ( A ) cDNAs encoding the 195-kD protein that were sequenced are depicted in schematic form. The ov 6D1a cDNA was isolated from an ovary expression library, and the partial cDNAs were isolated from an 8–12-h cDNA library. The Em 10C cDNA appears to extend to the 5′ end of the transcript whereas the Em 10A cDNA extends to the poly (A) tail. The sequence data are available from GenBank/ EMBL/DDBJ under the accession number AF041382 . ( B ) An alignment of the entire amino acid sequence of D-CLIP-190 ( uppercase ) and CLIP-170 ( lowercase ) is shown. Identities ( vertical lines ), conservative substitutions ( two dots ), and less conservative substitutions ( one dot ) are determined by the Genetic Computer Group program Bestfit ( Devereux et al., 1984 ). The first 48 amino acids of D-CLIP-190 do not align with CLIP-170. The amino terminus contains two repeats, rep1 and rep2, ( black lines ) that have been demonstrated to mediate binding of CLIP-170 to microtubules. The central region of both proteins is predicted to be an extended region of coiled coil ( arrows indicate start and end points; Lupas et al., 1991 ). The predicted coiled-coil domain of D-CLIP-190 is interrupted after ∼100 amino acids by four prolines ( asterisks ), which are thought to disrupt α helices. The carboxy-terminal domain has two conserved sequences: a metal-binding motif ( shaded ) and a cysteine-rich sequence of 23 amino acids that does not conform to any previously recognized motif ( dotted line ).

    Techniques Used: Sequencing, Isolation, Expressing, cDNA Library Assay, Cross-linking Immunoprecipitation, Binding Assay

    10) Product Images from "A Class VI Unconventional Myosin Is Associated with a Homologue of a Microtubule-binding Protein, Cytoplasmic Linker Protein-170, in Neurons and at the Posterior Pole of Drosophila Embryos "

    Article Title: A Class VI Unconventional Myosin Is Associated with a Homologue of a Microtubule-binding Protein, Cytoplasmic Linker Protein-170, in Neurons and at the Posterior Pole of Drosophila Embryos

    Journal: The Journal of Cell Biology

    doi:

    The 6D1a clone isolated using the α195-kD protein antibody corresponds to the gene that encodes the 195-kD protein. Duplicate immunoblots of identical samples are shown in each panel. On each blot are IPs from 0–3-h embryos using α95F myosin antibody ( IP-α95F ), 0–3-h embryo extract ( 0–3 hr E E ) and bacterial extracts containing the 6D1a-maltose–binding protein fusion protein ( 6D1a– MBP ). ( A ) Antiserum from a mouse immunized with the 195-kD protein gel purified after immunoprecipitation ( α195 kD ). ( B ) Antiserum from a mouse immunized with the 6D1a–GST fusion protein ( α6D1a–GST ). ( C ) Preimmune sera from mice immunized with the 6D1a–GST fusion protein PI ( α6D1a-GST ). ( D ) Anti–95F myosin monoclonal antibody ( α95F ). The proteins are labeled with arrowheads and numbers as in Fig. 1 .
    Figure Legend Snippet: The 6D1a clone isolated using the α195-kD protein antibody corresponds to the gene that encodes the 195-kD protein. Duplicate immunoblots of identical samples are shown in each panel. On each blot are IPs from 0–3-h embryos using α95F myosin antibody ( IP-α95F ), 0–3-h embryo extract ( 0–3 hr E E ) and bacterial extracts containing the 6D1a-maltose–binding protein fusion protein ( 6D1a– MBP ). ( A ) Antiserum from a mouse immunized with the 195-kD protein gel purified after immunoprecipitation ( α195 kD ). ( B ) Antiserum from a mouse immunized with the 6D1a–GST fusion protein ( α6D1a–GST ). ( C ) Preimmune sera from mice immunized with the 6D1a–GST fusion protein PI ( α6D1a-GST ). ( D ) Anti–95F myosin monoclonal antibody ( α95F ). The proteins are labeled with arrowheads and numbers as in Fig. 1 .

    Techniques Used: Isolation, Western Blot, Binding Assay, Purification, Immunoprecipitation, Mouse Assay, Labeling

    11) Product Images from "Control of cotton fibre elongation by a homeodomain transcription factor GhHOX3"

    Article Title: Control of cotton fibre elongation by a homeodomain transcription factor GhHOX3

    Journal: Nature Communications

    doi: 10.1038/ncomms6519

    GhRDL1 and GhEXPA1 are direct targets of GhHOX3. ( a – d ) Quantitative reverse transcription-PCR analysis of GhRDL1 and GhEXPA1 transcripts in cotton fibre of the WT, co-suppression (5–8) and overexpression (ox1-1) plants. ( e – h ) GhHOX3 directly binds to GhRDL1 and GhEXPA1 promoters. Data are shown as mean±s.e.m. ( n =3). ( e – g ) EMSA of GhHOX3 binding to L1-box from the GhRDL1 and GhEXPA1 promoters. The 6 × fragments of GhRDL1 and GhEXPA1 promoters containing the intact (upper) or the mutated (lower) L1-box ( e ) were incubated with gradient concentrations of maltose-binding protein (MBP)-GhHOX3 fusion protein ( f ). Labelled GhRDL1 and GhEXPA1 were incubated with MBP-GhHOX3 to compete with different concentrations of cold probes of intact or mutated L1-box ( g ). ( h ) Yeast one-hybrid assay of protein–DNA interaction, the 6 × fragments described in e were used. ( i – l ) Expression levels of GhHOX3 and two downstream genes in ovule (O) and/or fibre (F), which were taken from GhHOX3 -silenced line 5–8 and the WT cotton at 2 DPA and cultured in vitro with addition of the hormone GA 3 (1 μM) for 6 days. GhEXPA2 , expressed at a nearly equal level in ovule and fibre, was analysed as a control of GA treatments. Data are shown as mean±s.e.m. ( n =3).
    Figure Legend Snippet: GhRDL1 and GhEXPA1 are direct targets of GhHOX3. ( a – d ) Quantitative reverse transcription-PCR analysis of GhRDL1 and GhEXPA1 transcripts in cotton fibre of the WT, co-suppression (5–8) and overexpression (ox1-1) plants. ( e – h ) GhHOX3 directly binds to GhRDL1 and GhEXPA1 promoters. Data are shown as mean±s.e.m. ( n =3). ( e – g ) EMSA of GhHOX3 binding to L1-box from the GhRDL1 and GhEXPA1 promoters. The 6 × fragments of GhRDL1 and GhEXPA1 promoters containing the intact (upper) or the mutated (lower) L1-box ( e ) were incubated with gradient concentrations of maltose-binding protein (MBP)-GhHOX3 fusion protein ( f ). Labelled GhRDL1 and GhEXPA1 were incubated with MBP-GhHOX3 to compete with different concentrations of cold probes of intact or mutated L1-box ( g ). ( h ) Yeast one-hybrid assay of protein–DNA interaction, the 6 × fragments described in e were used. ( i – l ) Expression levels of GhHOX3 and two downstream genes in ovule (O) and/or fibre (F), which were taken from GhHOX3 -silenced line 5–8 and the WT cotton at 2 DPA and cultured in vitro with addition of the hormone GA 3 (1 μM) for 6 days. GhEXPA2 , expressed at a nearly equal level in ovule and fibre, was analysed as a control of GA treatments. Data are shown as mean±s.e.m. ( n =3).

    Techniques Used: Polymerase Chain Reaction, Over Expression, Binding Assay, Incubation, Y1H Assay, Expressing, Cell Culture, In Vitro

    12) Product Images from "Plk2 attachment to NSF induces homeostatic removal of GluA2 during chronic overexcitation"

    Article Title: Plk2 attachment to NSF induces homeostatic removal of GluA2 during chronic overexcitation

    Journal: Nature neuroscience

    doi: 10.1038/nn.2624

    Plk2 binding NSF is sufficient for decreased surface GluA2 (a) Lysates of COS-7 cells expressing NSF and GFP or GFP-PBind were subjected to immunoprecipitation (IP) with GFP antibodies and immunoblotted (IB) as indicated. Input, 5% of lysate for IP. Full-length blots are presented in Supplementary Fig. 9 . (b) Pulldown of brain lysates with MBP or MBP-PBind and analyzed by IB for NSF and by Coomassie stain for MBP fusion proteins. Note MBP contains a 73aa polylinker C-terminal tail and therefore runs larger than MBP-PBind. (c) Cultured hippocampal neurons expressing GFP or GFP-PBind as indicated were immunostained for sGluA2 (bottom, violet) and GFP or Plk2 (middle, green). Arrows indicate transfected neurons. Colocalization appears white in merged images (top). Magnified views of representative dendrites at bottom. Scale bars, 10 μm (wide view), 5 μm (magnified). Surface GluA2 immunofluorescence intensities (in arbitrary units) were 82.8±7.3 for GFP, 18.6±3.3 for GFP-PBind; p=4.7×10 -10 ; N=15–20 neurons. (d) Pep-PBind or scrambled control peptide (pep-scr) were analyzed for direct binding to His 6 -NSF by surface plasmon resonance. Averages of peak values were 89.4±7.6 response units for pep-PBind; -1.3±1.1 for pep-scr (negative value due to slight rundown during the experiment); p=0.0003, N=3). (e) Normalized AMPAR EPSCs peak amplitude vs time (pep-PBind, N=9, point 1 vs. point 2: p=0.0001; pep-scr, N=6, point 1 vs. point 2: p=0.17). (f) Representative EPSCs from individual neurons recorded at indicated times in the peak amplitude vs. time plot in the presence of intracellular pep-PBind (top) or pep-scr (bottom).
    Figure Legend Snippet: Plk2 binding NSF is sufficient for decreased surface GluA2 (a) Lysates of COS-7 cells expressing NSF and GFP or GFP-PBind were subjected to immunoprecipitation (IP) with GFP antibodies and immunoblotted (IB) as indicated. Input, 5% of lysate for IP. Full-length blots are presented in Supplementary Fig. 9 . (b) Pulldown of brain lysates with MBP or MBP-PBind and analyzed by IB for NSF and by Coomassie stain for MBP fusion proteins. Note MBP contains a 73aa polylinker C-terminal tail and therefore runs larger than MBP-PBind. (c) Cultured hippocampal neurons expressing GFP or GFP-PBind as indicated were immunostained for sGluA2 (bottom, violet) and GFP or Plk2 (middle, green). Arrows indicate transfected neurons. Colocalization appears white in merged images (top). Magnified views of representative dendrites at bottom. Scale bars, 10 μm (wide view), 5 μm (magnified). Surface GluA2 immunofluorescence intensities (in arbitrary units) were 82.8±7.3 for GFP, 18.6±3.3 for GFP-PBind; p=4.7×10 -10 ; N=15–20 neurons. (d) Pep-PBind or scrambled control peptide (pep-scr) were analyzed for direct binding to His 6 -NSF by surface plasmon resonance. Averages of peak values were 89.4±7.6 response units for pep-PBind; -1.3±1.1 for pep-scr (negative value due to slight rundown during the experiment); p=0.0003, N=3). (e) Normalized AMPAR EPSCs peak amplitude vs time (pep-PBind, N=9, point 1 vs. point 2: p=0.0001; pep-scr, N=6, point 1 vs. point 2: p=0.17). (f) Representative EPSCs from individual neurons recorded at indicated times in the peak amplitude vs. time plot in the presence of intracellular pep-PBind (top) or pep-scr (bottom).

    Techniques Used: Binding Assay, Expressing, Immunoprecipitation, Staining, Cell Culture, Transfection, Immunofluorescence, SPR Assay

    13) Product Images from "Decreased expression of BRCA1 in SK-BR-3 cells is the result of aberrant activation of the GABP Beta promoter by an NRF-1-containing complex"

    Article Title: Decreased expression of BRCA1 in SK-BR-3 cells is the result of aberrant activation of the GABP Beta promoter by an NRF-1-containing complex

    Journal: Molecular Cancer

    doi: 10.1186/1476-4598-10-62

    NRF-1 loss attenuates GABPβ promoter activity and GABPβ/BRCA1 expression; NRF-1 is consistent between cell lines . ( a ) The transcriptional activity of the GABPβ promoter constructs -268, which contains the NRF-1 binding site, and -251 which does not, was assessed in MCF-7 cells via dual luciferase assay in the presence of siRNA against GAPDH (siGAPDH, negative control) and NRF-1 (siNRF-1). Promoter activity is expressed as relative light units. ( b ) The protein levels of NRF-1, GABPβ, GABPα, BRCA1 and TBP (internal control) were assessed by Western blot in whole cell lysates prepared from MCF-7 cells treated with siGAPDH or siNRF-1. ( c ) NRF-1 levels were determined by Western blot in MCF-7 and SK-BR-3 whole cell lysates. TBP was used as an internal control. Apparent molecular weight markers (kDa) are indicated to the right of the panels. ( d ) The activity of two GABPβ promoter constructs, Gb-270 multimer (which contains a triple repeat of the Gb-270 sequence specified in Figure 6) and -268 (see part a ), was examined via dual luciferase assay in MCF-7 and SK-BR-3 cells following exogenous NRF-1 expression. Promoter activation by NRF-1 is expressed as a fold relative to empty vector controls in each cell line. ( e ) A ChIP assay was performed using MCF-7 and SK-BR-3 chromatin and antibodies against acetylated histone H3K9 (acH3), haemagglutinin (HA, negative control), RNA polymerase II (RNA pol II), histone deacetylase I (HDAC), NRF-1 and Oct-4 (transcription factor, negative control). PCR products obtained using primers specific to the GABPβ promoter (refer to Methods) are shown.
    Figure Legend Snippet: NRF-1 loss attenuates GABPβ promoter activity and GABPβ/BRCA1 expression; NRF-1 is consistent between cell lines . ( a ) The transcriptional activity of the GABPβ promoter constructs -268, which contains the NRF-1 binding site, and -251 which does not, was assessed in MCF-7 cells via dual luciferase assay in the presence of siRNA against GAPDH (siGAPDH, negative control) and NRF-1 (siNRF-1). Promoter activity is expressed as relative light units. ( b ) The protein levels of NRF-1, GABPβ, GABPα, BRCA1 and TBP (internal control) were assessed by Western blot in whole cell lysates prepared from MCF-7 cells treated with siGAPDH or siNRF-1. ( c ) NRF-1 levels were determined by Western blot in MCF-7 and SK-BR-3 whole cell lysates. TBP was used as an internal control. Apparent molecular weight markers (kDa) are indicated to the right of the panels. ( d ) The activity of two GABPβ promoter constructs, Gb-270 multimer (which contains a triple repeat of the Gb-270 sequence specified in Figure 6) and -268 (see part a ), was examined via dual luciferase assay in MCF-7 and SK-BR-3 cells following exogenous NRF-1 expression. Promoter activation by NRF-1 is expressed as a fold relative to empty vector controls in each cell line. ( e ) A ChIP assay was performed using MCF-7 and SK-BR-3 chromatin and antibodies against acetylated histone H3K9 (acH3), haemagglutinin (HA, negative control), RNA polymerase II (RNA pol II), histone deacetylase I (HDAC), NRF-1 and Oct-4 (transcription factor, negative control). PCR products obtained using primers specific to the GABPβ promoter (refer to Methods) are shown.

    Techniques Used: Activity Assay, Expressing, Construct, Binding Assay, Luciferase, Negative Control, Western Blot, Molecular Weight, Sequencing, Activation Assay, Plasmid Preparation, Chromatin Immunoprecipitation, Histone Deacetylase Assay, Polymerase Chain Reaction

    NRF-1 binds to the GABPβ promoter . ( a ) An oligonucleotide with a known NRF-1 binding site (RC4) [ 22 ] was used as a probe in an EMSA with MCF-7 nuclear extracts and Gb-270 as a cold competitor. NRF-1 binding (NRF-1), non-specific (NS) binding and free probe (F) are indicated. ( b ) Recombinant NRF-1 (rNRF-1) was prepared as a fusion with the maltose binding protein. Decreasing amounts of recombinant protein (5, 2.6, 1.3, 0.26, 0.13 and 0.03 μg) were used in an EMSA with RC4 and Gb-270 probes. Binding complexes as indicated above. ( c ) A ChIP assay was performed using MCF-7 chromatin and antibodies against acetylated histone H3K9 (acH3), haemagglutinin (HA, negative control), NRF-1 and Oct-4 (transcription factor, negative control). PCR products obtained using primers specific to the GABPβ promoter (refer to Methods) are shown.
    Figure Legend Snippet: NRF-1 binds to the GABPβ promoter . ( a ) An oligonucleotide with a known NRF-1 binding site (RC4) [ 22 ] was used as a probe in an EMSA with MCF-7 nuclear extracts and Gb-270 as a cold competitor. NRF-1 binding (NRF-1), non-specific (NS) binding and free probe (F) are indicated. ( b ) Recombinant NRF-1 (rNRF-1) was prepared as a fusion with the maltose binding protein. Decreasing amounts of recombinant protein (5, 2.6, 1.3, 0.26, 0.13 and 0.03 μg) were used in an EMSA with RC4 and Gb-270 probes. Binding complexes as indicated above. ( c ) A ChIP assay was performed using MCF-7 chromatin and antibodies against acetylated histone H3K9 (acH3), haemagglutinin (HA, negative control), NRF-1 and Oct-4 (transcription factor, negative control). PCR products obtained using primers specific to the GABPβ promoter (refer to Methods) are shown.

    Techniques Used: Binding Assay, Recombinant, Chromatin Immunoprecipitation, Negative Control, Polymerase Chain Reaction

    GABPα and β subunit protein and mRNA levels are decreased in the SK-BR-3 cell line . ( a ) Western blot analysis of whole cell lysates from MCF-7, T-47D and SK-BR-3 cells was carried out using antibodies to GABPα, GABPβ and the blots were then reprobed with anti-γ-tubulin as an internal control. Apparent molecular weight markers (kDa) are indicated to the right of the panels. ( b ) The relative transcript levels of the GABP subunits in MCF-7 (M), T-47D (T) and SK-BR-3 (S) cells were examined by semi-quantitative RT-PCR. Specific products were amplified from equal amounts of RT product from the cell lines indicated using primer sets for GABPα, GABPβ and GAPDH as an internal control. Products were separated on a 1.5% agarose gel with 100 bp ladder in leftmost lane. ( c ) Quantitative RT-PCR analysis of GABP beta-41 subunit mRNA was carried out on the indicated cell lines. Levels are expressed in relation to the 184hTERT cell line.
    Figure Legend Snippet: GABPα and β subunit protein and mRNA levels are decreased in the SK-BR-3 cell line . ( a ) Western blot analysis of whole cell lysates from MCF-7, T-47D and SK-BR-3 cells was carried out using antibodies to GABPα, GABPβ and the blots were then reprobed with anti-γ-tubulin as an internal control. Apparent molecular weight markers (kDa) are indicated to the right of the panels. ( b ) The relative transcript levels of the GABP subunits in MCF-7 (M), T-47D (T) and SK-BR-3 (S) cells were examined by semi-quantitative RT-PCR. Specific products were amplified from equal amounts of RT product from the cell lines indicated using primer sets for GABPα, GABPβ and GAPDH as an internal control. Products were separated on a 1.5% agarose gel with 100 bp ladder in leftmost lane. ( c ) Quantitative RT-PCR analysis of GABP beta-41 subunit mRNA was carried out on the indicated cell lines. Levels are expressed in relation to the 184hTERT cell line.

    Techniques Used: Western Blot, Molecular Weight, Quantitative RT-PCR, Amplification, Agarose Gel Electrophoresis

    14) Product Images from "KIAA1199 interacts with glycogen phosphorylase kinase β-subunit (PHKB) to promote glycogen breakdown and cancer cell survival"

    Article Title: KIAA1199 interacts with glycogen phosphorylase kinase β-subunit (PHKB) to promote glycogen breakdown and cancer cell survival

    Journal: Oncotarget

    doi:

    Construction of MBP-KIAA1199 fusion protein and detection KIAA1199 binding protein using a pull down assay (A) The schematic structure of five fragments of KIAA1199 used to fuse with MBP. The numbers represent the positions of the amino acids. All these fragments consisted of approximately 300 amino acids. (B) A pull down assay was performed using the fusion proteins with TU-KATO III whole cell lysate. The candidates were separated using SDS-PAGE, visualized using silver staining, and then analyzed using mass spectrometry. (C) An immunoprecipitation analysis was performed to confirm the interaction of COPA and PHKB with KIAA1199 in TU-KATO III cells.
    Figure Legend Snippet: Construction of MBP-KIAA1199 fusion protein and detection KIAA1199 binding protein using a pull down assay (A) The schematic structure of five fragments of KIAA1199 used to fuse with MBP. The numbers represent the positions of the amino acids. All these fragments consisted of approximately 300 amino acids. (B) A pull down assay was performed using the fusion proteins with TU-KATO III whole cell lysate. The candidates were separated using SDS-PAGE, visualized using silver staining, and then analyzed using mass spectrometry. (C) An immunoprecipitation analysis was performed to confirm the interaction of COPA and PHKB with KIAA1199 in TU-KATO III cells.

    Techniques Used: Binding Assay, Pull Down Assay, SDS Page, Silver Staining, Mass Spectrometry, Immunoprecipitation

    15) Product Images from "Comparative study of Arabidopsis PBS1 and a wheat PBS1 homolog helps understand the mechanism of PBS1 functioning in innate immunity"

    Article Title: Comparative study of Arabidopsis PBS1 and a wheat PBS1 homolog helps understand the mechanism of PBS1 functioning in innate immunity

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-05904-x

    TaPBS1 is localized to the plasma membrane. ( a ) The subcellular localization of TaPBS1 in Arabidopsis protoplasts, wheat protoplasts, and N . benthamiana leaves. TaPBS1 was transiently expressed in wheat protoplasts, Arabidopsis protoplasts or N . benthamiana leaves, and then subjected to laser confocal imaging. Chloroplasts were visualized with the autofluorescence of chlorophyll. ( b ) The plasmolyzed N . benthamiana epidermal cells expressing TaPBS1-GFP (arrows) demonstrated the PM localization of TaPBS1. N . benthamiana leaves infiltrated with A . tumefaciens carrying the TaPBS1-GFP construct were mounted in 1 M sucrose. ( c ) TaPBS1-GFP is co-localized with BSK1-RFP, a PM-localized fusion protein, at the PM. ( d ) Alanine substitutions of predicted myristoylation (glycine-2) and palmitoylation (cysteine-3/6) residues affected the PM localization of TaPBS1. TaPBS1 G2AC3/6A -GFP was transiently expressed in wheat protoplasts, Arabidopsis protoplasts or N . benthamiana leaves. ( e ) Co-localization analysis of TaPBS1 G2AC3/6A -GFP and BSK1-RFP.
    Figure Legend Snippet: TaPBS1 is localized to the plasma membrane. ( a ) The subcellular localization of TaPBS1 in Arabidopsis protoplasts, wheat protoplasts, and N . benthamiana leaves. TaPBS1 was transiently expressed in wheat protoplasts, Arabidopsis protoplasts or N . benthamiana leaves, and then subjected to laser confocal imaging. Chloroplasts were visualized with the autofluorescence of chlorophyll. ( b ) The plasmolyzed N . benthamiana epidermal cells expressing TaPBS1-GFP (arrows) demonstrated the PM localization of TaPBS1. N . benthamiana leaves infiltrated with A . tumefaciens carrying the TaPBS1-GFP construct were mounted in 1 M sucrose. ( c ) TaPBS1-GFP is co-localized with BSK1-RFP, a PM-localized fusion protein, at the PM. ( d ) Alanine substitutions of predicted myristoylation (glycine-2) and palmitoylation (cysteine-3/6) residues affected the PM localization of TaPBS1. TaPBS1 G2AC3/6A -GFP was transiently expressed in wheat protoplasts, Arabidopsis protoplasts or N . benthamiana leaves. ( e ) Co-localization analysis of TaPBS1 G2AC3/6A -GFP and BSK1-RFP.

    Techniques Used: Imaging, Expressing, Construct

    Cleavage of TaPBS1 by AvrPphB in Arabidopsis protoplasts. TaPBS1 could be cleaved by AvrPphB. FLAG-tagged PBS1, TaPBS1, or PBL2 was expressed together with AvrPphB-HA in Arabidopsis protoplasts. Protein cleavage was detected by Western blot analysis with an anti-FLAG antibody. AvrPphB-HA was detected by Western blot analysis with an anti-HA antibody.
    Figure Legend Snippet: Cleavage of TaPBS1 by AvrPphB in Arabidopsis protoplasts. TaPBS1 could be cleaved by AvrPphB. FLAG-tagged PBS1, TaPBS1, or PBL2 was expressed together with AvrPphB-HA in Arabidopsis protoplasts. Protein cleavage was detected by Western blot analysis with an anti-FLAG antibody. AvrPphB-HA was detected by Western blot analysis with an anti-HA antibody.

    Techniques Used: Western Blot

    Flagellin induces TaPBS1 phosphorylation in vivo . ( a ) Flg22 induces a mobility shift of TaPBS1 expressed in wheat protoplasts. TaPBS1-FLAG was transfected in wheat protoplasts. Then the wheat protoplasts were incubated for 8 h and treated with 1 μM flg22 for 10 min before harvesting. For CIP (calf intestinal alkaline phosphatase) treatment, 1 μM CIP was applied in the reactions containing TaPBS1 proteins and incubated at 37 °C for 1 h. Proteins were separated with SDS-PAGE, and TaPBS1 was detected by Western blot analysis with an anti-FLAG antibody. ( b ) Flg22 induces a mobility shift of TaPBS1 expressed in Arabidopsis protoplasts. TaPBS1 or PBS1 was expressed in Arabidopsis protoplasts and was analyzed as above. ( c ) The flg22-induced TaPBS1 phosphorylation in Arabidopsis protoplasts depends on BAK1. Protoplasts were isolated from col-0 or bak1 mutant plants and were transfected with FLAG-tagged TaPBS1 , PBS1 , or BIK1 . ( d ) The flg22-induced TaPBS1 phosphorylation in Arabidopsis protoplasts depends on its PM localization. Arabidopsis protoplasts were transfected with TaPBS1 or TaPBS1 G2AC3 / 6A and treated with 1 μM flg22 for 10 min before harvesting. The protein mobility shift was analyzed by Western blotting. ( e ) The kinase activity is not required for the flg22-induced TaPBS1/PBS1 phosphorylation. Arabidopsis protoplasts were transfected with TaPBS1 , TaPBS1Km , PBS1 , PBS1Km , BIK1 or BIK1Km and were treated with 1 μM flg22 for 10 min before harvesting.
    Figure Legend Snippet: Flagellin induces TaPBS1 phosphorylation in vivo . ( a ) Flg22 induces a mobility shift of TaPBS1 expressed in wheat protoplasts. TaPBS1-FLAG was transfected in wheat protoplasts. Then the wheat protoplasts were incubated for 8 h and treated with 1 μM flg22 for 10 min before harvesting. For CIP (calf intestinal alkaline phosphatase) treatment, 1 μM CIP was applied in the reactions containing TaPBS1 proteins and incubated at 37 °C for 1 h. Proteins were separated with SDS-PAGE, and TaPBS1 was detected by Western blot analysis with an anti-FLAG antibody. ( b ) Flg22 induces a mobility shift of TaPBS1 expressed in Arabidopsis protoplasts. TaPBS1 or PBS1 was expressed in Arabidopsis protoplasts and was analyzed as above. ( c ) The flg22-induced TaPBS1 phosphorylation in Arabidopsis protoplasts depends on BAK1. Protoplasts were isolated from col-0 or bak1 mutant plants and were transfected with FLAG-tagged TaPBS1 , PBS1 , or BIK1 . ( d ) The flg22-induced TaPBS1 phosphorylation in Arabidopsis protoplasts depends on its PM localization. Arabidopsis protoplasts were transfected with TaPBS1 or TaPBS1 G2AC3 / 6A and treated with 1 μM flg22 for 10 min before harvesting. The protein mobility shift was analyzed by Western blotting. ( e ) The kinase activity is not required for the flg22-induced TaPBS1/PBS1 phosphorylation. Arabidopsis protoplasts were transfected with TaPBS1 , TaPBS1Km , PBS1 , PBS1Km , BIK1 or BIK1Km and were treated with 1 μM flg22 for 10 min before harvesting.

    Techniques Used: In Vivo, Mobility Shift, Transfection, Incubation, SDS Page, Western Blot, Isolation, Mutagenesis, Activity Assay

    TaPBS1 associates with the CC domain of RPS5. TaPBS1/PBS1-FLAG and RPS5-CC-HA were co-expressed in Arabidopsis protoplasts, and TaPBS1/PBS1 was precipitated with an anti-FLAG antibody. The associated proteins were analyzed by Western blotting with an anti-HA antibody. GFP-FLAG was used as a negative control.
    Figure Legend Snippet: TaPBS1 associates with the CC domain of RPS5. TaPBS1/PBS1-FLAG and RPS5-CC-HA were co-expressed in Arabidopsis protoplasts, and TaPBS1/PBS1 was precipitated with an anti-FLAG antibody. The associated proteins were analyzed by Western blotting with an anti-HA antibody. GFP-FLAG was used as a negative control.

    Techniques Used: Western Blot, Negative Control

    In vitro analysis of TaPBS1 autophosphorylation. An in vitro protein phosphorylation assay was performed by incubating MBP, MBP-TaPBS1, MBP-TaPBS1Km (K132A), MBP-PBS1, MBP-PBS1Km (K115N), MBP-BIK1, and MBP-BIK1Km (K105A) in the presence of ATP. Proteins were separated with SDS-PAGE, and the protein phosphorylation was detected by Western blot analysis with an anti-phospho-Thr (pThr) antibody (upper panel). The protein loading control was shown by Coomassie brilliant blue staining (CBB, lower panel).
    Figure Legend Snippet: In vitro analysis of TaPBS1 autophosphorylation. An in vitro protein phosphorylation assay was performed by incubating MBP, MBP-TaPBS1, MBP-TaPBS1Km (K132A), MBP-PBS1, MBP-PBS1Km (K115N), MBP-BIK1, and MBP-BIK1Km (K105A) in the presence of ATP. Proteins were separated with SDS-PAGE, and the protein phosphorylation was detected by Western blot analysis with an anti-phospho-Thr (pThr) antibody (upper panel). The protein loading control was shown by Coomassie brilliant blue staining (CBB, lower panel).

    Techniques Used: In Vitro, Phosphorylation Assay, SDS Page, Western Blot, Staining

    Phylogenetic analysis and multiple alignment of TaPBS1 and related proteins. ( a ) Amino acid sequence alignment of TaPBS1 with putative PBS1 orthologs from different plant species. Identical and similar amino acid residues are shown on black and gray backgrounds, respectively. The conserved kinase subdomains are labeled with Roman numbers above the aligned sequences. The AvrPphB cleavage site GDK and the SEMPH/STRPH motif are also labeled. ( b ) Phylogenetic analysis of TaPBS1 and Arabidopsis PBS1 paralogs. A neighbor-joining phylogenetic tree was constructed based on the deduced amino acid sequences of TaPBS1, PBS1 and 29 PBL proteins in Arabidopsis using MEGA5.0.3 software. ( c ) Phylogenetic analysis of PBS1 orthologs from different plant species. Ca : Cicer arietinum ; Gm : Glycine max ; Cs : Cucumis sativus ; Pt : Populus trichocarpa ; Rc : Ricinus communis ; Tc : Theobroma cacao ; Pr . p : Prunus persica ; Fv : Fragaria vesca ; Vv : Vitis vinifera ; Sl : Solanum lycopersicum ; At : Arabidopsis thaliana ; Cr : Capsella rubella ; Pp : Physcomitrella patens ; Ta : Triticum aestivum ; Os : Oryza sativa ; Si : Setaria italica ; Zm : Zea mays ; Sb : Sorghum bicolor .
    Figure Legend Snippet: Phylogenetic analysis and multiple alignment of TaPBS1 and related proteins. ( a ) Amino acid sequence alignment of TaPBS1 with putative PBS1 orthologs from different plant species. Identical and similar amino acid residues are shown on black and gray backgrounds, respectively. The conserved kinase subdomains are labeled with Roman numbers above the aligned sequences. The AvrPphB cleavage site GDK and the SEMPH/STRPH motif are also labeled. ( b ) Phylogenetic analysis of TaPBS1 and Arabidopsis PBS1 paralogs. A neighbor-joining phylogenetic tree was constructed based on the deduced amino acid sequences of TaPBS1, PBS1 and 29 PBL proteins in Arabidopsis using MEGA5.0.3 software. ( c ) Phylogenetic analysis of PBS1 orthologs from different plant species. Ca : Cicer arietinum ; Gm : Glycine max ; Cs : Cucumis sativus ; Pt : Populus trichocarpa ; Rc : Ricinus communis ; Tc : Theobroma cacao ; Pr . p : Prunus persica ; Fv : Fragaria vesca ; Vv : Vitis vinifera ; Sl : Solanum lycopersicum ; At : Arabidopsis thaliana ; Cr : Capsella rubella ; Pp : Physcomitrella patens ; Ta : Triticum aestivum ; Os : Oryza sativa ; Si : Setaria italica ; Zm : Zea mays ; Sb : Sorghum bicolor .

    Techniques Used: Sequencing, Labeling, Construct, Software

    The introduction of the SEMPH motif into TaPBS1 enables it to activate RPS5 in transient assay. ( a ) The replacement of the STRPH motif with the SEMPH motif in TaPBS1 resulted into the RPS5-mediated HR in N . benthamiana following the cleavage of TaPBS1 SEMPH by AvrPphB. TaPBS1 or TaPBS1 SEMPH was co-expressed with RPS5 and AvrPphB in N . benthamiana leaves. HR assays were performed as above. ( b ) The protein expression control for the above assays. ( c ) Quantification of HRs induced in N . benthamiana leaves. HR was quantified by measuring the biomass loss rate of N . benthamiana leaves infiltrated with A . tumefaciens carrying indicated constructs. Results are the means ± SD of three independent repeats. Statistical significance was determined using a Student’s t -test: ** P
    Figure Legend Snippet: The introduction of the SEMPH motif into TaPBS1 enables it to activate RPS5 in transient assay. ( a ) The replacement of the STRPH motif with the SEMPH motif in TaPBS1 resulted into the RPS5-mediated HR in N . benthamiana following the cleavage of TaPBS1 SEMPH by AvrPphB. TaPBS1 or TaPBS1 SEMPH was co-expressed with RPS5 and AvrPphB in N . benthamiana leaves. HR assays were performed as above. ( b ) The protein expression control for the above assays. ( c ) Quantification of HRs induced in N . benthamiana leaves. HR was quantified by measuring the biomass loss rate of N . benthamiana leaves infiltrated with A . tumefaciens carrying indicated constructs. Results are the means ± SD of three independent repeats. Statistical significance was determined using a Student’s t -test: ** P

    Techniques Used: Expressing, Construct

    16) Product Images from "The Arabidopsis Transthyretin-Like Protein Is a Potential Substrate of BRASSINOSTEROID-INSENSITIVE 1"

    Article Title: The Arabidopsis Transthyretin-Like Protein Is a Potential Substrate of BRASSINOSTEROID-INSENSITIVE 1

    Journal:

    doi: 10.1105/tpc.104.023903

    The N Terminus of TTL Is Crucial for TTL–BRI1 Interaction
    Figure Legend Snippet: The N Terminus of TTL Is Crucial for TTL–BRI1 Interaction

    Techniques Used:

    TTL Is a Putative Substrate for BRI1.
    Figure Legend Snippet: TTL Is a Putative Substrate for BRI1.

    Techniques Used:

    Identification of TTL as a BRI1-Interacting Protein by Yeast Two-Hybrid Screening.
    Figure Legend Snippet: Identification of TTL as a BRI1-Interacting Protein by Yeast Two-Hybrid Screening.

    Techniques Used: Two Hybrid Screening

    17) Product Images from "Cloning and Expression of Two Related Connexins from the Perch Retina Define a Distinct Subgroup of the Connexin Family"

    Article Title: Cloning and Expression of Two Related Connexins from the Perch Retina Define a Distinct Subgroup of the Connexin Family

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.18-19-07625.1998

    Structure of the Cx35 and Cx34.7 genes. A , PCR amplification of Cx35 and Cx34.7 introns from hybrid bass genomic DNA. Introns were amplified by long PCR, as described in Materials and Methods. Amplification of Cx35 fragments from 100 ng of genomic DNA is shown in lane 1 , and control amplification from ∼1 pg of cDNA clone pcx7 is shown in lane 2 . The genomic product is ∼900 bp larger than the control. Amplification of Cx34.7 fragments from 100 ng of genomic DNA is shown in lane 3 , with control amplification from ∼1 pg of cDNA clone pcx1 in lane 4 ; a 20 kb product was obtained in the genomic amplification. The ∼4 kb product in lane 3 was cloned, sequenced, and found to be spurious. B , Gene maps of the known sequences of perch Cx35 and Cx34.7 genes based on sequence analysis of the intron PCR products. Regions comprising the cDNA sequences are enclosed by wide rectangles , with the hatched portion representing the coding sequence. Introns, indicated by the narrow bars , occur at the same location within the coding region as was described previously for skate Cx35. Known restriction sites within the cDNA sequences are coded as follows: Bam HI ( B ); Hin dIII ( H ); Nco I ( N ); Pst I ( P ). C , DNA sequences at the intron splice junctions for Cx35 and Cx34.7. Intron sequences are in lowercase letter, and the cDNA sequences are in uppercase letters. Both sets of intron splice junctions conform to the consensus sequence AGgt … agGA.
    Figure Legend Snippet: Structure of the Cx35 and Cx34.7 genes. A , PCR amplification of Cx35 and Cx34.7 introns from hybrid bass genomic DNA. Introns were amplified by long PCR, as described in Materials and Methods. Amplification of Cx35 fragments from 100 ng of genomic DNA is shown in lane 1 , and control amplification from ∼1 pg of cDNA clone pcx7 is shown in lane 2 . The genomic product is ∼900 bp larger than the control. Amplification of Cx34.7 fragments from 100 ng of genomic DNA is shown in lane 3 , with control amplification from ∼1 pg of cDNA clone pcx1 in lane 4 ; a 20 kb product was obtained in the genomic amplification. The ∼4 kb product in lane 3 was cloned, sequenced, and found to be spurious. B , Gene maps of the known sequences of perch Cx35 and Cx34.7 genes based on sequence analysis of the intron PCR products. Regions comprising the cDNA sequences are enclosed by wide rectangles , with the hatched portion representing the coding sequence. Introns, indicated by the narrow bars , occur at the same location within the coding region as was described previously for skate Cx35. Known restriction sites within the cDNA sequences are coded as follows: Bam HI ( B ); Hin dIII ( H ); Nco I ( N ); Pst I ( P ). C , DNA sequences at the intron splice junctions for Cx35 and Cx34.7. Intron sequences are in lowercase letter, and the cDNA sequences are in uppercase letters. Both sets of intron splice junctions conform to the consensus sequence AGgt … agGA.

    Techniques Used: Polymerase Chain Reaction, Amplification, Clone Assay, Sequencing

    18) Product Images from "Arabidopsis RopGAPs Are a Novel Family of Rho GTPase-Activating Proteins that Require the Cdc42/Rac-Interactive Binding Motif for Rop-Specific GTPase Stimulation 1"

    Article Title: Arabidopsis RopGAPs Are a Novel Family of Rho GTPase-Activating Proteins that Require the Cdc42/Rac-Interactive Binding Motif for Rop-Specific GTPase Stimulation 1

    Journal: Plant Physiology

    doi:

    ). Sequence alignment was performed by using the Clustal W program. The conserved motifs or domains shown were obtained from the GenBank database by using the BLAST search. A.t., Arabidopsis; D.d., Dictyostelium discoideum ; S.c. Saccharomyces cerevisiae ; H.s., human; C.e., Caenorhabditis elegans . A, Align- ment of predicted amino acid sequences of RopGAPs. B, Alignment of the GAP-like domain from RopGAP1 with various Rho GAPs. C, Alignment of the CRIB-like motif with known CRIB motifs from Cdc42/Rac effector proteins. D, Alignment of src homology domain 3-binding motifs from RopGAPs and other signaling proteins.
    Figure Legend Snippet: ). Sequence alignment was performed by using the Clustal W program. The conserved motifs or domains shown were obtained from the GenBank database by using the BLAST search. A.t., Arabidopsis; D.d., Dictyostelium discoideum ; S.c. Saccharomyces cerevisiae ; H.s., human; C.e., Caenorhabditis elegans . A, Align- ment of predicted amino acid sequences of RopGAPs. B, Alignment of the GAP-like domain from RopGAP1 with various Rho GAPs. C, Alignment of the CRIB-like motif with known CRIB motifs from Cdc42/Rac effector proteins. D, Alignment of src homology domain 3-binding motifs from RopGAPs and other signaling proteins.

    Techniques Used: Sequencing, Binding Assay

    19) Product Images from "Multifaceted Recognition of Vertebrate Rev1 by Translesion Polymerases ? and ? *"

    Article Title: Multifaceted Recognition of Vertebrate Rev1 by Translesion Polymerases ? and ? *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.380998

    Structure of the mRev1 CTD. A , stereo view of backbone traces from the structural ensemble of the mRev1 CTD, with helices colored in red and loops in gray. B , ribbon diagram. Conserved hydrophobic residues are shown in the stick model ( left panel ). The
    Figure Legend Snippet: Structure of the mRev1 CTD. A , stereo view of backbone traces from the structural ensemble of the mRev1 CTD, with helices colored in red and loops in gray. B , ribbon diagram. Conserved hydrophobic residues are shown in the stick model ( left panel ). The

    Techniques Used:

    Structure of the mRev1 CTD-Pol κ RIR complex. A , stereo view of backbone traces from the structural ensemble of the mRev1 CTD-Pol κ RIR complex, with helices colored in red , loops colored in gray , and a six-residue hairpin loop that undergoes
    Figure Legend Snippet: Structure of the mRev1 CTD-Pol κ RIR complex. A , stereo view of backbone traces from the structural ensemble of the mRev1 CTD-Pol κ RIR complex, with helices colored in red , loops colored in gray , and a six-residue hairpin loop that undergoes

    Techniques Used:

    Interactions between the mRev1 CTD and mRev7 probed by yeast two-hybrid assays. A , mRev1 CTD interacts with mRev7. Plasmids containing the mRev1 CTD fused to the GAL4 DNA-binding domain ( BD , upper panel ) or the activation domain ( AD , lower panel ) were
    Figure Legend Snippet: Interactions between the mRev1 CTD and mRev7 probed by yeast two-hybrid assays. A , mRev1 CTD interacts with mRev7. Plasmids containing the mRev1 CTD fused to the GAL4 DNA-binding domain ( BD , upper panel ) or the activation domain ( AD , lower panel ) were

    Techniques Used: Binding Assay, Activation Assay

    mRev7 binds to the mRev1 CTD primarily through a surface centered at the α2–α3 loop and the N-terminal part of α3. A , interactions of the GAL4 DNA-binding domain-fused mRev1 CTD harboring indicated mutations with GAL4 activation
    Figure Legend Snippet: mRev7 binds to the mRev1 CTD primarily through a surface centered at the α2–α3 loop and the N-terminal part of α3. A , interactions of the GAL4 DNA-binding domain-fused mRev1 CTD harboring indicated mutations with GAL4 activation

    Techniques Used: Binding Assay, Activation Assay

    20) Product Images from "Modulation of RNA polymerase II phosphorylation downstream of pathogen perception orchestrates plant immunity"

    Article Title: Modulation of RNA polymerase II phosphorylation downstream of pathogen perception orchestrates plant immunity

    Journal: Cell host & microbe

    doi: 10.1016/j.chom.2014.10.018

    MAPKs phosphorylate CDKCs in flagellin signaling (A) flg22 induces CDKC mobility shift in protoplasts. Protoplasts were expressed with CDKC; 1-HA or CDKC;2-HA and treated with 100 nM flg22 for 15 min. Total proteins were separated in a regular 10% SDS-PAGE gel (top) or supplemented with 50 μM phos-tag (Wako chemicals USA, Inc.) (middle). The loading control with α-H3 is shown on the bottom. (B) CDKC;1 mobility shift was removed by CIP treatment. Total proteins were separated in a SDS-PAGE gel supplemented with 50 μM phos-tag (top). (C) CDKC;1 mobility shift is blocked by MKK inhibitor PD184161 in protoplasts. 7.5 μM PD184161 was added 1 hr before flg22 treatment. (D) MAPK phosphatase (MKP) blocks CDKC;1 mobility shift. Protoplasts were co-expressed with CDKC;1-HA and MKP. In C D, top is a regular SDS-PAGE and middle is phos-tag gel. (E) MPK3 phosphorylates GST-CDKC;1 and GST-CDKC;2 fusion proteins. FLAG epitope-tagged MPKs were expressed in protoplasts treated with 100 nM flg22, and immunoprecipitated for in vitro kinase assay with GST-CDKC;1 or GST-CDKC;2 as substrate. The reactions with myelin basic protein and GST protein as substrates are shown as controls. The protein loading of substrates is shown by CBB staining. (F) MPKs phosphorylate CDKCs at S94 in vitro. The recombinant HIS-MPK3 and HIS-MPK6 proteins were activated by constitutively active MKK5 DD , and used to phosphorylate GST-CDKCs and their S94A mutants. The phosphorylation was detected by autoradiograph and the protein loading is shown by CBB staining. * in E F indicates the expected position of GST-CDKC. (G) CDKC;1 and CDKC;2 interact with MPK3 by Co-IP assay. FLAG epitope-tagged CDKC and HA epitope-tagged MPK3 were co-expressed in Col-0 protoplasts. The proteins were immunoprecipitated with α-FLAG agarose beads, immune-blotted with α-HA or α-FLAG antibody. The input of MPK3 and CDKCs is shown by WB. (H) CDKC;1 S94 is phosphorylated by MPK3 as shown with MS analysis. The graph indicates the sequence of a doubly charged peptide ion at m/z 469.72 that matches to EIVTpSPGR of CDKC;1. ).
    Figure Legend Snippet: MAPKs phosphorylate CDKCs in flagellin signaling (A) flg22 induces CDKC mobility shift in protoplasts. Protoplasts were expressed with CDKC; 1-HA or CDKC;2-HA and treated with 100 nM flg22 for 15 min. Total proteins were separated in a regular 10% SDS-PAGE gel (top) or supplemented with 50 μM phos-tag (Wako chemicals USA, Inc.) (middle). The loading control with α-H3 is shown on the bottom. (B) CDKC;1 mobility shift was removed by CIP treatment. Total proteins were separated in a SDS-PAGE gel supplemented with 50 μM phos-tag (top). (C) CDKC;1 mobility shift is blocked by MKK inhibitor PD184161 in protoplasts. 7.5 μM PD184161 was added 1 hr before flg22 treatment. (D) MAPK phosphatase (MKP) blocks CDKC;1 mobility shift. Protoplasts were co-expressed with CDKC;1-HA and MKP. In C D, top is a regular SDS-PAGE and middle is phos-tag gel. (E) MPK3 phosphorylates GST-CDKC;1 and GST-CDKC;2 fusion proteins. FLAG epitope-tagged MPKs were expressed in protoplasts treated with 100 nM flg22, and immunoprecipitated for in vitro kinase assay with GST-CDKC;1 or GST-CDKC;2 as substrate. The reactions with myelin basic protein and GST protein as substrates are shown as controls. The protein loading of substrates is shown by CBB staining. (F) MPKs phosphorylate CDKCs at S94 in vitro. The recombinant HIS-MPK3 and HIS-MPK6 proteins were activated by constitutively active MKK5 DD , and used to phosphorylate GST-CDKCs and their S94A mutants. The phosphorylation was detected by autoradiograph and the protein loading is shown by CBB staining. * in E F indicates the expected position of GST-CDKC. (G) CDKC;1 and CDKC;2 interact with MPK3 by Co-IP assay. FLAG epitope-tagged CDKC and HA epitope-tagged MPK3 were co-expressed in Col-0 protoplasts. The proteins were immunoprecipitated with α-FLAG agarose beads, immune-blotted with α-HA or α-FLAG antibody. The input of MPK3 and CDKCs is shown by WB. (H) CDKC;1 S94 is phosphorylated by MPK3 as shown with MS analysis. The graph indicates the sequence of a doubly charged peptide ion at m/z 469.72 that matches to EIVTpSPGR of CDKC;1. ).

    Techniques Used: Mobility Shift, SDS Page, FLAG-tag, Immunoprecipitation, In Vitro, Kinase Assay, Staining, Recombinant, Autoradiography, Co-Immunoprecipitation Assay, Western Blot, Mass Spectrometry, Sequencing

    CPL3 is a CTD phosphatase (A) MPK3-activated CDKCs induce GST-CTD phosphorylation in vitro. MPK3-HA was expressed in protoplasts treated with 100 nM flg22, immunoprecipitated with α-HA agarose beads, and incubated with MBP-CDKC proteins. The phosphorylated CDKC proteins were collected by centrifugation as supernatant (MPK3-HA conjugated beads were in pellets) and used to phosphorylate GST-CTD. The CTD phosphorylation was analyzed by WB with specific antibodies. (B) CDKC;1 S94A and CDKC;2 S94A reduce the ability to phosphorylate GST-CTD. (C) CPL3 dephosphorylates CDKC; 1-activated GST-CTD in vitro. CDKC;1-HA and CYCT1;3-HA were expressed in protoplasts, immunoprecipitated to phosphorylate GST-CTD. The phosphorylated GST-CTD was dephosphorylated by MBP-CPL3N (N), MBP-CPL3C (C) and MBP-FCPH. CTD phosphorylation was detected by WB with specific antibodies. CTD loading is shown by WB with α-GST antibody. (D) CPL3 dephosphorylates CDKC;2-activated GST-CTD in vitro . (E) CPL3 interacts with GST-CTD in vitro. Pull-down assay was performed by incubating MBP-CPL3 together with glutathione beads containing CTD or phosphorylated CTD (pCTD). The HA-tagged CPL3 proteins were detected with an α-HA WB after glutathione bead pull-down (PD). The input control is shown by WB. (F) CTD and CPL3C interact in yeast. The interactions between pGADT7-CTD and pGBDT7-CPL3C, pGBDT7-CPL3N and pGBDT7-FCPH were tested in SD medium without histidine, leucine and tryptophan (SD-H-L-T) supplemented with 1 mM 3AT. EV is the empty vector pGADT7. (G) CPL3C D933A and CPL3C DD1064AA lose phosphatase activity. (H) Enhanced CTD Ser2 phosphorylation in cpl3-4 mutant seedling. One-week-old seedlings of WT and cpl3-4 were treated with or without 1 μM flg22 for 30 min. ).
    Figure Legend Snippet: CPL3 is a CTD phosphatase (A) MPK3-activated CDKCs induce GST-CTD phosphorylation in vitro. MPK3-HA was expressed in protoplasts treated with 100 nM flg22, immunoprecipitated with α-HA agarose beads, and incubated with MBP-CDKC proteins. The phosphorylated CDKC proteins were collected by centrifugation as supernatant (MPK3-HA conjugated beads were in pellets) and used to phosphorylate GST-CTD. The CTD phosphorylation was analyzed by WB with specific antibodies. (B) CDKC;1 S94A and CDKC;2 S94A reduce the ability to phosphorylate GST-CTD. (C) CPL3 dephosphorylates CDKC; 1-activated GST-CTD in vitro. CDKC;1-HA and CYCT1;3-HA were expressed in protoplasts, immunoprecipitated to phosphorylate GST-CTD. The phosphorylated GST-CTD was dephosphorylated by MBP-CPL3N (N), MBP-CPL3C (C) and MBP-FCPH. CTD phosphorylation was detected by WB with specific antibodies. CTD loading is shown by WB with α-GST antibody. (D) CPL3 dephosphorylates CDKC;2-activated GST-CTD in vitro . (E) CPL3 interacts with GST-CTD in vitro. Pull-down assay was performed by incubating MBP-CPL3 together with glutathione beads containing CTD or phosphorylated CTD (pCTD). The HA-tagged CPL3 proteins were detected with an α-HA WB after glutathione bead pull-down (PD). The input control is shown by WB. (F) CTD and CPL3C interact in yeast. The interactions between pGADT7-CTD and pGBDT7-CPL3C, pGBDT7-CPL3N and pGBDT7-FCPH were tested in SD medium without histidine, leucine and tryptophan (SD-H-L-T) supplemented with 1 mM 3AT. EV is the empty vector pGADT7. (G) CPL3C D933A and CPL3C DD1064AA lose phosphatase activity. (H) Enhanced CTD Ser2 phosphorylation in cpl3-4 mutant seedling. One-week-old seedlings of WT and cpl3-4 were treated with or without 1 μM flg22 for 30 min. ).

    Techniques Used: In Vitro, Immunoprecipitation, Incubation, Centrifugation, Western Blot, Pull Down Assay, Plasmid Preparation, Activity Assay, Mutagenesis

    21) Product Images from "MAGE-A1 interacts with adaptor SKIP and the deacetylase HDAC1 to repress transcription"

    Article Title: MAGE-A1 interacts with adaptor SKIP and the deacetylase HDAC1 to repress transcription

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkh735

    Interaction between deleted forms of MAGE-A1 and SKIP in the yeast two-hybrid system. Truncated MAGE-A1 sequences were inserted in vector pGBT9 and SKIP in vector pACT2. These constructs were transformed in yeast PJ69-4A cells. β-Galactosidase
    Figure Legend Snippet: Interaction between deleted forms of MAGE-A1 and SKIP in the yeast two-hybrid system. Truncated MAGE-A1 sequences were inserted in vector pGBT9 and SKIP in vector pACT2. These constructs were transformed in yeast PJ69-4A cells. β-Galactosidase

    Techniques Used: Plasmid Preparation, Construct, Transformation Assay

    22) Product Images from "Molecular mechanism of a COOH-terminal gating determinant in the ROMK channel revealed by a Bartter's disease mutation"

    Article Title: Molecular mechanism of a COOH-terminal gating determinant in the ROMK channel revealed by a Bartter's disease mutation

    Journal: The Journal of Physiology

    doi: 10.1113/jphysiol.2002.027581

    Cytoplasmic NH 2 - and COOH-terminal domains of Kir1.1 interact in vitro A , cartoon illustrating the cytoplasmic domains of Kir1.1 and the corresponding bacterial fusion proteins. The NH 2 -domain was made as a GST fusion and the COOH-domain was made as a MBP fusion. B , purified recombinant domains resolved by SDS-PAGE and visualized by Coomassie Brillant Blue staining. C , gel overlay assay: GST alone or GST fusions of the Shaker B cytoplasmic NH 2 -terminus (GST-ShB) or the Kir1.1 N-terminus (GST-ROMKN) were resolved by SDS-PAGE, transferred to nitrocellulose, renatured, visualized by Ponceau staining (left) and then blotted with either the MBP protein alone (right) or MBP fusion of the Kir1.1 COOH-terminal domain (MBP-ROMKC, middle). After extensive washes, bound MBP protein was detected using an anti-MBP antibody.
    Figure Legend Snippet: Cytoplasmic NH 2 - and COOH-terminal domains of Kir1.1 interact in vitro A , cartoon illustrating the cytoplasmic domains of Kir1.1 and the corresponding bacterial fusion proteins. The NH 2 -domain was made as a GST fusion and the COOH-domain was made as a MBP fusion. B , purified recombinant domains resolved by SDS-PAGE and visualized by Coomassie Brillant Blue staining. C , gel overlay assay: GST alone or GST fusions of the Shaker B cytoplasmic NH 2 -terminus (GST-ShB) or the Kir1.1 N-terminus (GST-ROMKN) were resolved by SDS-PAGE, transferred to nitrocellulose, renatured, visualized by Ponceau staining (left) and then blotted with either the MBP protein alone (right) or MBP fusion of the Kir1.1 COOH-terminal domain (MBP-ROMKC, middle). After extensive washes, bound MBP protein was detected using an anti-MBP antibody.

    Techniques Used: In Vitro, Purification, Recombinant, SDS Page, Staining, Overlay Assay

    Residues 332-361 form an intrasteric pH-regulatory domain To delimit the COOH-terminal pH-gating determinant, the pH sensitivity of the COOH-terminal truncation mutants were determined. In contrast to Kir1.1a 351X, the pH sensitivities of both Kir1.1a 361X and Kir1.1a 388X were identical to wild-type channel ( A ). These data identify amino acids 332-361 as an essential pH-dependent gating element ( B ). Consistent with an intrasteric regulatory role of this domain, injection of a synthetic peptide corresponding to amino acids 332-362 partially restored the pH sensitivity (p K a = 6.88 ± 0.03, n = 15) of Kir1.1a 351X. The peptide had no effect on wild-type channel ( n = 8). The p K a of wild-type channel in the presence of the peptide was 6.56 ± 0.03. •, 351X + peptide; ▪, wild-type + peptide superimposed on 351X or wild-type titration curves (dotted lines) ( C ).
    Figure Legend Snippet: Residues 332-361 form an intrasteric pH-regulatory domain To delimit the COOH-terminal pH-gating determinant, the pH sensitivity of the COOH-terminal truncation mutants were determined. In contrast to Kir1.1a 351X, the pH sensitivities of both Kir1.1a 361X and Kir1.1a 388X were identical to wild-type channel ( A ). These data identify amino acids 332-361 as an essential pH-dependent gating element ( B ). Consistent with an intrasteric regulatory role of this domain, injection of a synthetic peptide corresponding to amino acids 332-362 partially restored the pH sensitivity (p K a = 6.88 ± 0.03, n = 15) of Kir1.1a 351X. The peptide had no effect on wild-type channel ( n = 8). The p K a of wild-type channel in the presence of the peptide was 6.56 ± 0.03. •, 351X + peptide; ▪, wild-type + peptide superimposed on 351X or wild-type titration curves (dotted lines) ( C ).

    Techniques Used: Injection, Titration

    Channel properties of Kir1.1 COOH-terminal truncation mutants A , cartoon of Kir1.1 highlighting the cytoplasmic COOH-terminal domain of interest. A series of progressively shorter deletion mutants, from the Bartter's truncation (331X) to the PDZ binding site (388X), were studied. Amino acid 391 is the last amino acid in Kir1.1, B ). The upper panel summarizes the single-channel open probability ( P o ) and single-channel conductance (γ) of the mutants. The lower panel summarizes the whole-cell potassium currents of the mutants relative to wild-type at −100 mV (* P
    Figure Legend Snippet: Channel properties of Kir1.1 COOH-terminal truncation mutants A , cartoon of Kir1.1 highlighting the cytoplasmic COOH-terminal domain of interest. A series of progressively shorter deletion mutants, from the Bartter's truncation (331X) to the PDZ binding site (388X), were studied. Amino acid 391 is the last amino acid in Kir1.1, B ). The upper panel summarizes the single-channel open probability ( P o ) and single-channel conductance (γ) of the mutants. The lower panel summarizes the whole-cell potassium currents of the mutants relative to wild-type at −100 mV (* P

    Techniques Used: Binding Assay

    COOH-terminal truncation induces an aberrant gating mode Diary plots of single-channel open probability, P o of wild-type Kir1.1a ( A) vs. the truncated mutant, Kir1.1a 351X (B) . Shown are plots of P o measured in 5 s intervals over the duration of a 3 min recording. Representative channel records corresponding to the indicated time are shown in a and b . Single-channel recordings were obtained in the cell-attached mode ( V m = −80 mV) from oocytes injected with either wild-type or mutant channel cRNA.
    Figure Legend Snippet: COOH-terminal truncation induces an aberrant gating mode Diary plots of single-channel open probability, P o of wild-type Kir1.1a ( A) vs. the truncated mutant, Kir1.1a 351X (B) . Shown are plots of P o measured in 5 s intervals over the duration of a 3 min recording. Representative channel records corresponding to the indicated time are shown in a and b . Single-channel recordings were obtained in the cell-attached mode ( V m = −80 mV) from oocytes injected with either wild-type or mutant channel cRNA.

    Techniques Used: Mutagenesis, Injection

    23) Product Images from "Pag1p, a Novel Protein Associated with Protein Kinase Cbk1p, Is Required for Cell Morphogenesis and Proliferation in Saccharomyces cerevisiae"

    Article Title: Pag1p, a Novel Protein Associated with Protein Kinase Cbk1p, Is Required for Cell Morphogenesis and Proliferation in Saccharomyces cerevisiae

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.01-07-0365

    Overexpression of an extracellular protein, Sim1p, s uppresses the growth defects of both pag1Δ and cbk1Δ . (A) SIM1 is a dosage-dependent suppressor of pag1Δ. pag1Δ [2 μ URA3 PAG1 ] (NY2336) with the HIS3 -containing plasmids shown in the figure streaked onto a 5-FOA plate and incubated at 25°C. (B) A multicopy [2 μ URA3 SIM1 ] plasmid suppresses the lethality of cbk1Δ . Tetrads were dissected onto a YPD plate and incubated at 25°C. Photo was taken 4 d later. G-418 resistant colonies ( cbk1Δ ) are circled. They are Ura + and cannot grow on a 5-FOA plate. (C) pag1Δ and cbk1Δ cells with a multicopy SIM1 plasmid display defects in cell separation and polarized growth. Wild-type (NY2355), pag1Δ (NY2356), and cbk1Δ (NY2357) homozygous diploid cells containing a [2 μ URA3 SIM1 ] plasmid growing exponentially in SC-URA at 30°C were fixed and photographed. (D) Sim1p is a secreted protein. Cells were grown to log phase in synthetic media. Lysates were made by vortexing with glass beads. Proteins secreted into the medium were precipitated with trichloroacetic acid. Samples corresponding to materials from cultures containing 0.4 OD 600 unit cells were separated by SDS-PAGE. Western blot was probed with antibodies against Sim1p and a cytosolic protein, alcohol dehydrogenase (ADH).
    Figure Legend Snippet: Overexpression of an extracellular protein, Sim1p, s uppresses the growth defects of both pag1Δ and cbk1Δ . (A) SIM1 is a dosage-dependent suppressor of pag1Δ. pag1Δ [2 μ URA3 PAG1 ] (NY2336) with the HIS3 -containing plasmids shown in the figure streaked onto a 5-FOA plate and incubated at 25°C. (B) A multicopy [2 μ URA3 SIM1 ] plasmid suppresses the lethality of cbk1Δ . Tetrads were dissected onto a YPD plate and incubated at 25°C. Photo was taken 4 d later. G-418 resistant colonies ( cbk1Δ ) are circled. They are Ura + and cannot grow on a 5-FOA plate. (C) pag1Δ and cbk1Δ cells with a multicopy SIM1 plasmid display defects in cell separation and polarized growth. Wild-type (NY2355), pag1Δ (NY2356), and cbk1Δ (NY2357) homozygous diploid cells containing a [2 μ URA3 SIM1 ] plasmid growing exponentially in SC-URA at 30°C were fixed and photographed. (D) Sim1p is a secreted protein. Cells were grown to log phase in synthetic media. Lysates were made by vortexing with glass beads. Proteins secreted into the medium were precipitated with trichloroacetic acid. Samples corresponding to materials from cultures containing 0.4 OD 600 unit cells were separated by SDS-PAGE. Western blot was probed with antibodies against Sim1p and a cytosolic protein, alcohol dehydrogenase (ADH).

    Techniques Used: Over Expression, Incubation, Plasmid Preparation, SDS Page, Western Blot

    24) Product Images from "Phosphorylation of Trihelix Transcriptional Repressor ASR3 by MAP KINASE4 Negatively Regulates Arabidopsis Immunity"

    Article Title: Phosphorylation of Trihelix Transcriptional Repressor ASR3 by MAP KINASE4 Negatively Regulates Arabidopsis Immunity

    Journal: The Plant Cell

    doi: 10.1105/tpc.114.134809

    ASR3 Globally Regulates flg22-Induced Gene Expression.
    Figure Legend Snippet: ASR3 Globally Regulates flg22-Induced Gene Expression.

    Techniques Used: Expressing

    flg22 Perception Induces ASR3 Phosphorylation.
    Figure Legend Snippet: flg22 Perception Induces ASR3 Phosphorylation.

    Techniques Used:

    Overexpression of ASR3 Compromises Disease Resistance to Virulent Bacterial Pathogens.
    Figure Legend Snippet: Overexpression of ASR3 Compromises Disease Resistance to Virulent Bacterial Pathogens.

    Techniques Used: Over Expression

    ASR3 Is a Transcriptional Repressor.
    Figure Legend Snippet: ASR3 Is a Transcriptional Repressor.

    Techniques Used:

    The flg22-Induced ASR3 Phosphorylation Occurs at Thr-189.
    Figure Legend Snippet: The flg22-Induced ASR3 Phosphorylation Occurs at Thr-189.

    Techniques Used:

    MPK4 Phosphorylates and Interacts with ASR3.
    Figure Legend Snippet: MPK4 Phosphorylates and Interacts with ASR3.

    Techniques Used:

    The asr3 Mutant Displays Enhanced Disease Resistance and Immune Gene Activation.
    Figure Legend Snippet: The asr3 Mutant Displays Enhanced Disease Resistance and Immune Gene Activation.

    Techniques Used: Mutagenesis, Activation Assay

    Phosphorylation of ASR3 by MPK4 Enhances Its DNA Binding Activity.
    Figure Legend Snippet: Phosphorylation of ASR3 by MPK4 Enhances Its DNA Binding Activity.

    Techniques Used: Binding Assay, Activity Assay

    25) Product Images from "Comparative analyses of ubiquitin-like ATG8 and cysteine protease ATG4 autophagy genes in the plant lineage and cross-kingdom processing of ATG8 by ATG4"

    Article Title: Comparative analyses of ubiquitin-like ATG8 and cysteine protease ATG4 autophagy genes in the plant lineage and cross-kingdom processing of ATG8 by ATG4

    Journal: Autophagy

    doi: 10.1080/15548627.2016.1217373

    HsLC3A directly binds to yeast, Arabidopsis , and human ATG4s. (A) C-HsLC3A-ShR and catalytic resistant C-HsLC3A G120A -ShR were used for in vitro affinity isolation with various ATG4 homologs. The yeast and Arabidopsis ATG4s affinity isolated C-HsLC3A-ShR but HsATG4B failed to affinity isolate HsLC3A due to kinetic activity of HsATG4B (left, middle panel). All tested ATG4 homologs affinity isolated the catalytic resistant C-HsLC3A G120A -ShR (right, middle panel). Anti-MBP and Coomassie staining were used for inputs of ATG4 and the ATG8 substrates, respectively. Asterisks indicate degraded ATG4s. (B) C-HsLC3A G120A -ShR is not affinity isolated by the MBP-alone control (bottom panel). Same amount of C-HsLC3A G120A -ShR was used for affinity isolation as shown in (A). Anti-MBP was used for inputs. Asterisks indicate nonspecific binding products with amylose resin.
    Figure Legend Snippet: HsLC3A directly binds to yeast, Arabidopsis , and human ATG4s. (A) C-HsLC3A-ShR and catalytic resistant C-HsLC3A G120A -ShR were used for in vitro affinity isolation with various ATG4 homologs. The yeast and Arabidopsis ATG4s affinity isolated C-HsLC3A-ShR but HsATG4B failed to affinity isolate HsLC3A due to kinetic activity of HsATG4B (left, middle panel). All tested ATG4 homologs affinity isolated the catalytic resistant C-HsLC3A G120A -ShR (right, middle panel). Anti-MBP and Coomassie staining were used for inputs of ATG4 and the ATG8 substrates, respectively. Asterisks indicate degraded ATG4s. (B) C-HsLC3A G120A -ShR is not affinity isolated by the MBP-alone control (bottom panel). Same amount of C-HsLC3A G120A -ShR was used for affinity isolation as shown in (A). Anti-MBP was used for inputs. Asterisks indicate nonspecific binding products with amylose resin.

    Techniques Used: In Vitro, Isolation, Activity Assay, Staining, Binding Assay

    Nicotiana benthamiana plant ATG4 efficiently process yeast Atg8 but weakly process human LC3A. (A) C-ScAtg8-ShR (lane 1), C-HsLC3A-ShR (lane 2), and C-HsLC3A-ShR with HsATG4B (lane 3) were expressed in N. benthamiana plant leaves and cleavage byproduct (ShR) was detected with anti-ShR antibodies (top panel). The cleaved ShR byproduct was observed in C-ScAtg8-ShR-expressing tissue (lane 1) but not in C-HsLC3A-ShR (lane 2). Coexpression of C-HsLC3A-ShR with HsATG4B resulted in accumulation of the cleaved ShR byproduct (lane 3). Arrow and arrowhead indicate full-length ATG8 sensors and cleaved byproducts, respectively. Anti-MYC was used to detect the HsATG4B input (middle panel, lane 3). Anti-PEPC was used for input loading control (bottom panel). (B to D) C-ScAtg8-ShR sensor was processed by endogenous N. benthamiana ATG4s and the mature form of C-ScAtg8 was incorporated into autophagic bodies in the vacuole (B). Coexpression of C-HsLC3A-ShR with HsATG4B showed enhanced accumulation of autophagic bodies in the vacuole (D) compared to the expression of C-HsLC3A-ShR alone (C) in N. benthamiana leaves. Scale bar: 20 µm. (E) Quantification of autophagic bodies observed in (B to D). One-way ANOVA test indicates a statistically different number of autophagic bodies accumulated in the vacuole of cells expressing ScATG8 alone and HsLC3A with HsATG4B compared to HsLC3A alone. Lowercase letters indicate statistical differences ( P
    Figure Legend Snippet: Nicotiana benthamiana plant ATG4 efficiently process yeast Atg8 but weakly process human LC3A. (A) C-ScAtg8-ShR (lane 1), C-HsLC3A-ShR (lane 2), and C-HsLC3A-ShR with HsATG4B (lane 3) were expressed in N. benthamiana plant leaves and cleavage byproduct (ShR) was detected with anti-ShR antibodies (top panel). The cleaved ShR byproduct was observed in C-ScAtg8-ShR-expressing tissue (lane 1) but not in C-HsLC3A-ShR (lane 2). Coexpression of C-HsLC3A-ShR with HsATG4B resulted in accumulation of the cleaved ShR byproduct (lane 3). Arrow and arrowhead indicate full-length ATG8 sensors and cleaved byproducts, respectively. Anti-MYC was used to detect the HsATG4B input (middle panel, lane 3). Anti-PEPC was used for input loading control (bottom panel). (B to D) C-ScAtg8-ShR sensor was processed by endogenous N. benthamiana ATG4s and the mature form of C-ScAtg8 was incorporated into autophagic bodies in the vacuole (B). Coexpression of C-HsLC3A-ShR with HsATG4B showed enhanced accumulation of autophagic bodies in the vacuole (D) compared to the expression of C-HsLC3A-ShR alone (C) in N. benthamiana leaves. Scale bar: 20 µm. (E) Quantification of autophagic bodies observed in (B to D). One-way ANOVA test indicates a statistically different number of autophagic bodies accumulated in the vacuole of cells expressing ScATG8 alone and HsLC3A with HsATG4B compared to HsLC3A alone. Lowercase letters indicate statistical differences ( P

    Techniques Used: Expressing

    Human ATG4B can cleave cross-kingdom ATG8s in vitro. Purified recombinant maltose binding protein (MBP) fused to different ATG4s (MBP-ATG4) and MBP-alone were incubated with various C-ATG8- or LC3A-ShR synthetic substrates as described in the Materials and Methods section. The reaction mixtures were separated on SDS-PAGE gel and blots were probed with anti-ShR. Arrows and arrowheads indicate full-length synthetic substrate (C-ATG8-ShR) and cleaved byproduct ShR, respectively. The yeast Atg4 (A), Arabidopsis ATG4a (B), and tomato ATG4 (C) could process both yeast and plant ATG8s but not human HsLC3A. HsATG4B could process all species ATG8 synthetic substrates (D). The tomato ATG4 mutant (SlATG4 178-188Δ ) in which the plant specific domain is deleted decreased the efficiency of processing of plant ATG8s and completely inhibited processing of yeast Atg8 (E). Sc, Saccharomyces cerevisiae; Hs, Homo sapiens; At, Arabidopsis thaliana; Sl, Solanum lycopersicum .
    Figure Legend Snippet: Human ATG4B can cleave cross-kingdom ATG8s in vitro. Purified recombinant maltose binding protein (MBP) fused to different ATG4s (MBP-ATG4) and MBP-alone were incubated with various C-ATG8- or LC3A-ShR synthetic substrates as described in the Materials and Methods section. The reaction mixtures were separated on SDS-PAGE gel and blots were probed with anti-ShR. Arrows and arrowheads indicate full-length synthetic substrate (C-ATG8-ShR) and cleaved byproduct ShR, respectively. The yeast Atg4 (A), Arabidopsis ATG4a (B), and tomato ATG4 (C) could process both yeast and plant ATG8s but not human HsLC3A. HsATG4B could process all species ATG8 synthetic substrates (D). The tomato ATG4 mutant (SlATG4 178-188Δ ) in which the plant specific domain is deleted decreased the efficiency of processing of plant ATG8s and completely inhibited processing of yeast Atg8 (E). Sc, Saccharomyces cerevisiae; Hs, Homo sapiens; At, Arabidopsis thaliana; Sl, Solanum lycopersicum .

    Techniques Used: In Vitro, Purification, Recombinant, Binding Assay, Incubation, SDS Page, Mutagenesis

    26) Product Images from "Reovirus ?NS Protein Is Required for Nucleation of Viral Assembly Complexes and Formation of Viral Inclusions"

    Article Title: Reovirus ?NS Protein Is Required for Nucleation of Viral Assembly Complexes and Formation of Viral Inclusions

    Journal: Journal of Virology

    doi: 10.1128/JVI.75.3.1459-1475.2001

    Subcellular localization of ςNS and μ2 proteins in cells infected with T3D, determined at different times postinfection. L cells were infected with T3D at an MOI of 10 PFU per cell and incubated at 37°C for the time periods shown. Cells were stained for ςNS by using a ςNS-specific polyclonal antiserum directly conjugated to Alexa Fluor 546 and for μ2 by using a μ2-specific polyclonal antiserum directly conjugated to Alexa Fluor 488. Images were obtained by using a confocal microscope. The ςNS protein is colored green, and the μ2 protein is colored red. A DIC image of each field was obtained. In the merged image, colocalization of ςNS and μ2 is indicated by the yellow color. Images were processed using Adobe Photoshop. Bars, 25 μm.
    Figure Legend Snippet: Subcellular localization of ςNS and μ2 proteins in cells infected with T3D, determined at different times postinfection. L cells were infected with T3D at an MOI of 10 PFU per cell and incubated at 37°C for the time periods shown. Cells were stained for ςNS by using a ςNS-specific polyclonal antiserum directly conjugated to Alexa Fluor 546 and for μ2 by using a μ2-specific polyclonal antiserum directly conjugated to Alexa Fluor 488. Images were obtained by using a confocal microscope. The ςNS protein is colored green, and the μ2 protein is colored red. A DIC image of each field was obtained. In the merged image, colocalization of ςNS and μ2 is indicated by the yellow color. Images were processed using Adobe Photoshop. Bars, 25 μm.

    Techniques Used: Infection, Incubation, Staining, Microscopy

    Subcellular localization of reovirus ςNS and μ2 proteins in cells infected with reovirus strain T3D. L cells were infected with T3D at an MOI of 10 PFU per cell and incubated at 37°C for 18 h. Cells were stained for ςNS by using ςNS-specific MAb 2H7 (B) and for μ2 by using a μ2-specific polyclonal antiserum (C) as primary antibodies followed by Alexa Fluor 546 goat anti-mouse IgG and Alexa Fluor 488 goat anti-rabbit IgG, respectively, as secondary antibodies. Images were obtained by using a confocal microscope. The ςNS protein is colored green, and the μ2 protein is colored red. (A) A DIC image of the field was obtained. (D) In the merged image, colocalization of ςNS and μ2 is indicated by the yellow color. The arrow indicates a viral inclusion in which three different zones of viral proteins are evident: a red (μ2) center, a yellow (ςNS and μ2) intermediate zone, and a narrow peripheral zone of green (ςNS). Images were processed using Adobe Photoshop. Bars, 25 μm.
    Figure Legend Snippet: Subcellular localization of reovirus ςNS and μ2 proteins in cells infected with reovirus strain T3D. L cells were infected with T3D at an MOI of 10 PFU per cell and incubated at 37°C for 18 h. Cells were stained for ςNS by using ςNS-specific MAb 2H7 (B) and for μ2 by using a μ2-specific polyclonal antiserum (C) as primary antibodies followed by Alexa Fluor 546 goat anti-mouse IgG and Alexa Fluor 488 goat anti-rabbit IgG, respectively, as secondary antibodies. Images were obtained by using a confocal microscope. The ςNS protein is colored green, and the μ2 protein is colored red. (A) A DIC image of the field was obtained. (D) In the merged image, colocalization of ςNS and μ2 is indicated by the yellow color. The arrow indicates a viral inclusion in which three different zones of viral proteins are evident: a red (μ2) center, a yellow (ςNS and μ2) intermediate zone, and a narrow peripheral zone of green (ςNS). Images were processed using Adobe Photoshop. Bars, 25 μm.

    Techniques Used: Infection, Incubation, Staining, Microscopy

    Subcellular localization of reovirus ςNS and μ1/μ1C proteins in cells infected with reovirus strain T3D. L cells were infected with T3D at an MOI of 10 PFU per cell and incubated at 37°C for 18 h. Cells were stained for ςNS by using a ςNS-specific polyclonal antiserum (B) and for μ1/μ1C by using μ1/μ1C-specific MAb 8H6 (C) as primary antibodies followed by Alexa Fluor 488 goat anti-rabbit IgG and Alexa Fluor 546 goat anti-mouse IgG, respectively, as secondary antibodies. Images were obtained by using a confocal microscope. The ςNS protein is colored green, and the μ1/μ1C protein is colored red. (A) A DIC image of the field was obtained. (D) In the merged image, colocalization of ςNS and μ1/μ1C is indicated by the yellow color. Images were processed using Adobe Photoshop. Bars, 25 μm.
    Figure Legend Snippet: Subcellular localization of reovirus ςNS and μ1/μ1C proteins in cells infected with reovirus strain T3D. L cells were infected with T3D at an MOI of 10 PFU per cell and incubated at 37°C for 18 h. Cells were stained for ςNS by using a ςNS-specific polyclonal antiserum (B) and for μ1/μ1C by using μ1/μ1C-specific MAb 8H6 (C) as primary antibodies followed by Alexa Fluor 488 goat anti-rabbit IgG and Alexa Fluor 546 goat anti-mouse IgG, respectively, as secondary antibodies. Images were obtained by using a confocal microscope. The ςNS protein is colored green, and the μ1/μ1C protein is colored red. (A) A DIC image of the field was obtained. (D) In the merged image, colocalization of ςNS and μ1/μ1C is indicated by the yellow color. Images were processed using Adobe Photoshop. Bars, 25 μm.

    Techniques Used: Infection, Incubation, Staining, Microscopy

    Stability of reovirus ςNS protein in cells infected with T3D or tsE320 at a nonpermissive temperature. L cells were either mock infected or infected with either T3D or tsE320 at an MOI of 10 PFU per cell and incubated at 39.5°C. At 6 h postinfection, cells were pulse-labeled with [ 35 S]methionine-[ 35 S]cysteine for 1 h and then incubated in the absence of radiolabeled methionine-cysteine for the time periods shown. The ςNS protein was immunoprecipitated from cell lysates by using polyclonal ςNS-specific antiserum, resolved by SDS-PAGE, visualized by autoradiography, and quantitated by phosphorimager analysis. (A) Representative autoradiogram. (B) Band densities corresponding to ςNS protein, quantitated with a phosphorimager and normalized to the 0-h time point. The results are presented as the mean relative protein units for three independent experiments. Error bars indicate standard deviations of the means.
    Figure Legend Snippet: Stability of reovirus ςNS protein in cells infected with T3D or tsE320 at a nonpermissive temperature. L cells were either mock infected or infected with either T3D or tsE320 at an MOI of 10 PFU per cell and incubated at 39.5°C. At 6 h postinfection, cells were pulse-labeled with [ 35 S]methionine-[ 35 S]cysteine for 1 h and then incubated in the absence of radiolabeled methionine-cysteine for the time periods shown. The ςNS protein was immunoprecipitated from cell lysates by using polyclonal ςNS-specific antiserum, resolved by SDS-PAGE, visualized by autoradiography, and quantitated by phosphorimager analysis. (A) Representative autoradiogram. (B) Band densities corresponding to ςNS protein, quantitated with a phosphorimager and normalized to the 0-h time point. The results are presented as the mean relative protein units for three independent experiments. Error bars indicate standard deviations of the means.

    Techniques Used: Infection, Incubation, Labeling, Immunoprecipitation, SDS Page, Autoradiography

    Subcellular localization of ςNS and reovirus proteins in cells infected with wt T3D or mutant tsE320 at a nonpermissive temperature. L cells were infected with either T3D (A to D) or tsE320 (E to H) at an MOI of 10 PFU per cell and incubated at 37°C (T3D) or 39.5°C ( tsE320 ) for 24 h. Cells were stained for ςNS by using ςNS-specific MAb 2H7 (B and F) and for reovirus proteins by using a polyclonal antiserum raised against T3D (C and G) as primary antibodies followed by Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 546 goat anti-rabbit IgG, respectively, as secondary antibodies. Images were obtained by using a confocal microscope. The ςNS protein is colored green, and the reovirus proteins are colored red. (A and E) A DIC image of each field was obtained. (D and H) In the merged images, colocalization of ςNS and reovirus proteins is indicated by the yellow color. Images were processed using Adobe Photoshop. Bars, 25 μm.
    Figure Legend Snippet: Subcellular localization of ςNS and reovirus proteins in cells infected with wt T3D or mutant tsE320 at a nonpermissive temperature. L cells were infected with either T3D (A to D) or tsE320 (E to H) at an MOI of 10 PFU per cell and incubated at 37°C (T3D) or 39.5°C ( tsE320 ) for 24 h. Cells were stained for ςNS by using ςNS-specific MAb 2H7 (B and F) and for reovirus proteins by using a polyclonal antiserum raised against T3D (C and G) as primary antibodies followed by Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 546 goat anti-rabbit IgG, respectively, as secondary antibodies. Images were obtained by using a confocal microscope. The ςNS protein is colored green, and the reovirus proteins are colored red. (A and E) A DIC image of each field was obtained. (D and H) In the merged images, colocalization of ςNS and reovirus proteins is indicated by the yellow color. Images were processed using Adobe Photoshop. Bars, 25 μm.

    Techniques Used: Infection, Mutagenesis, Incubation, Staining, Microscopy

    27) Product Images from "GATA-4 and Nkx-2.5 Coactivate Nkx-2 DNA Binding Targets: Role for Regulating Early Cardiac Gene Expression"

    Article Title: GATA-4 and Nkx-2.5 Coactivate Nkx-2 DNA Binding Targets: Role for Regulating Early Cardiac Gene Expression

    Journal: Molecular and Cellular Biology

    doi:

    Working model for activation of Nkx-2.5 by interaction with GATA-4. GATA-4 interacts with Nkx-2.5 and induces a conformational change resulting in increased affinity for the Nkx-2.5 binding site (NKE) and increased accessibility of the activation domain (+++). (A) Isolated GATA-4 protein; (B) isolated Nkx-2.5 protein. The model postulates that in the inactivated state the Nkx-2.5 homeodomain (HD) has a low affinity for NKE due to steric hindrance by the C-terminal domain, possibly via hydrophobic interactions with the N terminus of Nkx-2.5. In addition, it is possible that the C-terminal–N-terminal interactions keep the activation domain inaccessible to the transcriptional machinery. (C) Interaction with GATA-4 would then remove this negative effect of the C terminus on Nkx-2.5 transcriptional activity. (D) NKE binding by activated Nkx-2.5–GATA-4 complexes. It is also possible that GATA-4 leaves the complex before or after NKE binding (data not shown). ZF1 and ZF2, zinc fingers 1 and 2, respectively.
    Figure Legend Snippet: Working model for activation of Nkx-2.5 by interaction with GATA-4. GATA-4 interacts with Nkx-2.5 and induces a conformational change resulting in increased affinity for the Nkx-2.5 binding site (NKE) and increased accessibility of the activation domain (+++). (A) Isolated GATA-4 protein; (B) isolated Nkx-2.5 protein. The model postulates that in the inactivated state the Nkx-2.5 homeodomain (HD) has a low affinity for NKE due to steric hindrance by the C-terminal domain, possibly via hydrophobic interactions with the N terminus of Nkx-2.5. In addition, it is possible that the C-terminal–N-terminal interactions keep the activation domain inaccessible to the transcriptional machinery. (C) Interaction with GATA-4 would then remove this negative effect of the C terminus on Nkx-2.5 transcriptional activity. (D) NKE binding by activated Nkx-2.5–GATA-4 complexes. It is also possible that GATA-4 leaves the complex before or after NKE binding (data not shown). ZF1 and ZF2, zinc fingers 1 and 2, respectively.

    Techniques Used: Activation Assay, Binding Assay, Isolation, Activity Assay, Zinc-Fingers

    Physical interaction between Nkx-2.5 and GATA-4 is mediated by the C-terminal zinc finger (ZF2) of GATA-4. Ten microliters of reticulocyte lysate containing in vitro-translated, 35 S-labeled wild-type (Wt) GATA-4 (lanes 1 to 3), an N-terminally truncated GATA-4 mutant (ΔN) (lanes 4 to 6), GATA-4 with both zinc fingers (lanes 7 to 9), GATA-4 with only zinc finger 2 (lanes 10 to 12), or GATA-4 with only zinc finger 1 (ZF1) (lanes 13 to 15) was incubated with approximately 10 μg of MBP (lanes 2, 5, 8, 11, and 14) or MBP–Nkx-2.5 (lanes 3, 6, 9, 12, and 15). The beads were washed extensively, and the bound proteins were resolved on SDS–10% polyacrylamide gels and visualized by autoradiography. Lanes 1, 4, 7, 10, and 13 contained 10% of the in vitro translation volume used for binding reactions. After being washed, bound proteins were visualized by fluorography.
    Figure Legend Snippet: Physical interaction between Nkx-2.5 and GATA-4 is mediated by the C-terminal zinc finger (ZF2) of GATA-4. Ten microliters of reticulocyte lysate containing in vitro-translated, 35 S-labeled wild-type (Wt) GATA-4 (lanes 1 to 3), an N-terminally truncated GATA-4 mutant (ΔN) (lanes 4 to 6), GATA-4 with both zinc fingers (lanes 7 to 9), GATA-4 with only zinc finger 2 (lanes 10 to 12), or GATA-4 with only zinc finger 1 (ZF1) (lanes 13 to 15) was incubated with approximately 10 μg of MBP (lanes 2, 5, 8, 11, and 14) or MBP–Nkx-2.5 (lanes 3, 6, 9, 12, and 15). The beads were washed extensively, and the bound proteins were resolved on SDS–10% polyacrylamide gels and visualized by autoradiography. Lanes 1, 4, 7, 10, and 13 contained 10% of the in vitro translation volume used for binding reactions. After being washed, bound proteins were visualized by fluorography.

    Techniques Used: In Vitro, Labeling, Mutagenesis, Zinc-Fingers, Incubation, Autoradiography, Binding Assay

    GATA-4 facilitates DNA binding of full-length Nkx-2.5 but not the homeodomain. (A) End-labeled double-stranded A20 oligonucleotide probe was incubated with 10 or 25 ng of MBP–Nkx-2.5 homeodomain (HD), either with 250 ng of MBP–GATA-4 (lanes 3 and 7, respectively) or without MBP–GATA-4 (lanes 2 and 5, respectively), in 1× binding buffer. Lanes 4 and 6 contained only MBP–GATA-4 and A20 probe. The faint bands in lane 1 are due to spillover from lane 2. (B) MBP–Nkx-2.5 (10 or 25 ng of purified protein) was incubated with (lanes 7 and 8, respectively) or without (lanes 3 and 4, respectively) 250 ng of MBP–GATA-4 for 15 min at 30°C in 1× binding buffer. End-labeled double-stranded NKE (0.02 pmol) was added to the protein mixtures, which were subsequently incubated for an additional 15 min. Under identical conditions, 10 ng (lane 1), 25 ng (lane 2), or 250 ng (lane 5) of MBP and 250 ng of MBP–GATA-4 (lane 6) did not bind the labeled NKE probe. DNA-protein complexes are indicated by arrowheads. The gel in panel B was exposed overnight, and the one in panel A was exposed for 4 days at −70°C.
    Figure Legend Snippet: GATA-4 facilitates DNA binding of full-length Nkx-2.5 but not the homeodomain. (A) End-labeled double-stranded A20 oligonucleotide probe was incubated with 10 or 25 ng of MBP–Nkx-2.5 homeodomain (HD), either with 250 ng of MBP–GATA-4 (lanes 3 and 7, respectively) or without MBP–GATA-4 (lanes 2 and 5, respectively), in 1× binding buffer. Lanes 4 and 6 contained only MBP–GATA-4 and A20 probe. The faint bands in lane 1 are due to spillover from lane 2. (B) MBP–Nkx-2.5 (10 or 25 ng of purified protein) was incubated with (lanes 7 and 8, respectively) or without (lanes 3 and 4, respectively) 250 ng of MBP–GATA-4 for 15 min at 30°C in 1× binding buffer. End-labeled double-stranded NKE (0.02 pmol) was added to the protein mixtures, which were subsequently incubated for an additional 15 min. Under identical conditions, 10 ng (lane 1), 25 ng (lane 2), or 250 ng (lane 5) of MBP and 250 ng of MBP–GATA-4 (lane 6) did not bind the labeled NKE probe. DNA-protein complexes are indicated by arrowheads. The gel in panel B was exposed overnight, and the one in panel A was exposed for 4 days at −70°C.

    Techniques Used: Binding Assay, Labeling, Incubation, Purification

    Synergistic activation of the αCA promoter and the NKE multimerized A20(3) promoter by Nkx-2.5 and GATA-4. (A) CV-1 cells were transfected with luciferase reporter genes (1 μg of DNA) containing the αCA promoter, the −100-bp deletion mutant of αCA containing a serum response element (αCA–Del-100), and the simian virus 40 (SV40) early promoter. Cotransfectants contained CMV promoter-directed expression vectors driving Nkx-2.5 or GATA-4 (400 μg of DNA) or the empty vector pCG (1 μg of DNA). Luciferase activity was assayed 3 days after transfection. The bars represent the averages of data from two independent transfections, and the error bars represent the standard deviations of corrected luciferase activity relative to that of the pCG vector in a typical experiment. (B) Reporter vectors contained either 58 bp of the αCA promoter upstream of the transcriptional start site (aCA-58) or the A20 Nkx-2.5 binding site cloned in triplicate upstream of aCA-58 [A20(3)]. Nkx-2.5pm is a mutant of Nkx-2.5 with greatly decreased DNA binding affinity.
    Figure Legend Snippet: Synergistic activation of the αCA promoter and the NKE multimerized A20(3) promoter by Nkx-2.5 and GATA-4. (A) CV-1 cells were transfected with luciferase reporter genes (1 μg of DNA) containing the αCA promoter, the −100-bp deletion mutant of αCA containing a serum response element (αCA–Del-100), and the simian virus 40 (SV40) early promoter. Cotransfectants contained CMV promoter-directed expression vectors driving Nkx-2.5 or GATA-4 (400 μg of DNA) or the empty vector pCG (1 μg of DNA). Luciferase activity was assayed 3 days after transfection. The bars represent the averages of data from two independent transfections, and the error bars represent the standard deviations of corrected luciferase activity relative to that of the pCG vector in a typical experiment. (B) Reporter vectors contained either 58 bp of the αCA promoter upstream of the transcriptional start site (aCA-58) or the A20 Nkx-2.5 binding site cloned in triplicate upstream of aCA-58 [A20(3)]. Nkx-2.5pm is a mutant of Nkx-2.5 with greatly decreased DNA binding affinity.

    Techniques Used: Activation Assay, Transfection, Luciferase, Mutagenesis, Expressing, Plasmid Preparation, Activity Assay, Binding Assay, Clone Assay

    28) Product Images from "Abl Interactor 1 Binds to Sos and Inhibits Epidermal Growth Factor- and v-Abl-Induced Activation of Extracellular Signal-Regulated Kinases"

    Article Title: Abl Interactor 1 Binds to Sos and Inhibits Epidermal Growth Factor- and v-Abl-Induced Activation of Extracellular Signal-Regulated Kinases

    Journal: Molecular and Cellular Biology

    doi:

    Tyrosine phosphorylation of endogenous Abi-1 induced by v-Abl. (A) Serum-starved D5 cells were left at 39°C (nonpermissive temperature) or shifted to 32°C (permissive temperature) for 1 h. Abi-1 was immunoprecipitated from cell lysates and immunoblotted with antiphosphotyrosine antibody (αPtyr) (top). The membranes were reprobed with anti-Abi-1 antibodies to confirm equal levels of protein loading (bottom). The arrows indicate the position of Abi-1. N and P denote nonpermissive and permissive temperatures, respectively. WB, Western blot. (B) Growth factor-deprived parental BAF/3 cells and BAF/3 cells stably transfected with p160 v- abl were left untreated or were stimulated with IL-3 (50 ng/ml). Abi-1 was immunoprecipitated and immunoblotted as described for panel A. IP, immunoprecipitate.
    Figure Legend Snippet: Tyrosine phosphorylation of endogenous Abi-1 induced by v-Abl. (A) Serum-starved D5 cells were left at 39°C (nonpermissive temperature) or shifted to 32°C (permissive temperature) for 1 h. Abi-1 was immunoprecipitated from cell lysates and immunoblotted with antiphosphotyrosine antibody (αPtyr) (top). The membranes were reprobed with anti-Abi-1 antibodies to confirm equal levels of protein loading (bottom). The arrows indicate the position of Abi-1. N and P denote nonpermissive and permissive temperatures, respectively. WB, Western blot. (B) Growth factor-deprived parental BAF/3 cells and BAF/3 cells stably transfected with p160 v- abl were left untreated or were stimulated with IL-3 (50 ng/ml). Abi-1 was immunoprecipitated and immunoblotted as described for panel A. IP, immunoprecipitate.

    Techniques Used: Immunoprecipitation, Western Blot, Stable Transfection, Transfection

    In vitro binding of the Abi-1 SH3 domain to Sos. (A) MBP and MBP-Abi SH3 immobilized on amylose resin were incubated with BALB/c3T3 cell lysate. Bound proteins were immunoblotted with antibodies that recognize both Sos1 and Sos2 (αSos1/2) (top). The membrane was stripped and reprobed with anti-Sos1 (middle) and anti-Sos2 (bottom) antibodies. Total cell lysate and anti-Sos1 and -Sos2 immunoprecipitate (IP) from BALB/c3T3 cells were included for reference. WB, Western blot. (B) COS cells were transiently transfected with plasmids encoding HA-tagged DOKL, Abi(Δ1–85), or Sos2(874–1297) [Sos2(C)]. Cell lysates were incubated with MBP and MBP-Abi SH3 as described for panel A. Total cell lysates and bound proteins were probed with anti-HA antibody (upper). HA-Sos2(874–1297) expression in the total cell lysate was detected upon longer exposure. A fraction of the resin eluate was stained with Coomassie brilliant blue to confirm roughly equivalent amounts of MBP and MBP-Abi SH3 (lower).
    Figure Legend Snippet: In vitro binding of the Abi-1 SH3 domain to Sos. (A) MBP and MBP-Abi SH3 immobilized on amylose resin were incubated with BALB/c3T3 cell lysate. Bound proteins were immunoblotted with antibodies that recognize both Sos1 and Sos2 (αSos1/2) (top). The membrane was stripped and reprobed with anti-Sos1 (middle) and anti-Sos2 (bottom) antibodies. Total cell lysate and anti-Sos1 and -Sos2 immunoprecipitate (IP) from BALB/c3T3 cells were included for reference. WB, Western blot. (B) COS cells were transiently transfected with plasmids encoding HA-tagged DOKL, Abi(Δ1–85), or Sos2(874–1297) [Sos2(C)]. Cell lysates were incubated with MBP and MBP-Abi SH3 as described for panel A. Total cell lysates and bound proteins were probed with anti-HA antibody (upper). HA-Sos2(874–1297) expression in the total cell lysate was detected upon longer exposure. A fraction of the resin eluate was stained with Coomassie brilliant blue to confirm roughly equivalent amounts of MBP and MBP-Abi SH3 (lower).

    Techniques Used: In Vitro, Binding Assay, Incubation, Western Blot, Transfection, Expressing, Staining

    Interaction between Abi-1 and Sos in COS cells. (A) COS cells were transiently transfected with plasmids encoding myc-tagged GM-CSF receptor α chain, c-Abl, Abi-1, or AbiΔ328–356. Sos1 and Sos2 were immunoprecipitated from cell lysates. Precipitated proteins (top) were immunoblotted with anti-myc antibody (αmyc). The membrane from the top blot was reprobed with anti-Sos1 and -Sos2 antibodies to confirm equal levels of protein loading (middle). Total cell lysates were probed with anti-myc antibody to confirm expression of myc-tagged proteins (bottom). IP, immunoprecipitate; WB, Western blot. (B) Schematic representation of full-length (FL) Abi-1 and deletion mutant proteins used in panel C. The homeobox-like domain, proline-rich motifs, polyproline region, and SH3 domain are indicated. (C) COS cells were transiently transfected with plasmids encoding myc-tagged full-length Abi-1 or the indicated deletion mutant proteins. Proteins were immunoprecipitated from cell lysates with anti-Sos1 and -Sos2 antibodies and were analyzed as described for panel A. The asterisk indicates the position of the immunoglobulin heavy chain.
    Figure Legend Snippet: Interaction between Abi-1 and Sos in COS cells. (A) COS cells were transiently transfected with plasmids encoding myc-tagged GM-CSF receptor α chain, c-Abl, Abi-1, or AbiΔ328–356. Sos1 and Sos2 were immunoprecipitated from cell lysates. Precipitated proteins (top) were immunoblotted with anti-myc antibody (αmyc). The membrane from the top blot was reprobed with anti-Sos1 and -Sos2 antibodies to confirm equal levels of protein loading (middle). Total cell lysates were probed with anti-myc antibody to confirm expression of myc-tagged proteins (bottom). IP, immunoprecipitate; WB, Western blot. (B) Schematic representation of full-length (FL) Abi-1 and deletion mutant proteins used in panel C. The homeobox-like domain, proline-rich motifs, polyproline region, and SH3 domain are indicated. (C) COS cells were transiently transfected with plasmids encoding myc-tagged full-length Abi-1 or the indicated deletion mutant proteins. Proteins were immunoprecipitated from cell lysates with anti-Sos1 and -Sos2 antibodies and were analyzed as described for panel A. The asterisk indicates the position of the immunoglobulin heavy chain.

    Techniques Used: Transfection, Immunoprecipitation, Expressing, Western Blot, Mutagenesis

    Association of Abi-1 and Grb2 in COS cells. (A) COS cells were transiently cotransfected with the indicated HA-Sos1 and HA–Abi-1 plasmids. Grb2 was immunoprecipitated from cell lysates. Precipitated proteins (top) and total cell lysates (bottom) were immunoblotted with anti-HA antibody (αHA). The membrane from the top blot was reprobed with anti-Grb2 antibody to confirm equal levels of protein loading (middle). IP, immunoprecipitate; WB, Western blot. (B) COS cells were transiently cotransfected with the indicated HA-Sos1 and HA–Abi-1 plasmids. Serum-starved cells were left untreated or were stimulated with EGF (100 ng/ml) for 2 or 10 min. Cell lysates were prepared and incubated with anti-EGFR antibody. Precipitated proteins (top) and total cell lysates (bottom) were immunoblotted with anti-HA antibody. The membrane from the top blot was reprobed with anti-EGFR antibody to confirm equal levels of protein loading. FL, full-length. (C) COS cells were transiently transfected with the indicated myc–Abi-1 plasmids. Grb2 was immunoprecipitated from cell lysates. Precipitated proteins (top) and total cell lysates (bottom) were immunoblotted with anti-myc antibody. The membrane from the top blot was reprobed with anti-Grb2 antibody to confirm equal levels of protein loading (middle).
    Figure Legend Snippet: Association of Abi-1 and Grb2 in COS cells. (A) COS cells were transiently cotransfected with the indicated HA-Sos1 and HA–Abi-1 plasmids. Grb2 was immunoprecipitated from cell lysates. Precipitated proteins (top) and total cell lysates (bottom) were immunoblotted with anti-HA antibody (αHA). The membrane from the top blot was reprobed with anti-Grb2 antibody to confirm equal levels of protein loading (middle). IP, immunoprecipitate; WB, Western blot. (B) COS cells were transiently cotransfected with the indicated HA-Sos1 and HA–Abi-1 plasmids. Serum-starved cells were left untreated or were stimulated with EGF (100 ng/ml) for 2 or 10 min. Cell lysates were prepared and incubated with anti-EGFR antibody. Precipitated proteins (top) and total cell lysates (bottom) were immunoblotted with anti-HA antibody. The membrane from the top blot was reprobed with anti-EGFR antibody to confirm equal levels of protein loading. FL, full-length. (C) COS cells were transiently transfected with the indicated myc–Abi-1 plasmids. Grb2 was immunoprecipitated from cell lysates. Precipitated proteins (top) and total cell lysates (bottom) were immunoblotted with anti-myc antibody. The membrane from the top blot was reprobed with anti-Grb2 antibody to confirm equal levels of protein loading (middle).

    Techniques Used: Immunoprecipitation, Western Blot, Incubation, Transfection

    Tyrosine phosphorylation of endogenous Abi-1 induced by serum stimulation. Serum-starved BALB/c3T3 cells were stimulated with 20% FBS for the indicated times (in minutes). Abi-1 was immunoprecipitated from cell lysates with anti-Abi-1 antibodies (αAbi) and immunoblotted with antiphosphotyrosine antibody (top). The membrane was reprobed with anti-Abi-1 antibodies to confirm equal levels of protein loading (bottom). The arrows indicate the position of Abi-1. PI, preimmune serum control; IP, immunoprecipitate; WB, Western blot.
    Figure Legend Snippet: Tyrosine phosphorylation of endogenous Abi-1 induced by serum stimulation. Serum-starved BALB/c3T3 cells were stimulated with 20% FBS for the indicated times (in minutes). Abi-1 was immunoprecipitated from cell lysates with anti-Abi-1 antibodies (αAbi) and immunoblotted with antiphosphotyrosine antibody (top). The membrane was reprobed with anti-Abi-1 antibodies to confirm equal levels of protein loading (bottom). The arrows indicate the position of Abi-1. PI, preimmune serum control; IP, immunoprecipitate; WB, Western blot.

    Techniques Used: Immunoprecipitation, Western Blot

    Inhibition of v-Abl-induced Erk2 activation by Abi-1. 293T cells were transiently cotransfected with plasmids encoding HA-Erk2, wild-type (WT) p160 v- abl , kinase-dead (KD) p160 v- abl , or v-Src, and full-length HA–Abi-1 (FL) or HA–AbiΔ1–85 (Δ1–85). Transfected cells were serum starved in 0.2% FBS for 24 h before lysis. Cell lysates were probed with antibodies against active Erks (αActive Erk) (top), HA (middle), and Abl (bottom). Expression of v-Src was not detected by our methods. WB, Western blot.
    Figure Legend Snippet: Inhibition of v-Abl-induced Erk2 activation by Abi-1. 293T cells were transiently cotransfected with plasmids encoding HA-Erk2, wild-type (WT) p160 v- abl , kinase-dead (KD) p160 v- abl , or v-Src, and full-length HA–Abi-1 (FL) or HA–AbiΔ1–85 (Δ1–85). Transfected cells were serum starved in 0.2% FBS for 24 h before lysis. Cell lysates were probed with antibodies against active Erks (αActive Erk) (top), HA (middle), and Abl (bottom). Expression of v-Src was not detected by our methods. WB, Western blot.

    Techniques Used: Inhibition, Activation Assay, Transfection, Lysis, Expressing, Western Blot

    29) Product Images from "Reovirus Growth in Cell Culture Does Not Require the Full Complement of Viral Proteins: Identification of a ?1s-Null Mutant"

    Article Title: Reovirus Growth in Cell Culture Does Not Require the Full Complement of Viral Proteins: Identification of a ?1s-Null Mutant

    Journal: Journal of Virology

    doi:

    (A) Linear depiction of the S1 gene and its protein products, ς1 and ς1s. Arrows indicate point mutations found in the S1 gene segment of T3C84-MA. (B) Nucleotide sequences of the first nine codons of the ς1s ORF of T3C84 and T3C84-MA. Amino acids in the single-letter code are shown above the corresponding nucleotide sequences in the S1 gene. A stop codon is shown at codon 7 in the T3C84-MA ς1s sequence.
    Figure Legend Snippet: (A) Linear depiction of the S1 gene and its protein products, ς1 and ς1s. Arrows indicate point mutations found in the S1 gene segment of T3C84-MA. (B) Nucleotide sequences of the first nine codons of the ς1s ORF of T3C84 and T3C84-MA. Amino acids in the single-letter code are shown above the corresponding nucleotide sequences in the S1 gene. A stop codon is shown at codon 7 in the T3C84-MA ς1s sequence.

    Techniques Used: Sequencing

    (A) Schematic of full-length and truncated forms of ς1s expressed as fusion proteins with MBP. Sequences of the T3D ς1s ORF were cloned into the pMAL-c2 vector. MBP fusion proteins containing β-galactosidase, full-length ς1s (MBP-ς1s/1–120), or truncations of ς1s (MBP-ς1s/1–84 and MBP-ς1s/1–42) were expressed in E. coli and purified by affinity chromatography using an amylose resin. (B) Immunoblot of MBP-ς1s fusion proteins using MAbs 2F4 and 3E2. The upper gel shows the MBP-β-galactosidase fusion protein as a control and the three MBP-ς1s fusion proteins after electrophoresis in a 10% polyacrylamide gel and staining with Coomassie blue (1 μg of protein per lane). The lower two gels are immunoblots of the same four proteins (20 ng of protein per lane) using MAbs 2F4 and 3E2 (5 μg per ml). Molecular size markers are given in kilodaltons.
    Figure Legend Snippet: (A) Schematic of full-length and truncated forms of ς1s expressed as fusion proteins with MBP. Sequences of the T3D ς1s ORF were cloned into the pMAL-c2 vector. MBP fusion proteins containing β-galactosidase, full-length ς1s (MBP-ς1s/1–120), or truncations of ς1s (MBP-ς1s/1–84 and MBP-ς1s/1–42) were expressed in E. coli and purified by affinity chromatography using an amylose resin. (B) Immunoblot of MBP-ς1s fusion proteins using MAbs 2F4 and 3E2. The upper gel shows the MBP-β-galactosidase fusion protein as a control and the three MBP-ς1s fusion proteins after electrophoresis in a 10% polyacrylamide gel and staining with Coomassie blue (1 μg of protein per lane). The lower two gels are immunoblots of the same four proteins (20 ng of protein per lane) using MAbs 2F4 and 3E2 (5 μg per ml). Molecular size markers are given in kilodaltons.

    Techniques Used: Clone Assay, Plasmid Preparation, Purification, Affinity Chromatography, Electrophoresis, Staining, Western Blot

    Time course of ς1s and μ1/μ1C expression during reovirus infection of L cells. Cells were infected with either T3D, T3C84, or T3C84-MA at an MOI of 10 PFU per cell and harvested at the time points indicated. Cytoplasmic extracts were prepared and electrophoresed in a 14% polyacrylamide gel (100 μg of protein per lane), transferred to nitrocellulose, and blotted with either anti-ς1s MAb 2F4 (5 μg per ml) or anti-μ1/μ1C MAb 8H6 (5 μg per ml).
    Figure Legend Snippet: Time course of ς1s and μ1/μ1C expression during reovirus infection of L cells. Cells were infected with either T3D, T3C84, or T3C84-MA at an MOI of 10 PFU per cell and harvested at the time points indicated. Cytoplasmic extracts were prepared and electrophoresed in a 14% polyacrylamide gel (100 μg of protein per lane), transferred to nitrocellulose, and blotted with either anti-ς1s MAb 2F4 (5 μg per ml) or anti-μ1/μ1C MAb 8H6 (5 μg per ml).

    Techniques Used: Expressing, Infection

    Detection of ς1s and ςNS expression in reovirus-infected L cells by immunofluorescence staining. Cells grown on glass coverslips were mock-infected or infected with reovirus strains T3D, T3C84, or T3C84-MA at an MOI of 10 PFU per cell and fixed in methanol-acetone 12 h postinfection. Cells were incubated with anti-ς1s MAb 2F4 (50 μg per ml), followed by biotinylated goat anti-mouse IgG2a (1:1,000). Cells then were incubated with streptavidin-conjugated Cy2 and anti-ςNS MAb 2H7 cross-linked to Cy3 (10 μg per ml). Green fluorescence indicates ς1s; red fluorescence indicates ςNS. Immunofluorescence was visualized with a Zeiss LSM 410 confocal microscope.
    Figure Legend Snippet: Detection of ς1s and ςNS expression in reovirus-infected L cells by immunofluorescence staining. Cells grown on glass coverslips were mock-infected or infected with reovirus strains T3D, T3C84, or T3C84-MA at an MOI of 10 PFU per cell and fixed in methanol-acetone 12 h postinfection. Cells were incubated with anti-ς1s MAb 2F4 (50 μg per ml), followed by biotinylated goat anti-mouse IgG2a (1:1,000). Cells then were incubated with streptavidin-conjugated Cy2 and anti-ςNS MAb 2H7 cross-linked to Cy3 (10 μg per ml). Green fluorescence indicates ς1s; red fluorescence indicates ςNS. Immunofluorescence was visualized with a Zeiss LSM 410 confocal microscope.

    Techniques Used: Expressing, Infection, Immunofluorescence, Staining, Incubation, Fluorescence, Microscopy

    30) Product Images from "The LIM-Only Protein PINCH Directly Interacts with Integrin-Linked Kinase and Is Recruited to Integrin-Rich Sites in Spreading Cells"

    Article Title: The LIM-Only Protein PINCH Directly Interacts with Integrin-Linked Kinase and Is Recruited to Integrin-Rich Sites in Spreading Cells

    Journal: Molecular and Cellular Biology

    doi:

    Subcellular localization of PINCH in cells spreading on fibronectin. (A) Immunoblotting with monoclonal antibody 25.9. Lane 1, CHO cell lysate (10 μg/lane); lane 2, MBP-PINCH (0.5 μg/lane); lane 3, MBP (0.5 μg/lane); lanes 4 and 8, His fusion protein containing PINCH LIM1 and LIM2 domains (residues 1 to 129); lanes 5 and 9, His fusion protein containing PINCH LIM1, LIM2, and LIM3 domains (residues 1 to 187); lanes 6 and 10, His fusion protein containing PINCH LIM1, LIM2, LIM3, and LIM4 domains (residues 1 to 248); lanes 7 and 11, His fusion protein containing PINCH LIM4 and LIM5 domains (residues 188 to 314). Lanes 4 through 7 were loaded at 0.1 μg/lane, and lanes 8 through 11 were loaded at 0.5 μg/lane. Lanes 1 through 7 were blotted with monoclonal antibody 25.9, and lanes 8 through 11 were stained with Coomassie blue. (B to E) Immunofluorescence staining of cells spreading on fibronectin. Rat mesangial cells were plated on fibronectin for 1 (B and C) or 4 (D and E) h, fixed, and double stained with mouse monoclonal anti-PINCH antibody (B and D) and rabbit anti-α5β1 integrin antibody (C and E) as described in Materials and Methods. The bar in panel C is 5 μm and applies to panels B through E.
    Figure Legend Snippet: Subcellular localization of PINCH in cells spreading on fibronectin. (A) Immunoblotting with monoclonal antibody 25.9. Lane 1, CHO cell lysate (10 μg/lane); lane 2, MBP-PINCH (0.5 μg/lane); lane 3, MBP (0.5 μg/lane); lanes 4 and 8, His fusion protein containing PINCH LIM1 and LIM2 domains (residues 1 to 129); lanes 5 and 9, His fusion protein containing PINCH LIM1, LIM2, and LIM3 domains (residues 1 to 187); lanes 6 and 10, His fusion protein containing PINCH LIM1, LIM2, LIM3, and LIM4 domains (residues 1 to 248); lanes 7 and 11, His fusion protein containing PINCH LIM4 and LIM5 domains (residues 188 to 314). Lanes 4 through 7 were loaded at 0.1 μg/lane, and lanes 8 through 11 were loaded at 0.5 μg/lane. Lanes 1 through 7 were blotted with monoclonal antibody 25.9, and lanes 8 through 11 were stained with Coomassie blue. (B to E) Immunofluorescence staining of cells spreading on fibronectin. Rat mesangial cells were plated on fibronectin for 1 (B and C) or 4 (D and E) h, fixed, and double stained with mouse monoclonal anti-PINCH antibody (B and D) and rabbit anti-α5β1 integrin antibody (C and E) as described in Materials and Methods. The bar in panel C is 5 μm and applies to panels B through E.

    Techniques Used: Staining, Immunofluorescence

    31) Product Images from "Arabidopsis INOSITOL TRANSPORTER2 Mediates H+ Symport of Different Inositol Epimers and Derivatives across the Plasma Membrane 1 Symport of Different Inositol Epimers and Derivatives across the Plasma Membrane 1 [C] Symport of Different Inositol Epimers and Derivatives across the Plasma Membrane 1 [C] [OA]"

    Article Title: Arabidopsis INOSITOL TRANSPORTER2 Mediates H+ Symport of Different Inositol Epimers and Derivatives across the Plasma Membrane 1 Symport of Different Inositol Epimers and Derivatives across the Plasma Membrane 1 [C] Symport of Different Inositol Epimers and Derivatives across the Plasma Membrane 1 [C] [OA]

    Journal: Plant Physiology

    doi: 10.1104/pp.107.109033

    Immunohistochemical detection of recombinant AtINT2 protein in western blots of yeast total membranes and in thin sections of AtINT2 -expressing yeast cells. A, Unpurified α AtINT2 (diluted 1:200 or 1:2,000) that had been raised against 26 amino acids from the AtINT2 C terminus labeled a 55-kD band in detergent extracts from yeast total membranes after gel electrophoresis and blotting to nitrocellulose filters (10 μ g lane −1 ; AtINT2 = AtINT2 -expressing cells; C = control cells). B, Incubation of thin sections with affinity-purified α AtINT2 and fluorescence-tagged second antibody yielded fluorescence only in AtINT2 -expressing cells (SSY39) but not in control cells (SSY40). Bars are 2 μ m. [See online article for color version of this figure.]
    Figure Legend Snippet: Immunohistochemical detection of recombinant AtINT2 protein in western blots of yeast total membranes and in thin sections of AtINT2 -expressing yeast cells. A, Unpurified α AtINT2 (diluted 1:200 or 1:2,000) that had been raised against 26 amino acids from the AtINT2 C terminus labeled a 55-kD band in detergent extracts from yeast total membranes after gel electrophoresis and blotting to nitrocellulose filters (10 μ g lane −1 ; AtINT2 = AtINT2 -expressing cells; C = control cells). B, Incubation of thin sections with affinity-purified α AtINT2 and fluorescence-tagged second antibody yielded fluorescence only in AtINT2 -expressing cells (SSY39) but not in control cells (SSY40). Bars are 2 μ m. [See online article for color version of this figure.]

    Techniques Used: Immunohistochemistry, Recombinant, Western Blot, Expressing, Labeling, Nucleic Acid Electrophoresis, Incubation, Affinity Purification, Fluorescence

    32) Product Images from "Arabidopsis Inositol Polyphosphate 6-/3-Kinase Is a Nuclear Protein That Complements a Yeast Mutant Lacking a Functional ArgR-Mcm1 Transcription Complex"

    Article Title: Arabidopsis Inositol Polyphosphate 6-/3-Kinase Is a Nuclear Protein That Complements a Yeast Mutant Lacking a Functional ArgR-Mcm1 Transcription Complex

    Journal: The Plant Cell

    doi: 10.1105/tpc.006676

    Protein Gel Blot Analysis of the MBP-AtIpk2β Fusion Protein. E. coli cells were transformed with either the empty vector pMAL-c2 (lane C) or plasmid pMAL-c2–AtIpk2β (lanes 1 and 2). Protein extracts were obtained from noninduced (−) or IPTG-induced (+) cells. Proteins (45 μg per lane) were detected using an antiserum that recognizes the MBP portion of the proteins. The positions of MBP (42 kD) and the MBP-AtIpk2β fusion protein (75 kD) are indicated by arrows.
    Figure Legend Snippet: Protein Gel Blot Analysis of the MBP-AtIpk2β Fusion Protein. E. coli cells were transformed with either the empty vector pMAL-c2 (lane C) or plasmid pMAL-c2–AtIpk2β (lanes 1 and 2). Protein extracts were obtained from noninduced (−) or IPTG-induced (+) cells. Proteins (45 μg per lane) were detected using an antiserum that recognizes the MBP portion of the proteins. The positions of MBP (42 kD) and the MBP-AtIpk2β fusion protein (75 kD) are indicated by arrows.

    Techniques Used: Western Blot, Transformation Assay, Plasmid Preparation

    33) Product Images from "A Hippo-like signalling pathway controls tracheal morphogenesis in Drosophila melanogaster"

    Article Title: A Hippo-like signalling pathway controls tracheal morphogenesis in Drosophila melanogaster

    Journal: Developmental cell

    doi: 10.1016/j.devcel.2018.09.024

    The Tricornered kinase is phosphorylated on its hydrophobic motif by GckIII, and regulates tracheal development. (A) Alignment of the hydrophobic motif of D. melanogaster (dm) and human (hs) NDR kinase sequences. Identical residues are highlighted in yellow. The positions of the regulatory hydrophobic motif phosphorylation sites are indicated. The hydrophobic motif phosphorylation site of Trc/NDR kinases is conserved from flies to humans. (B) Lysates of S2R+ cells transiently expressing wild-type (wt) or kinase-dead (kd) HA-GckIII were immunoprecipitated with anti-HA antibodies and incubated with a recombinant Mal-tagged hydrophobic motif fragment of Trc [Mal-Trc(HM)] in kinase assays. Subsequently, samples were examined by immunoblotting using the indicated antibodies. Relative molecular masses are shown in kDa for each blot. (C) Recombinant GST-tagged wild-type full-length MST kinases (MST [1], MST2 [2], MST3 [3], MST4 [4] or STK25 [25], with respective abbreviations in square brackets) were incubated with recombinant Mal-tagged hydrophobic motif fragments of human NDR1 or NDR2 [Mal-NDR1/2 (HM)]. Following kinase reactions, the samples were analysed by Western blotting using the indicated antibodies. Relative molecular masses are shown in kDa for each blot. (D-D”) A trc 1 terminal cell clone marked by GFP (green). Brightfield (BF) images are shown in (D’) and the merged image (D”). The * indicates a prominent transition zone dilation. The terminal cell also exhibits a gas-filling defect and so is outlined with a white dashed line in (C’ and C”). (E-E”) Terminal cell expressing a trc-RNAi ( trcRi ) transgene using btl-GAL4 (marked by GFP) and stained for Fas3 (red). Large dilations (*) in the transition zone were readily detected, and ectopic Fas3 is present in the transition zone dilation (arrowheads) of Tao-1 eta terminal cell clones and more distally as well (arrowheads). (F-F”) Terminal cells marked with GFP (green) expressing a trc-RNAi ( trcRi ) transgene using drm-GAL4 and stained for Coracle (magenta) displayed ectopic localization of Coracle in the transition zone (arrowheads) and distally (data not shown). Arrows mark intercellular junction. (G-G”) Terminal cell (tubes visualised with UV) expressing a trc-RNAi ( trcRi ) transgene using drm-GAL4 with Crumbs-GFP almost continuously lined the lumenal membrane. (H-H”) A trc 1 terminal cell clone [marked by GFP (green)] expressing a hyperactive GckIII transgene. A brightfield (BF) image is shown in (H’) and the merged image (H”). The * indicates a transition zone tube dilation; expression of hyperactive GckIII failed to rescue transition zone dilations in trc 1 cells. Scale bar = 20 μm in ( D-H ). .
    Figure Legend Snippet: The Tricornered kinase is phosphorylated on its hydrophobic motif by GckIII, and regulates tracheal development. (A) Alignment of the hydrophobic motif of D. melanogaster (dm) and human (hs) NDR kinase sequences. Identical residues are highlighted in yellow. The positions of the regulatory hydrophobic motif phosphorylation sites are indicated. The hydrophobic motif phosphorylation site of Trc/NDR kinases is conserved from flies to humans. (B) Lysates of S2R+ cells transiently expressing wild-type (wt) or kinase-dead (kd) HA-GckIII were immunoprecipitated with anti-HA antibodies and incubated with a recombinant Mal-tagged hydrophobic motif fragment of Trc [Mal-Trc(HM)] in kinase assays. Subsequently, samples were examined by immunoblotting using the indicated antibodies. Relative molecular masses are shown in kDa for each blot. (C) Recombinant GST-tagged wild-type full-length MST kinases (MST [1], MST2 [2], MST3 [3], MST4 [4] or STK25 [25], with respective abbreviations in square brackets) were incubated with recombinant Mal-tagged hydrophobic motif fragments of human NDR1 or NDR2 [Mal-NDR1/2 (HM)]. Following kinase reactions, the samples were analysed by Western blotting using the indicated antibodies. Relative molecular masses are shown in kDa for each blot. (D-D”) A trc 1 terminal cell clone marked by GFP (green). Brightfield (BF) images are shown in (D’) and the merged image (D”). The * indicates a prominent transition zone dilation. The terminal cell also exhibits a gas-filling defect and so is outlined with a white dashed line in (C’ and C”). (E-E”) Terminal cell expressing a trc-RNAi ( trcRi ) transgene using btl-GAL4 (marked by GFP) and stained for Fas3 (red). Large dilations (*) in the transition zone were readily detected, and ectopic Fas3 is present in the transition zone dilation (arrowheads) of Tao-1 eta terminal cell clones and more distally as well (arrowheads). (F-F”) Terminal cells marked with GFP (green) expressing a trc-RNAi ( trcRi ) transgene using drm-GAL4 and stained for Coracle (magenta) displayed ectopic localization of Coracle in the transition zone (arrowheads) and distally (data not shown). Arrows mark intercellular junction. (G-G”) Terminal cell (tubes visualised with UV) expressing a trc-RNAi ( trcRi ) transgene using drm-GAL4 with Crumbs-GFP almost continuously lined the lumenal membrane. (H-H”) A trc 1 terminal cell clone [marked by GFP (green)] expressing a hyperactive GckIII transgene. A brightfield (BF) image is shown in (H’) and the merged image (H”). The * indicates a transition zone tube dilation; expression of hyperactive GckIII failed to rescue transition zone dilations in trc 1 cells. Scale bar = 20 μm in ( D-H ). .

    Techniques Used: Expressing, Immunoprecipitation, Incubation, Recombinant, Microscale Thermophoresis, Western Blot, Staining, Clone Assay

    34) Product Images from "Bifurcation of Arabidopsis NLR Immune Signaling via Ca2+-Dependent Protein Kinases"

    Article Title: Bifurcation of Arabidopsis NLR Immune Signaling via Ca2+-Dependent Protein Kinases

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003127

    CPKs phosphorylate WRKYs and RBOHs. ( A ) Phosphorylation of WRKYs by CPK5 in vitro . MBP-WRKY fusion proteins were used as the substrates for GST-CPK5 in an in vitro kinase assay in the presence of 1 mM Ca 2+ . Phosphorylation was analyzed by autoradiography (top panel), and the protein loading was shown by Coomassie blue staining (CBS) (bottom panel). 5 m is a kinase-dead mutant of CPK5. ( B ) Phosphorylation of WRKYs by CPK11 in vitro . 11 m is a kinase-dead mutant of CPK11. ( C ) Phosphorylation of WRKY DNA binding domains by different CPKs in vitro . ( D ) T248 is required for WRKY48 DNA binding domain phosphorylation by CPKs in vitro . ( E ) WRKY48 T248 is phosphorylated by CPKs with MS analysis. Sequencing of a doubly charged peptide ion at m/z 531.22 that matches to CTpTVGCGVK of WRKY48. The confident b2 and b3 ions as well as y7 ion provide strong evidence for phosphorylation of the third Thr residue. ( F ) CPKacs phosphorylated RBOHD and RBOHF with an immunocomplex kinase assay. The FLAG-tagged CPKacs or the kinase-dead mutants (m) were expressed in protoplasts, and immunoprecipitated with an α-FLAG antibody for an in vitro kinase assay using GST-RBOHD or GST-RBOHF as a substrate. The proteins of RBOHD and RBOHF were shown, and the expression of individual CPKacs was detected by Western blot (bottom panel). ( G ) S148 is an essential phosphorylation site of RBOHD by CPKs in vitro . * indicates phosphorylated RBOHD. The numbers below indicate the relative phosphorylation level compared to WT RBOHD (set as 1) as quantified by Image J. The above experiments were repeated three times with similar results. The MS analysis was repeated twice.
    Figure Legend Snippet: CPKs phosphorylate WRKYs and RBOHs. ( A ) Phosphorylation of WRKYs by CPK5 in vitro . MBP-WRKY fusion proteins were used as the substrates for GST-CPK5 in an in vitro kinase assay in the presence of 1 mM Ca 2+ . Phosphorylation was analyzed by autoradiography (top panel), and the protein loading was shown by Coomassie blue staining (CBS) (bottom panel). 5 m is a kinase-dead mutant of CPK5. ( B ) Phosphorylation of WRKYs by CPK11 in vitro . 11 m is a kinase-dead mutant of CPK11. ( C ) Phosphorylation of WRKY DNA binding domains by different CPKs in vitro . ( D ) T248 is required for WRKY48 DNA binding domain phosphorylation by CPKs in vitro . ( E ) WRKY48 T248 is phosphorylated by CPKs with MS analysis. Sequencing of a doubly charged peptide ion at m/z 531.22 that matches to CTpTVGCGVK of WRKY48. The confident b2 and b3 ions as well as y7 ion provide strong evidence for phosphorylation of the third Thr residue. ( F ) CPKacs phosphorylated RBOHD and RBOHF with an immunocomplex kinase assay. The FLAG-tagged CPKacs or the kinase-dead mutants (m) were expressed in protoplasts, and immunoprecipitated with an α-FLAG antibody for an in vitro kinase assay using GST-RBOHD or GST-RBOHF as a substrate. The proteins of RBOHD and RBOHF were shown, and the expression of individual CPKacs was detected by Western blot (bottom panel). ( G ) S148 is an essential phosphorylation site of RBOHD by CPKs in vitro . * indicates phosphorylated RBOHD. The numbers below indicate the relative phosphorylation level compared to WT RBOHD (set as 1) as quantified by Image J. The above experiments were repeated three times with similar results. The MS analysis was repeated twice.

    Techniques Used: In Vitro, Kinase Assay, Autoradiography, Staining, Mutagenesis, Binding Assay, Mass Spectrometry, Sequencing, Immunoprecipitation, Expressing, Western Blot

    Synergism of CPKs and WRKYs on WRKY46 promoter activity. ( A ) Requirement of W-boxes for WRKY46 promoter activity in protoplasts. The WT or mutant WRKY46 promoter was co-transfected with avrRpt2 or a vector control. The scheme represents the positions of four W-boxes in the WRKY46 promoter. ( B ) Functional genomic screen of WRKYs in protoplasts. The representative WRKY from different groups were co-transfected with CPKac5 for the activation of WRKY46 promoter. The bottom panel shows the expression of individual HA epitope-tagged WRKYs detected by Western blot. ( C ) Synergistic activation of WRKY46 promoter by WRKY48 and specific CPKacs in protoplasts. ( D ) Synergistic activation of WRKY46 promoter by WRKY28 and specific CPKacs in protoplasts. “m” indicates the kinase-dead mutants of CPKacs. ( E ) Synergistic activation of WRKY46 promoter by WRKY8 and specific CPKacs in protoplasts. “m” indicates the kinase-dead mutants of CPKacs. The above experiments were repeated three times with similar results.
    Figure Legend Snippet: Synergism of CPKs and WRKYs on WRKY46 promoter activity. ( A ) Requirement of W-boxes for WRKY46 promoter activity in protoplasts. The WT or mutant WRKY46 promoter was co-transfected with avrRpt2 or a vector control. The scheme represents the positions of four W-boxes in the WRKY46 promoter. ( B ) Functional genomic screen of WRKYs in protoplasts. The representative WRKY from different groups were co-transfected with CPKac5 for the activation of WRKY46 promoter. The bottom panel shows the expression of individual HA epitope-tagged WRKYs detected by Western blot. ( C ) Synergistic activation of WRKY46 promoter by WRKY48 and specific CPKacs in protoplasts. ( D ) Synergistic activation of WRKY46 promoter by WRKY28 and specific CPKacs in protoplasts. “m” indicates the kinase-dead mutants of CPKacs. ( E ) Synergistic activation of WRKY46 promoter by WRKY8 and specific CPKacs in protoplasts. “m” indicates the kinase-dead mutants of CPKacs. The above experiments were repeated three times with similar results.

    Techniques Used: Activity Assay, Mutagenesis, Transfection, Plasmid Preparation, Functional Assay, Activation Assay, Expressing, Western Blot

    CPKs enhance WRKY binding to the W-boxes. ( A ) Subcellular localization of CPK5 in protoplasts. CPK5-GFP was co-transfected with avrRpt2 or a vector control, and CPK5-GFP localization was observed with a confocal microscope 12 hpt. The nucleus was indicated with a co-transfected nuclear-localized RFP. Bar = 50 µm. ( B ) Subcellular fractionation of CPK5 in protoplasts. CPK5-HA was co-transfected with avrRpt2 or a vector control. Total protein extracts (T) were separated into nuclear (N) and soluble (S) fractions. CPK5 expression was detected by Western blot with an α-HA antibody. The purity of the nuclear and soluble fractions was demonstrated with α-Histone H3 antibody and CBS for RuBisCO (RBC). ( C ) T248 was required for WRKY48 synergistic activation with CPKs on WRKY46 promoter in protoplasts. The protein expression of WRKY48 and its T248A mutant was shown in the insert. ( D ) CPK5 enhanced WRKY48 binding to the W-boxes in vitro . The recombinant WRKY48 protein was incubated with 32 P-labeled W-boxes or mutated W-boxes (mW-boxes) probe in a gel mobility shift assay. CPK phosphorylation of WRKY48 was performed prior to DNA binding assay. ( E ) WRKY48 bound to the endogenous WRKY46 promoter regions enriched with W-boxes in protoplasts. Fragment A to F were ChIP-PCRed with primers across WRKY46 promoter and gene body. W1 to W4 indicate the positions of W-boxes corresponding to Figure 3A . CAB1 is a control gene. +1 is the transcriptional start site. Data are shown as mean ± SD. The input control for each primer pair was shown on the bottom. ( F ) In vitro pull down of WRKYs and CPK5. MBP was the control for MBP-fused WRKY proteins with a HA tag. GST was the control for GST-fused CPK5 proteins. MBP-WRKY48-HA, MBP-WRKY8-HA or MBP proteins were incubated with GST or GST-CPK5 beads, and the beads were collected and washed for Western blot of immunoprecipitated proteins with an α-HA antibody. The above experiments were repeated three times with similar results.
    Figure Legend Snippet: CPKs enhance WRKY binding to the W-boxes. ( A ) Subcellular localization of CPK5 in protoplasts. CPK5-GFP was co-transfected with avrRpt2 or a vector control, and CPK5-GFP localization was observed with a confocal microscope 12 hpt. The nucleus was indicated with a co-transfected nuclear-localized RFP. Bar = 50 µm. ( B ) Subcellular fractionation of CPK5 in protoplasts. CPK5-HA was co-transfected with avrRpt2 or a vector control. Total protein extracts (T) were separated into nuclear (N) and soluble (S) fractions. CPK5 expression was detected by Western blot with an α-HA antibody. The purity of the nuclear and soluble fractions was demonstrated with α-Histone H3 antibody and CBS for RuBisCO (RBC). ( C ) T248 was required for WRKY48 synergistic activation with CPKs on WRKY46 promoter in protoplasts. The protein expression of WRKY48 and its T248A mutant was shown in the insert. ( D ) CPK5 enhanced WRKY48 binding to the W-boxes in vitro . The recombinant WRKY48 protein was incubated with 32 P-labeled W-boxes or mutated W-boxes (mW-boxes) probe in a gel mobility shift assay. CPK phosphorylation of WRKY48 was performed prior to DNA binding assay. ( E ) WRKY48 bound to the endogenous WRKY46 promoter regions enriched with W-boxes in protoplasts. Fragment A to F were ChIP-PCRed with primers across WRKY46 promoter and gene body. W1 to W4 indicate the positions of W-boxes corresponding to Figure 3A . CAB1 is a control gene. +1 is the transcriptional start site. Data are shown as mean ± SD. The input control for each primer pair was shown on the bottom. ( F ) In vitro pull down of WRKYs and CPK5. MBP was the control for MBP-fused WRKY proteins with a HA tag. GST was the control for GST-fused CPK5 proteins. MBP-WRKY48-HA, MBP-WRKY8-HA or MBP proteins were incubated with GST or GST-CPK5 beads, and the beads were collected and washed for Western blot of immunoprecipitated proteins with an α-HA antibody. The above experiments were repeated three times with similar results.

    Techniques Used: Binding Assay, Transfection, Plasmid Preparation, Microscopy, Fractionation, Expressing, Western Blot, Activation Assay, Mutagenesis, In Vitro, Recombinant, Incubation, Labeling, Mobility Shift, DNA Binding Assay, Chromatin Immunoprecipitation, Immunoprecipitation

    35) Product Images from "Antagonistic Rgg Regulators Mediate Quorum Sensing via Competitive DNA Binding in Streptococcus pyogenes"

    Article Title: Antagonistic Rgg Regulators Mediate Quorum Sensing via Competitive DNA Binding in Streptococcus pyogenes

    Journal: mBio

    doi: 10.1128/mBio.00333-12

    (A) Co-EMSA analysis of competitive DNA binding by MBP-Rgg2 and Rgg3 using various protein concentrations. (B) Co-EMSA analysis of competitive DNA binding by Rgg proteins in the presence of pure ( > 95%) sSHP-C8 peptides. sSHP3-revC8 was included as a control. All reaction mixtures contained 10 nM Pshp3 probe.
    Figure Legend Snippet: (A) Co-EMSA analysis of competitive DNA binding by MBP-Rgg2 and Rgg3 using various protein concentrations. (B) Co-EMSA analysis of competitive DNA binding by Rgg proteins in the presence of pure ( > 95%) sSHP-C8 peptides. sSHP3-revC8 was included as a control. All reaction mixtures contained 10 nM Pshp3 probe.

    Techniques Used: Binding Assay

    36) Product Images from "Control of cotton fibre elongation by a homeodomain transcription factor GhHOX3"

    Article Title: Control of cotton fibre elongation by a homeodomain transcription factor GhHOX3

    Journal: Nature Communications

    doi: 10.1038/ncomms6519

    The DELLA protein GhSLR1 binds to GhHOX3 and interferes with the GhHOX3–GhHD1 interaction. ( a ) Yeast two-hybrid assay. pGAD-GhHD1 combined with pGBK-GhHOX3 conferred yeast growth on SD/-Leu/-Trp/-His plates supplemented with 10 mM 3-amino-1,2,4-triazole (3-AT). ( b ) BiFc assay. GhHOX3 and GhHD1 were interchangeably fused to the carboxyl- and amino-terminal of firefly luciferase (LUC, LUCc and LUCn), transiently co-expressed, and LUCc or LUCn was co-expressed with each other or with each un-fused target protein as the control. Fluorescence signal intensities represent their binding activities. Top bar, heat map’s scale of the signal intensity. GhHOX3 interacted with GhHD1. Scale bar, 1 cm in b. ( c ) Coimmunoprecipition (CoIP) of transiently co-expressed cMyc-GhHOX3 and HA-GhHD1 in leaves of Nicotiana benthamiana . Soluble protein extracts before (input) and after (IP) immunoprecipitation with anti-cMyc antibody-conjugated beads were detected by immunoblot with anti-HA antibody. ( d ) Yeast two-hybrid assay. GhHOX3, but not GhHD1, bound to GhSLR1 at 10 mM 3-AT. ( e , f ) In vivo BiFc ( e ) and CoIP ( f ) assays. GhHOX3 interacted with GhSLR1. Scale bar, 1 cm in e . ( g ) Yeast three-hybrid assay showing the influence of GhSLR1 on GhHOX3–GhHD1 binding represented by β-galactosidase activity, and the GhSLR1 expression was suppressed by increasing Met concentrations (data are shown as mean±s.e.m., n =3). ( h ) Domain deletion assay. Top, GhHOX3 contains three conserved domains. Below, yeast two-hybrid detection. GhHOX3 fragment containing both the Leu-zipper (LZ) and the START domains interacted with both GhHD1 and GhSLR1 in yeast, whereas the GhHOX3 homeodomain (HD) did not.
    Figure Legend Snippet: The DELLA protein GhSLR1 binds to GhHOX3 and interferes with the GhHOX3–GhHD1 interaction. ( a ) Yeast two-hybrid assay. pGAD-GhHD1 combined with pGBK-GhHOX3 conferred yeast growth on SD/-Leu/-Trp/-His plates supplemented with 10 mM 3-amino-1,2,4-triazole (3-AT). ( b ) BiFc assay. GhHOX3 and GhHD1 were interchangeably fused to the carboxyl- and amino-terminal of firefly luciferase (LUC, LUCc and LUCn), transiently co-expressed, and LUCc or LUCn was co-expressed with each other or with each un-fused target protein as the control. Fluorescence signal intensities represent their binding activities. Top bar, heat map’s scale of the signal intensity. GhHOX3 interacted with GhHD1. Scale bar, 1 cm in b. ( c ) Coimmunoprecipition (CoIP) of transiently co-expressed cMyc-GhHOX3 and HA-GhHD1 in leaves of Nicotiana benthamiana . Soluble protein extracts before (input) and after (IP) immunoprecipitation with anti-cMyc antibody-conjugated beads were detected by immunoblot with anti-HA antibody. ( d ) Yeast two-hybrid assay. GhHOX3, but not GhHD1, bound to GhSLR1 at 10 mM 3-AT. ( e , f ) In vivo BiFc ( e ) and CoIP ( f ) assays. GhHOX3 interacted with GhSLR1. Scale bar, 1 cm in e . ( g ) Yeast three-hybrid assay showing the influence of GhSLR1 on GhHOX3–GhHD1 binding represented by β-galactosidase activity, and the GhSLR1 expression was suppressed by increasing Met concentrations (data are shown as mean±s.e.m., n =3). ( h ) Domain deletion assay. Top, GhHOX3 contains three conserved domains. Below, yeast two-hybrid detection. GhHOX3 fragment containing both the Leu-zipper (LZ) and the START domains interacted with both GhHD1 and GhSLR1 in yeast, whereas the GhHOX3 homeodomain (HD) did not.

    Techniques Used: Y2H Assay, Bimolecular Fluorescence Complementation Assay, Luciferase, Fluorescence, Binding Assay, Co-Immunoprecipitation Assay, Immunoprecipitation, In Vivo, Hybrid Assay, Activity Assay, Expressing, DNA Deletion Assay

    Transcriptional regulation of target genes by GhHOX3 and the effects of GhHD1 and GhSLR1. GhRDL1 and GhEXPA1 promoters were fused to the LUC reporter, respectively, and the promoter activities were determined by a transient dual-LUC assay in Nicotiana benthamiana . The relative LUC activities were normalized to the reference Renilla (REN) luciferase. The corresponding effector (+), empty vector (−) or neither (NA) were co-filtrated (data are presented as mean±s.e.m., n =3, * P
    Figure Legend Snippet: Transcriptional regulation of target genes by GhHOX3 and the effects of GhHD1 and GhSLR1. GhRDL1 and GhEXPA1 promoters were fused to the LUC reporter, respectively, and the promoter activities were determined by a transient dual-LUC assay in Nicotiana benthamiana . The relative LUC activities were normalized to the reference Renilla (REN) luciferase. The corresponding effector (+), empty vector (−) or neither (NA) were co-filtrated (data are presented as mean±s.e.m., n =3, * P

    Techniques Used: Luciferase, Plasmid Preparation

    37) Product Images from "The Solanum lycopersicum Zinc Finger2 Cysteine-2/Histidine-2 Repressor-Like Transcription Factor Regulates Development and Tolerance to Salinity in Tomato and Arabidopsis 1 Zinc Finger2 Cysteine-2/Histidine-2 Repressor-Like Transcription Factor Regulates Development and Tolerance to Salinity in Tomato and Arabidopsis 1 [W]"

    Article Title: The Solanum lycopersicum Zinc Finger2 Cysteine-2/Histidine-2 Repressor-Like Transcription Factor Regulates Development and Tolerance to Salinity in Tomato and Arabidopsis 1 Zinc Finger2 Cysteine-2/Histidine-2 Repressor-Like Transcription Factor Regulates Development and Tolerance to Salinity in Tomato and Arabidopsis 1 [W]

    Journal: Plant Physiology

    doi: 10.1104/pp.113.225920

    SlZF2 transcriptional properties. A, Transient expression of YFP-SlZF2 fusion protein in tomato leaf protoplasts: YFP-SlZF2 (Aa and Aa′), YFP control fluorescence (Ab and Ab′), and bright field/chlorophyll/YFP fluorescence respectively. B, SlZF2 transactivation ability. The SlZF2 coding region was fused to GAL4 DNA binding domain (DBD) into the pGBKT7 vector carrying the nutritional marker TRYPTOPHAN1 as the reporter gene. Yeasts were transformed with pGBKT7 empty vector (Ba and Ba′) or with the GAL4-DBD-SlZF2 construct (Bb and Bb′). Both constructs were transformed into the yeast strain Y8930 harboring the ß-galactosidase L , ADE2 , and HIS3 reporter genes. Yeasts were separately grown on synthetic dropout medium lacking either Trp (Ba and Bb), or A and His (Ba′ and Bb′). C, Position weight matrix representation of the three top-scoring 8 mers obtained in a seed-and-wobble algorithm. D, Box plot representation of signal intensities of the probes containing the elements indicated. Different combinations of the AGT modules in bipartite motifs are shown in blue, whereas their corresponding mutant versions are shown in red. Letters above the boxes represent different groups of statistical significance, relative to the motif ACTnnnAGT with highest median intensity, as follows: a, P > 0.05 (no significant differences); b, P
    Figure Legend Snippet: SlZF2 transcriptional properties. A, Transient expression of YFP-SlZF2 fusion protein in tomato leaf protoplasts: YFP-SlZF2 (Aa and Aa′), YFP control fluorescence (Ab and Ab′), and bright field/chlorophyll/YFP fluorescence respectively. B, SlZF2 transactivation ability. The SlZF2 coding region was fused to GAL4 DNA binding domain (DBD) into the pGBKT7 vector carrying the nutritional marker TRYPTOPHAN1 as the reporter gene. Yeasts were transformed with pGBKT7 empty vector (Ba and Ba′) or with the GAL4-DBD-SlZF2 construct (Bb and Bb′). Both constructs were transformed into the yeast strain Y8930 harboring the ß-galactosidase L , ADE2 , and HIS3 reporter genes. Yeasts were separately grown on synthetic dropout medium lacking either Trp (Ba and Bb), or A and His (Ba′ and Bb′). C, Position weight matrix representation of the three top-scoring 8 mers obtained in a seed-and-wobble algorithm. D, Box plot representation of signal intensities of the probes containing the elements indicated. Different combinations of the AGT modules in bipartite motifs are shown in blue, whereas their corresponding mutant versions are shown in red. Letters above the boxes represent different groups of statistical significance, relative to the motif ACTnnnAGT with highest median intensity, as follows: a, P > 0.05 (no significant differences); b, P

    Techniques Used: Expressing, Fluorescence, Binding Assay, Plasmid Preparation, Marker, Transformation Assay, Construct, Mutagenesis

    Effects of SlZF2 ectopic expression in tomato. A, Transfer DNA (T-DNA) insertion in tomato genome and expression of SlZF2 in Z4, Z5, and Z16 transgenic lines and wild-type (WT) plants were analyzed by PCR. B, Defaults of germination of SlZF2 medium supplemented with 100 mg/L kanamycin. C, Dwarfism of 35S :: SlZF2 tomato seedlings grown in vitro compared with wild-type tomatoes. D, Fully expanded leaf of transgenic (Da) and wild-type (Db) plants. E, Flowers of SlZF2 -transgenic (Ea, Eb, and Ec) or wild-type (Ed and Ee) plants. F, Details of 35S :: SlZF2 and wild-type tomato fruits (Fa and Fb, respectively) and according to a longitudinal section (Fc and Fd, respectively), and seeds of transgenic (Fe) and wild-type (Ff) tomatoes.
    Figure Legend Snippet: Effects of SlZF2 ectopic expression in tomato. A, Transfer DNA (T-DNA) insertion in tomato genome and expression of SlZF2 in Z4, Z5, and Z16 transgenic lines and wild-type (WT) plants were analyzed by PCR. B, Defaults of germination of SlZF2 medium supplemented with 100 mg/L kanamycin. C, Dwarfism of 35S :: SlZF2 tomato seedlings grown in vitro compared with wild-type tomatoes. D, Fully expanded leaf of transgenic (Da) and wild-type (Db) plants. E, Flowers of SlZF2 -transgenic (Ea, Eb, and Ec) or wild-type (Ed and Ee) plants. F, Details of 35S :: SlZF2 and wild-type tomato fruits (Fa and Fb, respectively) and according to a longitudinal section (Fc and Fd, respectively), and seeds of transgenic (Fe) and wild-type (Ff) tomatoes.

    Techniques Used: Expressing, Transgenic Assay, Polymerase Chain Reaction, In Vitro

    38) Product Images from "Transcription factor DUO1 generated by neo-functionalization is associated with evolution of sperm differentiation in plants"

    Article Title: Transcription factor DUO1 generated by neo-functionalization is associated with evolution of sperm differentiation in plants

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07728-3

    Characterization of DUO1 transcription factor activity. a Amino acid sequence alignment of regions A, B, and C among AtDUO1, MpDUO1, and MpR2R3-MYB21 (top). Dots indicate matching residues with AtDUO1. Asterisks indicate putative DNA-interacting residues in region C. Structural modeling of MpDUO1 in complex with DNA using SWISS-MODEL (bottom). The MpDUO1 MYB domain is overlaid onto the structure of the AMV v-MYB-DNA complex (PDB code: 1H8A). b in vivo transcriptional activation potentials of MpDUO1 and chimeras. Schematic diagram of constructs (left) are color-coded light blue (AtDUO1), dark blue (MpDUO1), and orange (MpR2R3-MYB21). DUO1 transcriptional activation potentials were measured by relative luciferase activity (right). n = 4 (upper), n = 8 (lower) (** p
    Figure Legend Snippet: Characterization of DUO1 transcription factor activity. a Amino acid sequence alignment of regions A, B, and C among AtDUO1, MpDUO1, and MpR2R3-MYB21 (top). Dots indicate matching residues with AtDUO1. Asterisks indicate putative DNA-interacting residues in region C. Structural modeling of MpDUO1 in complex with DNA using SWISS-MODEL (bottom). The MpDUO1 MYB domain is overlaid onto the structure of the AMV v-MYB-DNA complex (PDB code: 1H8A). b in vivo transcriptional activation potentials of MpDUO1 and chimeras. Schematic diagram of constructs (left) are color-coded light blue (AtDUO1), dark blue (MpDUO1), and orange (MpR2R3-MYB21). DUO1 transcriptional activation potentials were measured by relative luciferase activity (right). n = 4 (upper), n = 8 (lower) (** p

    Techniques Used: Activity Assay, Sequencing, In Vivo, Activation Assay, Construct, Luciferase

    39) Product Images from "Septin 7 Interacts with Centromere-associated Protein E and Is Required for Its Kinetochore Localization *"

    Article Title: Septin 7 Interacts with Centromere-associated Protein E and Is Required for Its Kinetochore Localization *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M710591200

    SEPT7 interacts with CENP-E in vivo . A , characterization of a SEPT7-specific mouse antibody. Lysates from MDCK cells transiently expressing GFP-tagged SEPT2, SEPT6, SEPT7, and FLAG-SEPT11 were separated on a SDS-PAGE and then subjected to Western blotting
    Figure Legend Snippet: SEPT7 interacts with CENP-E in vivo . A , characterization of a SEPT7-specific mouse antibody. Lysates from MDCK cells transiently expressing GFP-tagged SEPT2, SEPT6, SEPT7, and FLAG-SEPT11 were separated on a SDS-PAGE and then subjected to Western blotting

    Techniques Used: In Vivo, Expressing, SDS Page, Western Blot

    CENP-E interacts with SEPT2/6/7 complex by a direct contact with SEPT7. A , co-immunoprecipitation of CENP-E 2131–2701 and SEPT2/6/7/11 from transfected 293T cells. 293T cells co-transfected with GFP-CENP-E 2131–2701 (CENP-E) and FLAG-SEPT2/6/7
    Figure Legend Snippet: CENP-E interacts with SEPT2/6/7 complex by a direct contact with SEPT7. A , co-immunoprecipitation of CENP-E 2131–2701 and SEPT2/6/7/11 from transfected 293T cells. 293T cells co-transfected with GFP-CENP-E 2131–2701 (CENP-E) and FLAG-SEPT2/6/7

    Techniques Used: Immunoprecipitation, Transfection

    Repression of SEPT7 activates spindle assembly checkpoint. A , repression of SEPT7 altered the kinetochore localization of Hec1. MDCK cells were transfected with SEPT7 siRNA oligonucleotide ( SEPT7 siRNA ) and scramble control ( Scramble ). 48 h after the
    Figure Legend Snippet: Repression of SEPT7 activates spindle assembly checkpoint. A , repression of SEPT7 altered the kinetochore localization of Hec1. MDCK cells were transfected with SEPT7 siRNA oligonucleotide ( SEPT7 siRNA ) and scramble control ( Scramble ). 48 h after the

    Techniques Used: Transfection

    Depletion of SEPT7 destabilized CENP-E localization to the kinetochore and resulted in chromosomes misalignment. A , efficiency of siRNA on suppression of SEPT7. HeLa cells and MDCK cells were transfected with 100 n m SEPT7 siRNA for optimal suppression
    Figure Legend Snippet: Depletion of SEPT7 destabilized CENP-E localization to the kinetochore and resulted in chromosomes misalignment. A , efficiency of siRNA on suppression of SEPT7. HeLa cells and MDCK cells were transfected with 100 n m SEPT7 siRNA for optimal suppression

    Techniques Used: Transfection

    Identification and characterization of a novel SEPT7-CENP-E interaction. A , schematic drawing shows SEPT7 and its fragments that encode various bait clones used for yeast two-hybrid assay for CENP-E binding activity. B , yeast cells were co-transformed
    Figure Legend Snippet: Identification and characterization of a novel SEPT7-CENP-E interaction. A , schematic drawing shows SEPT7 and its fragments that encode various bait clones used for yeast two-hybrid assay for CENP-E binding activity. B , yeast cells were co-transformed

    Techniques Used: Clone Assay, Y2H Assay, Binding Assay, Activity Assay, Transformation Assay

    Depletion of SEPT2/7 releases the tension across sister kinetochores. A , immunofluorescence assay of control siRNA-treated HeLa cells (scramble), SEPT2 siRNA-treated cells, SEPT7 siRNA-treated cells, CENP-E siRNA-treated cells, SEPT2 and SEPT7 siRNA-treated
    Figure Legend Snippet: Depletion of SEPT2/7 releases the tension across sister kinetochores. A , immunofluorescence assay of control siRNA-treated HeLa cells (scramble), SEPT2 siRNA-treated cells, SEPT7 siRNA-treated cells, CENP-E siRNA-treated cells, SEPT2 and SEPT7 siRNA-treated

    Techniques Used: Immunofluorescence

    40) Product Images from "The DEFECTIVE IN ANTHER DEHISCENCE1 Gene Encodes a Novel Phospholipase A1 Catalyzing the Initial Step of Jasmonic Acid Biosynthesis, Which Synchronizes Pollen Maturation, Anther Dehiscence, and Flower Opening in Arabidopsis"

    Article Title: The DEFECTIVE IN ANTHER DEHISCENCE1 Gene Encodes a Novel Phospholipase A1 Catalyzing the Initial Step of Jasmonic Acid Biosynthesis, Which Synchronizes Pollen Maturation, Anther Dehiscence, and Flower Opening in Arabidopsis

    Journal: The Plant Cell

    doi: 10.1105/tpc.010192

    PLA1 Activity of the DAD1 Protein. (A) pH dependence of DAD1 activity. The MBP-DAD1dE protein was incubated with PC in phosphate buffers of various pH levels for 30 min at 25°C, and the released fatty acids were quantified. Data are expressed as the relative activities compared with the activity at pH 6.0, which was assigned a value of 1.0. (B) Substrate specificity of DAD1 activity. The MBP-DAD1dE protein and R. miehei lipase were incubated with PC, MGDG, or trilinolein (TG), and the released free fatty acids were quantified. Data are expressed as the relative activities compared with the maximum activity for each enzyme (i.e., the activity for PC was assigned a value of 1.0 for MBP-DAD1dE, and the activity for MGDG was assigned a value of 1.0 for R. miehei lipase). Closed bars, MBP-DAD1dE; open bars, R. miehei lipase. (C) and (D) Substrate specificity of DAD1 activity with respect to sn positions. (C) The MBP-DAD1dE protein was incubated with 1-palmitoyl-2-linoleoyl-PC. After incubation for the indicated times, the amounts of palmitic acid and linoleic acid in the free fatty acid fraction and in the lysoPC fraction were measured by gas chromatography. Open circles, palmitic acid in the free fatty acid fraction; closed squares, linoleic acid in the lysoPC fraction; open squares, linoleic acid in the free fatty acid fraction; closed circles, palmitic acid in the lysoPC fraction. (D) The MBP-DAD1dE protein was incubated with 1-palmitoyl-2- 14 C-linoleoyl-PC. As controls, MBP or buffer (no protein) was added in place of MBP-DAD1dE. After separation by thin layer chromatography, the radioactivity in the bands for 14 C-PC (open bars), 14 C-free fatty acid (hatched bars), and 14 C-lysoPC (closed bars) was quantified and expressed as a proportion of the sum of the three fractions.
    Figure Legend Snippet: PLA1 Activity of the DAD1 Protein. (A) pH dependence of DAD1 activity. The MBP-DAD1dE protein was incubated with PC in phosphate buffers of various pH levels for 30 min at 25°C, and the released fatty acids were quantified. Data are expressed as the relative activities compared with the activity at pH 6.0, which was assigned a value of 1.0. (B) Substrate specificity of DAD1 activity. The MBP-DAD1dE protein and R. miehei lipase were incubated with PC, MGDG, or trilinolein (TG), and the released free fatty acids were quantified. Data are expressed as the relative activities compared with the maximum activity for each enzyme (i.e., the activity for PC was assigned a value of 1.0 for MBP-DAD1dE, and the activity for MGDG was assigned a value of 1.0 for R. miehei lipase). Closed bars, MBP-DAD1dE; open bars, R. miehei lipase. (C) and (D) Substrate specificity of DAD1 activity with respect to sn positions. (C) The MBP-DAD1dE protein was incubated with 1-palmitoyl-2-linoleoyl-PC. After incubation for the indicated times, the amounts of palmitic acid and linoleic acid in the free fatty acid fraction and in the lysoPC fraction were measured by gas chromatography. Open circles, palmitic acid in the free fatty acid fraction; closed squares, linoleic acid in the lysoPC fraction; open squares, linoleic acid in the free fatty acid fraction; closed circles, palmitic acid in the lysoPC fraction. (D) The MBP-DAD1dE protein was incubated with 1-palmitoyl-2- 14 C-linoleoyl-PC. As controls, MBP or buffer (no protein) was added in place of MBP-DAD1dE. After separation by thin layer chromatography, the radioactivity in the bands for 14 C-PC (open bars), 14 C-free fatty acid (hatched bars), and 14 C-lysoPC (closed bars) was quantified and expressed as a proportion of the sum of the three fractions.

    Techniques Used: Activity Assay, Incubation, Gas Chromatography, Thin Layer Chromatography, Radioactivity

    Structures of DAD1 and Homologous Proteins Encoded in the Arabidopsis Genome. (A) The deduced amino acid sequence of DAD1. The serine and aspartic acid residues and two candidates for the histidine residue, which constitute the catalytic triad, are highlighted. The lipase consensus sequence is boxed. The closed triangle indicates the putative cleavage site of the predicted transit peptide. The asterisk indicates the N-terminal residue that is fused to the C terminus of MBP in the MBP-DAD1dE fusion protein. The open triangle indicates the position corresponding to the T-DNA insertion in genomic DNA of the dad1 mutant. (B) Amino acid alignment in the catalytic regions of the DAD1 protein, homologs identified from Arabidopsis databases, and Rhizomucor miehei lipase. Residues identical in more than half of the sequences are highlighted. The Arabidopsis sequences are classified into three classes, class I (I), class II (II), and class III (III), based on their similarities and the presence of N-terminal stretches. The serine and aspartic acid residues (closed arrowheads) and two candidates for the histidine residue (open arrowheads), which constitute the putative catalytic triad, are indicated. Boxed amino acid residues in R. miehei ). For At2g30550, we supplemented the database sequence with the in-frame 82–amino acid stretch continued on the N terminus. (C) Scheme of DAD1 and its Arabidopsis homologs showing predicted transit peptides (closed boxes), predicted mitochondrial signal sequence (hatched box), and catalytic regions (open boxes) indicated in (B) . Classes I, II, and III are indicated.
    Figure Legend Snippet: Structures of DAD1 and Homologous Proteins Encoded in the Arabidopsis Genome. (A) The deduced amino acid sequence of DAD1. The serine and aspartic acid residues and two candidates for the histidine residue, which constitute the catalytic triad, are highlighted. The lipase consensus sequence is boxed. The closed triangle indicates the putative cleavage site of the predicted transit peptide. The asterisk indicates the N-terminal residue that is fused to the C terminus of MBP in the MBP-DAD1dE fusion protein. The open triangle indicates the position corresponding to the T-DNA insertion in genomic DNA of the dad1 mutant. (B) Amino acid alignment in the catalytic regions of the DAD1 protein, homologs identified from Arabidopsis databases, and Rhizomucor miehei lipase. Residues identical in more than half of the sequences are highlighted. The Arabidopsis sequences are classified into three classes, class I (I), class II (II), and class III (III), based on their similarities and the presence of N-terminal stretches. The serine and aspartic acid residues (closed arrowheads) and two candidates for the histidine residue (open arrowheads), which constitute the putative catalytic triad, are indicated. Boxed amino acid residues in R. miehei ). For At2g30550, we supplemented the database sequence with the in-frame 82–amino acid stretch continued on the N terminus. (C) Scheme of DAD1 and its Arabidopsis homologs showing predicted transit peptides (closed boxes), predicted mitochondrial signal sequence (hatched box), and catalytic regions (open boxes) indicated in (B) . Classes I, II, and III are indicated.

    Techniques Used: Sequencing, Mutagenesis

    Related Articles

    Clone Assay:

    Article Title: Molecular Cloning and Characterization of Taurocyamine Kinase from Clonorchis sinensis: A Candidate Chemotherapeutic Target
    Article Snippet: .. Coding region of C. sinensis PK cDNA of D1D2 was cloned into XbaI/PstI site of pMAL-c2 (New England Biolabs, Ipswich, MA, USA). .. Maltose binding protein (MBP)-C. sinensis PK fusion protein was expressed in E. coli TB1 cells by induction with 1 mM IPTG at 25°C for 24 h. The cells were resuspended and sonicated in 5× TE buffer.

    Article Title: The tapetal AHL family protein TEK determines nexine formation in the pollen wall
    Article Snippet: .. Electrophoretic mobility shift assay To obtain purified AMS protein for the EMSA experiments, the full-length fragment of the AMS gene was amplified using the primer pairs AMSpMAL-F and AMSpMAL-R, and cloned into the pMAL-p5X vector (NEB, USA) to produce the (Maltose Binding Protein) MBP-AMS construct. ..

    Article Title: Immunoreactivity of the Mycobacterium avium subsp. paratuberculosis 19-kDa lipoprotein
    Article Snippet: .. Cloning and expression of the M. avium subsp. paratuberculosis 19-kDa gene A maltose binding protein (MBP) fusion of the M. avium subsp. paratuberculosis 19-kDa sequence (MBP-19 kDa) was constructed using the pMAL-c2 vector (New England Biolabs, Beverly, MA). .. To amplify the 19-kDa coding region from M. avium subsp. paratuberculosis , primers were designed directly from the MAP0261c sequence.

    Article Title: DER containing two consecutive GTP-binding domains plays an essential role in chloroplast ribosomal RNA processing and ribosome biogenesis in higher plants
    Article Snippet: .. To purify MBP:CTD for RNA-binding assays, the NbDER cDNA fragment corresponding to amino acid residues 520–651 was amplified by PCR and cloned into the pMALTM c2 or pMAL-c2 vector (New England Biolabs). .. RNA binding assay To prepare 16S and 23S rRNA, the cDNAs encoding full-length 16S and 23S rRNA were cloned into the pGEM T-easy vector.

    Article Title: Transcription factor DUO1 generated by neo-functionalization is associated with evolution of sperm differentiation in plants
    Article Snippet: .. For the protein-binding DNA microarray assay, synthesized Escherichia coli -codon-optimized fragments (ThermoFisher Scientific) encoding MYB domains of AtDUO1, MpDUO1, Chimera 4, 5, 6 as well as MpR2R3-MYB21 and KflMYB were cloned into pDONR221 and then transferred to the destination vector pMAL-C2 vector (New England Biolabs) through Gateway technology, generating the in-frame fusion of Maltose Binding Protein (MBP) and MYB DNA-binding domain. .. Transgenic Arabidopsis plants were generated using the floral dip method and T1 transgenic plants were screened based on each selection marker of the construct.

    Article Title: Stimulation of NeuroD activity by huntingtin and huntingtin-associated proteins HAP1 and MLK2
    Article Snippet: .. The MBP-MLK2 expression vector was generated by cloning a cDNA fragment corresponding to amino acids 1–457 of the human MLK2 protein into the pMAL-c2 plasmid (New England Biolabs), downstream of and in-frame with the MBP affinity tag. .. A kinase-dead version of MBP-MLK2 (K125E) was generated by using the QuikChange site-directed mutagenesis kit (Stratagene) as directed by the manufacturer.

    Amplification:

    Article Title: The tapetal AHL family protein TEK determines nexine formation in the pollen wall
    Article Snippet: .. Electrophoretic mobility shift assay To obtain purified AMS protein for the EMSA experiments, the full-length fragment of the AMS gene was amplified using the primer pairs AMSpMAL-F and AMSpMAL-R, and cloned into the pMAL-p5X vector (NEB, USA) to produce the (Maltose Binding Protein) MBP-AMS construct. ..

    Article Title: DER containing two consecutive GTP-binding domains plays an essential role in chloroplast ribosomal RNA processing and ribosome biogenesis in higher plants
    Article Snippet: .. To purify MBP:CTD for RNA-binding assays, the NbDER cDNA fragment corresponding to amino acid residues 520–651 was amplified by PCR and cloned into the pMALTM c2 or pMAL-c2 vector (New England Biolabs). .. RNA binding assay To prepare 16S and 23S rRNA, the cDNAs encoding full-length 16S and 23S rRNA were cloned into the pGEM T-easy vector.

    Synthesized:

    Article Title: Transcription factor DUO1 generated by neo-functionalization is associated with evolution of sperm differentiation in plants
    Article Snippet: .. For the protein-binding DNA microarray assay, synthesized Escherichia coli -codon-optimized fragments (ThermoFisher Scientific) encoding MYB domains of AtDUO1, MpDUO1, Chimera 4, 5, 6 as well as MpR2R3-MYB21 and KflMYB were cloned into pDONR221 and then transferred to the destination vector pMAL-C2 vector (New England Biolabs) through Gateway technology, generating the in-frame fusion of Maltose Binding Protein (MBP) and MYB DNA-binding domain. .. Transgenic Arabidopsis plants were generated using the floral dip method and T1 transgenic plants were screened based on each selection marker of the construct.

    Protein Binding:

    Article Title: Transcription factor DUO1 generated by neo-functionalization is associated with evolution of sperm differentiation in plants
    Article Snippet: .. For the protein-binding DNA microarray assay, synthesized Escherichia coli -codon-optimized fragments (ThermoFisher Scientific) encoding MYB domains of AtDUO1, MpDUO1, Chimera 4, 5, 6 as well as MpR2R3-MYB21 and KflMYB were cloned into pDONR221 and then transferred to the destination vector pMAL-C2 vector (New England Biolabs) through Gateway technology, generating the in-frame fusion of Maltose Binding Protein (MBP) and MYB DNA-binding domain. .. Transgenic Arabidopsis plants were generated using the floral dip method and T1 transgenic plants were screened based on each selection marker of the construct.

    Incubation:

    Article Title: CTGC motifs within the HIV core promoter specify Tat-responsive pre-initiation complexes
    Article Snippet: .. For RNase H directed degradation of 7SK RNA within the nuclear extracts used in EMSA, nuclear extracts were first incubated with anti-sense oligonucleotide and 10 U of RNase H (NEB) for 1 h at 30°C in binding buffer. ..

    Electrophoretic Mobility Shift Assay:

    Article Title: The tapetal AHL family protein TEK determines nexine formation in the pollen wall
    Article Snippet: .. Electrophoretic mobility shift assay To obtain purified AMS protein for the EMSA experiments, the full-length fragment of the AMS gene was amplified using the primer pairs AMSpMAL-F and AMSpMAL-R, and cloned into the pMAL-p5X vector (NEB, USA) to produce the (Maltose Binding Protein) MBP-AMS construct. ..

    Purification:

    Article Title: The tapetal AHL family protein TEK determines nexine formation in the pollen wall
    Article Snippet: .. Electrophoretic mobility shift assay To obtain purified AMS protein for the EMSA experiments, the full-length fragment of the AMS gene was amplified using the primer pairs AMSpMAL-F and AMSpMAL-R, and cloned into the pMAL-p5X vector (NEB, USA) to produce the (Maltose Binding Protein) MBP-AMS construct. ..

    Sequencing:

    Article Title: Immunoreactivity of the Mycobacterium avium subsp. paratuberculosis 19-kDa lipoprotein
    Article Snippet: .. Cloning and expression of the M. avium subsp. paratuberculosis 19-kDa gene A maltose binding protein (MBP) fusion of the M. avium subsp. paratuberculosis 19-kDa sequence (MBP-19 kDa) was constructed using the pMAL-c2 vector (New England Biolabs, Beverly, MA). .. To amplify the 19-kDa coding region from M. avium subsp. paratuberculosis , primers were designed directly from the MAP0261c sequence.

    Microarray:

    Article Title: Transcription factor DUO1 generated by neo-functionalization is associated with evolution of sperm differentiation in plants
    Article Snippet: .. For the protein-binding DNA microarray assay, synthesized Escherichia coli -codon-optimized fragments (ThermoFisher Scientific) encoding MYB domains of AtDUO1, MpDUO1, Chimera 4, 5, 6 as well as MpR2R3-MYB21 and KflMYB were cloned into pDONR221 and then transferred to the destination vector pMAL-C2 vector (New England Biolabs) through Gateway technology, generating the in-frame fusion of Maltose Binding Protein (MBP) and MYB DNA-binding domain. .. Transgenic Arabidopsis plants were generated using the floral dip method and T1 transgenic plants were screened based on each selection marker of the construct.

    Affinity Magnetic Separation:

    Article Title: The tapetal AHL family protein TEK determines nexine formation in the pollen wall
    Article Snippet: .. Electrophoretic mobility shift assay To obtain purified AMS protein for the EMSA experiments, the full-length fragment of the AMS gene was amplified using the primer pairs AMSpMAL-F and AMSpMAL-R, and cloned into the pMAL-p5X vector (NEB, USA) to produce the (Maltose Binding Protein) MBP-AMS construct. ..

    Construct:

    Article Title: The tapetal AHL family protein TEK determines nexine formation in the pollen wall
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    Article Title: Immunoreactivity of the Mycobacterium avium subsp. paratuberculosis 19-kDa lipoprotein
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    Article Title: Chibby forms a homodimer through a heptad repeat of leucine residues in its C-terminal coiled-coil motif
    Article Snippet: .. To construct MBP-Cby mutants, the cDNA inserts were PCR-amplified, digested with Bgl II and Xho I, and ligated into pMAL-c2 (New England Biolabs). .. For synthetic Renilla luciferase (hRluc) protein-fragment-assisted complementation assays [ ], cDNAs encoding Cby, GFP, Jun or Fos were amplified by PCR using plasmid templates, and ligated in-frame with the N-terminal portion (amino acids 1–239) or the C-terminal portion (amino acids 240–321) of hRluc into the pJCH510 or pJCH511 vector [ ].

    Expressing:

    Article Title: Immunoreactivity of the Mycobacterium avium subsp. paratuberculosis 19-kDa lipoprotein
    Article Snippet: .. Cloning and expression of the M. avium subsp. paratuberculosis 19-kDa gene A maltose binding protein (MBP) fusion of the M. avium subsp. paratuberculosis 19-kDa sequence (MBP-19 kDa) was constructed using the pMAL-c2 vector (New England Biolabs, Beverly, MA). .. To amplify the 19-kDa coding region from M. avium subsp. paratuberculosis , primers were designed directly from the MAP0261c sequence.

    Article Title: Stimulation of NeuroD activity by huntingtin and huntingtin-associated proteins HAP1 and MLK2
    Article Snippet: .. The MBP-MLK2 expression vector was generated by cloning a cDNA fragment corresponding to amino acids 1–457 of the human MLK2 protein into the pMAL-c2 plasmid (New England Biolabs), downstream of and in-frame with the MBP affinity tag. .. A kinase-dead version of MBP-MLK2 (K125E) was generated by using the QuikChange site-directed mutagenesis kit (Stratagene) as directed by the manufacturer.

    Polymerase Chain Reaction:

    Article Title: DER containing two consecutive GTP-binding domains plays an essential role in chloroplast ribosomal RNA processing and ribosome biogenesis in higher plants
    Article Snippet: .. To purify MBP:CTD for RNA-binding assays, the NbDER cDNA fragment corresponding to amino acid residues 520–651 was amplified by PCR and cloned into the pMALTM c2 or pMAL-c2 vector (New England Biolabs). .. RNA binding assay To prepare 16S and 23S rRNA, the cDNAs encoding full-length 16S and 23S rRNA were cloned into the pGEM T-easy vector.

    Article Title: Chibby forms a homodimer through a heptad repeat of leucine residues in its C-terminal coiled-coil motif
    Article Snippet: .. To construct MBP-Cby mutants, the cDNA inserts were PCR-amplified, digested with Bgl II and Xho I, and ligated into pMAL-c2 (New England Biolabs). .. For synthetic Renilla luciferase (hRluc) protein-fragment-assisted complementation assays [ ], cDNAs encoding Cby, GFP, Jun or Fos were amplified by PCR using plasmid templates, and ligated in-frame with the N-terminal portion (amino acids 1–239) or the C-terminal portion (amino acids 240–321) of hRluc into the pJCH510 or pJCH511 vector [ ].

    RNA Binding Assay:

    Article Title: DER containing two consecutive GTP-binding domains plays an essential role in chloroplast ribosomal RNA processing and ribosome biogenesis in higher plants
    Article Snippet: .. To purify MBP:CTD for RNA-binding assays, the NbDER cDNA fragment corresponding to amino acid residues 520–651 was amplified by PCR and cloned into the pMALTM c2 or pMAL-c2 vector (New England Biolabs). .. RNA binding assay To prepare 16S and 23S rRNA, the cDNAs encoding full-length 16S and 23S rRNA were cloned into the pGEM T-easy vector.

    Binding Assay:

    Article Title: CTGC motifs within the HIV core promoter specify Tat-responsive pre-initiation complexes
    Article Snippet: .. For RNase H directed degradation of 7SK RNA within the nuclear extracts used in EMSA, nuclear extracts were first incubated with anti-sense oligonucleotide and 10 U of RNase H (NEB) for 1 h at 30°C in binding buffer. ..

    Article Title: The tapetal AHL family protein TEK determines nexine formation in the pollen wall
    Article Snippet: .. Electrophoretic mobility shift assay To obtain purified AMS protein for the EMSA experiments, the full-length fragment of the AMS gene was amplified using the primer pairs AMSpMAL-F and AMSpMAL-R, and cloned into the pMAL-p5X vector (NEB, USA) to produce the (Maltose Binding Protein) MBP-AMS construct. ..

    Article Title: Immunoreactivity of the Mycobacterium avium subsp. paratuberculosis 19-kDa lipoprotein
    Article Snippet: .. Cloning and expression of the M. avium subsp. paratuberculosis 19-kDa gene A maltose binding protein (MBP) fusion of the M. avium subsp. paratuberculosis 19-kDa sequence (MBP-19 kDa) was constructed using the pMAL-c2 vector (New England Biolabs, Beverly, MA). .. To amplify the 19-kDa coding region from M. avium subsp. paratuberculosis , primers were designed directly from the MAP0261c sequence.

    Article Title: Transcription factor DUO1 generated by neo-functionalization is associated with evolution of sperm differentiation in plants
    Article Snippet: .. For the protein-binding DNA microarray assay, synthesized Escherichia coli -codon-optimized fragments (ThermoFisher Scientific) encoding MYB domains of AtDUO1, MpDUO1, Chimera 4, 5, 6 as well as MpR2R3-MYB21 and KflMYB were cloned into pDONR221 and then transferred to the destination vector pMAL-C2 vector (New England Biolabs) through Gateway technology, generating the in-frame fusion of Maltose Binding Protein (MBP) and MYB DNA-binding domain. .. Transgenic Arabidopsis plants were generated using the floral dip method and T1 transgenic plants were screened based on each selection marker of the construct.

    Plasmid Preparation:

    Article Title: The tapetal AHL family protein TEK determines nexine formation in the pollen wall
    Article Snippet: .. Electrophoretic mobility shift assay To obtain purified AMS protein for the EMSA experiments, the full-length fragment of the AMS gene was amplified using the primer pairs AMSpMAL-F and AMSpMAL-R, and cloned into the pMAL-p5X vector (NEB, USA) to produce the (Maltose Binding Protein) MBP-AMS construct. ..

    Article Title: Immunoreactivity of the Mycobacterium avium subsp. paratuberculosis 19-kDa lipoprotein
    Article Snippet: .. Cloning and expression of the M. avium subsp. paratuberculosis 19-kDa gene A maltose binding protein (MBP) fusion of the M. avium subsp. paratuberculosis 19-kDa sequence (MBP-19 kDa) was constructed using the pMAL-c2 vector (New England Biolabs, Beverly, MA). .. To amplify the 19-kDa coding region from M. avium subsp. paratuberculosis , primers were designed directly from the MAP0261c sequence.

    Article Title: DER containing two consecutive GTP-binding domains plays an essential role in chloroplast ribosomal RNA processing and ribosome biogenesis in higher plants
    Article Snippet: .. To purify MBP:CTD for RNA-binding assays, the NbDER cDNA fragment corresponding to amino acid residues 520–651 was amplified by PCR and cloned into the pMALTM c2 or pMAL-c2 vector (New England Biolabs). .. RNA binding assay To prepare 16S and 23S rRNA, the cDNAs encoding full-length 16S and 23S rRNA were cloned into the pGEM T-easy vector.

    Article Title: Transcription factor DUO1 generated by neo-functionalization is associated with evolution of sperm differentiation in plants
    Article Snippet: .. For the protein-binding DNA microarray assay, synthesized Escherichia coli -codon-optimized fragments (ThermoFisher Scientific) encoding MYB domains of AtDUO1, MpDUO1, Chimera 4, 5, 6 as well as MpR2R3-MYB21 and KflMYB were cloned into pDONR221 and then transferred to the destination vector pMAL-C2 vector (New England Biolabs) through Gateway technology, generating the in-frame fusion of Maltose Binding Protein (MBP) and MYB DNA-binding domain. .. Transgenic Arabidopsis plants were generated using the floral dip method and T1 transgenic plants were screened based on each selection marker of the construct.

    Article Title: Stimulation of NeuroD activity by huntingtin and huntingtin-associated proteins HAP1 and MLK2
    Article Snippet: .. The MBP-MLK2 expression vector was generated by cloning a cDNA fragment corresponding to amino acids 1–457 of the human MLK2 protein into the pMAL-c2 plasmid (New England Biolabs), downstream of and in-frame with the MBP affinity tag. .. A kinase-dead version of MBP-MLK2 (K125E) was generated by using the QuikChange site-directed mutagenesis kit (Stratagene) as directed by the manufacturer.

    Generated:

    Article Title: Stimulation of NeuroD activity by huntingtin and huntingtin-associated proteins HAP1 and MLK2
    Article Snippet: .. The MBP-MLK2 expression vector was generated by cloning a cDNA fragment corresponding to amino acids 1–457 of the human MLK2 protein into the pMAL-c2 plasmid (New England Biolabs), downstream of and in-frame with the MBP affinity tag. .. A kinase-dead version of MBP-MLK2 (K125E) was generated by using the QuikChange site-directed mutagenesis kit (Stratagene) as directed by the manufacturer.

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    New England Biolabs expression vector pmal c2
    Protein Gel Blot Analysis of the MBP-AtIpk2β Fusion Protein. E. coli cells were transformed with either the empty vector <t>pMAL-c2</t> (lane C) or plasmid pMAL-c2–AtIpk2β (lanes 1 and 2). Protein extracts were obtained from noninduced (−) or IPTG-induced (+) cells. Proteins (45 μg per lane) were detected using an antiserum that recognizes the MBP portion of the proteins. The positions of MBP (42 kD) and the MBP-AtIpk2β fusion protein (75 kD) are indicated by arrows.
    Expression Vector Pmal C2, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 88/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/expression vector pmal c2/product/New England Biolabs
    Average 88 stars, based on 50 article reviews
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    expression vector pmal c2 - by Bioz Stars, 2020-09
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    94
    New England Biolabs plasmid dna
    Proposed mechanism for the regulation of Wss1 protease activity by cysteine switch mechanism. ( I ) Mechanism of Wss1 activation by thiol-reactive electrophiles. (a) Modification of the regulatory cysteine by thiram (Th) or APMA displaces the cysteine from the active site Zn, activates the metalloprotease and induces in-cis Wss1 cleavage. (b) Activated Wss1 may also proteolyze other Wss1 molecules acting in-trans as endopeptidase or caboxypeptidase. (c) In-trans proteolysis results in gradual degradation of Wss1 pool, the most persistent fragment being a compact WLM domain. ( II ) Activation of Wss1 proteolysis by <t>ssDNA.</t> The <t>DNA</t> may act in two ways. (a) First, interaction of a positively charged WLM domain with DNA may induce conformational changes facilitating displacement of the negatively charged C-terminal peptide with an inhibitory cysteine from the active site. This may promote the initial event of Wss1 activation. The process is not efficient and can be reversed by thiols such as DTT and glutathione ( Figure 3D ). (b) Then, DNA may facilitate Wss1 intermolecular interaction and greatly promote in-trans proteolysis. (c) This results in rapid propagation of proteolytic activity and degradation of the Wss1 pool. ( III ) Cooperative mechanism. The DNA may induce Wss1 oligomerization (a), whereby initial in-cis cleavage (b) is followed by in-trans proteolysis of the whole oligomer (c). DOI: http://dx.doi.org/10.7554/eLife.06763.010
    Plasmid Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 666 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Protein Gel Blot Analysis of the MBP-AtIpk2β Fusion Protein. E. coli cells were transformed with either the empty vector pMAL-c2 (lane C) or plasmid pMAL-c2–AtIpk2β (lanes 1 and 2). Protein extracts were obtained from noninduced (−) or IPTG-induced (+) cells. Proteins (45 μg per lane) were detected using an antiserum that recognizes the MBP portion of the proteins. The positions of MBP (42 kD) and the MBP-AtIpk2β fusion protein (75 kD) are indicated by arrows.

    Journal: The Plant Cell

    Article Title: Arabidopsis Inositol Polyphosphate 6-/3-Kinase Is a Nuclear Protein That Complements a Yeast Mutant Lacking a Functional ArgR-Mcm1 Transcription Complex

    doi: 10.1105/tpc.006676

    Figure Lengend Snippet: Protein Gel Blot Analysis of the MBP-AtIpk2β Fusion Protein. E. coli cells were transformed with either the empty vector pMAL-c2 (lane C) or plasmid pMAL-c2–AtIpk2β (lanes 1 and 2). Protein extracts were obtained from noninduced (−) or IPTG-induced (+) cells. Proteins (45 μg per lane) were detected using an antiserum that recognizes the MBP portion of the proteins. The positions of MBP (42 kD) and the MBP-AtIpk2β fusion protein (75 kD) are indicated by arrows.

    Article Snippet: The AtIpk2 β coding sequence was excised from plasmid pCR-K-2 using EcoRI-XhoI restriction enzymes and subcloned into the expression vector pMAL-c2 (New England Biolabs, Schwalbach, Germany), which had been cut previously with EcoRI and SalI.

    Techniques: Western Blot, Transformation Assay, Plasmid Preparation

    Protein splicing of R. marinus DnaB. The R. marinus dnaB gene (complete or partial) was inserted into the expression plasmid vector pMAL-c2 to produce corresponding recombinant fusion proteins and to observe protein splicing. ( A ) Illustration of recombinant fusion proteins. Each fusion protein consists of the maltose-binding protein (MBP), the DnaB intein (solid box), and the DnaB exteins (hatched boxes) of different lengths, and some vector-encoded sequences (open boxs). Calculated molecular masses for the predicted protein products are listed. ( B ) Production and splicing of recombinant DnaB proteins. E. coli cells containing individual recombinant plasmids described above were induced by IPTG to produce the corresponding protein products. Total cellular proteins from induced cells were resolved by electrophoresis on SDS/polyacrylamide gels and visualized by Coomassie blue staining. Lane 1, cells transformed with pMAL and producing a 51-kDa protein, as a control. Lane 2, cells transformed with pMR1, but before IPTG induction. Lanes 3, 4, and 5, cells transformed with pMR1, pMR2, and pMR3, respectively, after IPTG induction. Lanes 6 and 7, same as lanes 3 and 4, respectively, but electrophoresed for a longer period of time. Letters S1, S2, and S3 mark positions of putative spliced proteins produced from pMR1, pMR2, and pMR3, respectively. Letter I marks position of putative excised intein. Letter P marks protein bands that may include precursor proteins and protein splicing intermediates.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: A DnaB intein in Rhodothermus marinus: Indication of recent intein homing across remotely related organisms

    doi:

    Figure Lengend Snippet: Protein splicing of R. marinus DnaB. The R. marinus dnaB gene (complete or partial) was inserted into the expression plasmid vector pMAL-c2 to produce corresponding recombinant fusion proteins and to observe protein splicing. ( A ) Illustration of recombinant fusion proteins. Each fusion protein consists of the maltose-binding protein (MBP), the DnaB intein (solid box), and the DnaB exteins (hatched boxes) of different lengths, and some vector-encoded sequences (open boxs). Calculated molecular masses for the predicted protein products are listed. ( B ) Production and splicing of recombinant DnaB proteins. E. coli cells containing individual recombinant plasmids described above were induced by IPTG to produce the corresponding protein products. Total cellular proteins from induced cells were resolved by electrophoresis on SDS/polyacrylamide gels and visualized by Coomassie blue staining. Lane 1, cells transformed with pMAL and producing a 51-kDa protein, as a control. Lane 2, cells transformed with pMR1, but before IPTG induction. Lanes 3, 4, and 5, cells transformed with pMR1, pMR2, and pMR3, respectively, after IPTG induction. Lanes 6 and 7, same as lanes 3 and 4, respectively, but electrophoresed for a longer period of time. Letters S1, S2, and S3 mark positions of putative spliced proteins produced from pMR1, pMR2, and pMR3, respectively. Letter I marks position of putative excised intein. Letter P marks protein bands that may include precursor proteins and protein splicing intermediates.

    Article Snippet: Recombinant plasmid pMR1 was constructed by cloning the 2.9-kbp Nco I– Bsi W I DNA fragment (blunt ended) into the expression plasmid vector pMAL-c2 (New England Biolabs) at its Sal I site (blunt ended).

    Techniques: Expressing, Plasmid Preparation, Recombinant, Binding Assay, Electrophoresis, Staining, Transformation Assay, Produced

    (A) Diagrammatic representation of the successive clones obtained during the cloning of the chimeric gene. The pMAL-c2 polylinker restriction enzyme cut sites employed in the cloning strategy are indicated. The asterisks at the Xmn I sites indicate that these restriction sites were lost after cloning. nt, nucleotides. (B) Deduced amino acid sequence of the chimeric protein. Regions encoded by the linker sequences are underlined. The position of the maltose-binding fusion protein (MBP) is also indicated.

    Journal: Journal of Clinical Microbiology

    Article Title: Multicomponent Chimeric Antigen for Serodiagnosis of Canine Visceral Leishmaniasis

    doi:

    Figure Lengend Snippet: (A) Diagrammatic representation of the successive clones obtained during the cloning of the chimeric gene. The pMAL-c2 polylinker restriction enzyme cut sites employed in the cloning strategy are indicated. The asterisks at the Xmn I sites indicate that these restriction sites were lost after cloning. nt, nucleotides. (B) Deduced amino acid sequence of the chimeric protein. Regions encoded by the linker sequences are underlined. The position of the maltose-binding fusion protein (MBP) is also indicated.

    Article Snippet: This amplified DNA was directly cloned into the Xmn I restriction site of the plasmid pMAL-c2* and sequenced with the no. 1237 malE primer (New England Biolabs).

    Techniques: Clone Assay, Sequencing, Binding Assay

    Proposed mechanism for the regulation of Wss1 protease activity by cysteine switch mechanism. ( I ) Mechanism of Wss1 activation by thiol-reactive electrophiles. (a) Modification of the regulatory cysteine by thiram (Th) or APMA displaces the cysteine from the active site Zn, activates the metalloprotease and induces in-cis Wss1 cleavage. (b) Activated Wss1 may also proteolyze other Wss1 molecules acting in-trans as endopeptidase or caboxypeptidase. (c) In-trans proteolysis results in gradual degradation of Wss1 pool, the most persistent fragment being a compact WLM domain. ( II ) Activation of Wss1 proteolysis by ssDNA. The DNA may act in two ways. (a) First, interaction of a positively charged WLM domain with DNA may induce conformational changes facilitating displacement of the negatively charged C-terminal peptide with an inhibitory cysteine from the active site. This may promote the initial event of Wss1 activation. The process is not efficient and can be reversed by thiols such as DTT and glutathione ( Figure 3D ). (b) Then, DNA may facilitate Wss1 intermolecular interaction and greatly promote in-trans proteolysis. (c) This results in rapid propagation of proteolytic activity and degradation of the Wss1 pool. ( III ) Cooperative mechanism. The DNA may induce Wss1 oligomerization (a), whereby initial in-cis cleavage (b) is followed by in-trans proteolysis of the whole oligomer (c). DOI: http://dx.doi.org/10.7554/eLife.06763.010

    Journal: eLife

    Article Title: Wss1 metalloprotease partners with Cdc48/Doa1 in processing genotoxic SUMO conjugates

    doi: 10.7554/eLife.06763

    Figure Lengend Snippet: Proposed mechanism for the regulation of Wss1 protease activity by cysteine switch mechanism. ( I ) Mechanism of Wss1 activation by thiol-reactive electrophiles. (a) Modification of the regulatory cysteine by thiram (Th) or APMA displaces the cysteine from the active site Zn, activates the metalloprotease and induces in-cis Wss1 cleavage. (b) Activated Wss1 may also proteolyze other Wss1 molecules acting in-trans as endopeptidase or caboxypeptidase. (c) In-trans proteolysis results in gradual degradation of Wss1 pool, the most persistent fragment being a compact WLM domain. ( II ) Activation of Wss1 proteolysis by ssDNA. The DNA may act in two ways. (a) First, interaction of a positively charged WLM domain with DNA may induce conformational changes facilitating displacement of the negatively charged C-terminal peptide with an inhibitory cysteine from the active site. This may promote the initial event of Wss1 activation. The process is not efficient and can be reversed by thiols such as DTT and glutathione ( Figure 3D ). (b) Then, DNA may facilitate Wss1 intermolecular interaction and greatly promote in-trans proteolysis. (c) This results in rapid propagation of proteolytic activity and degradation of the Wss1 pool. ( III ) Cooperative mechanism. The DNA may induce Wss1 oligomerization (a), whereby initial in-cis cleavage (b) is followed by in-trans proteolysis of the whole oligomer (c). DOI: http://dx.doi.org/10.7554/eLife.06763.010

    Article Snippet: When examining the effect of various additives on Wss1 refolding and activity, all molecules, except SDS (0.1% final concentration) were added directly into protein solution before dialysis: heparin (200 μg/ml sodium salt, Sigma–Aldrich), plasmid DNA (100 μg/ml pMAL-c2), and ssDNA (100 μg/ml M13mp18 single-stranded DNA, New England Biolabs).

    Techniques: Activity Assay, Activation Assay, Modification, Activated Clotting Time Assay

    SUMO-dependent extraction of proteins from the chromatin. ( A ) ssDNA-activated SUMO E3 ligase sumoylates DNA-bound protein and induces its dissociation. ( B ) Delay in dissociation results in SUMO chain formation through multiple rounds of protein sumoylation. Subsequent ubiqutylation b y STUbL promotes Cdc48/Npl4/Ufd1 loading, protein extraction and degradation via proteasome. ( C ) When the extraction is compromised (e.g., covalent protein–DNA adduct), the protein is processed by Cdc48/Wss1/Doa1 complex. Wss1 is targeted to sumoylated protein via its SIMs and promotes extension of SUMO chain that in return could further stimulate Wss1 accumulation and oligomerization at the site of DNA damage (Wss1 foci). Binding to ssDNA and oligomerization triggers metalloprotease activity of Wss1 and initiates substrate processing. The process is assisted by Cdc48 and Doa1 and finally ends in the vacuole. DOI: http://dx.doi.org/10.7554/eLife.06763.033

    Journal: eLife

    Article Title: Wss1 metalloprotease partners with Cdc48/Doa1 in processing genotoxic SUMO conjugates

    doi: 10.7554/eLife.06763

    Figure Lengend Snippet: SUMO-dependent extraction of proteins from the chromatin. ( A ) ssDNA-activated SUMO E3 ligase sumoylates DNA-bound protein and induces its dissociation. ( B ) Delay in dissociation results in SUMO chain formation through multiple rounds of protein sumoylation. Subsequent ubiqutylation b y STUbL promotes Cdc48/Npl4/Ufd1 loading, protein extraction and degradation via proteasome. ( C ) When the extraction is compromised (e.g., covalent protein–DNA adduct), the protein is processed by Cdc48/Wss1/Doa1 complex. Wss1 is targeted to sumoylated protein via its SIMs and promotes extension of SUMO chain that in return could further stimulate Wss1 accumulation and oligomerization at the site of DNA damage (Wss1 foci). Binding to ssDNA and oligomerization triggers metalloprotease activity of Wss1 and initiates substrate processing. The process is assisted by Cdc48 and Doa1 and finally ends in the vacuole. DOI: http://dx.doi.org/10.7554/eLife.06763.033

    Article Snippet: When examining the effect of various additives on Wss1 refolding and activity, all molecules, except SDS (0.1% final concentration) were added directly into protein solution before dialysis: heparin (200 μg/ml sodium salt, Sigma–Aldrich), plasmid DNA (100 μg/ml pMAL-c2), and ssDNA (100 μg/ml M13mp18 single-stranded DNA, New England Biolabs).

    Techniques: Protein Extraction, Binding Assay, Activity Assay