Structured Review

GE Healthcare glutathione sepharose beads
HELZ interacts with the poly(A)-binding protein (PABP). GST pull-down using <t>glutathione-sepharose</t> beads was conducted by incubating crude HeLa cell lysates with recombinant GST, GST-Paip2 106–127 and GST-HELZ 1023–1199 ( A ), GST and the indicated GST-HELZ fragments ( D ), or crude HEK293 cell lysates with GST and GST-PABP 554–636 ( C ). Hypoxic HeLa cell lysates were incubated with GST or GST-HELZ 1023–1199 ( B ). Eluates were subjected to SDS-PAGE and immunoblotting using the indicated antibodies.
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Images

1) Product Images from "The Putative RNA Helicase HELZ Promotes Cell Proliferation, Translation Initiation and Ribosomal Protein S6 Phosphorylation"

Article Title: The Putative RNA Helicase HELZ Promotes Cell Proliferation, Translation Initiation and Ribosomal Protein S6 Phosphorylation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0022107

HELZ interacts with the poly(A)-binding protein (PABP). GST pull-down using glutathione-sepharose beads was conducted by incubating crude HeLa cell lysates with recombinant GST, GST-Paip2 106–127 and GST-HELZ 1023–1199 ( A ), GST and the indicated GST-HELZ fragments ( D ), or crude HEK293 cell lysates with GST and GST-PABP 554–636 ( C ). Hypoxic HeLa cell lysates were incubated with GST or GST-HELZ 1023–1199 ( B ). Eluates were subjected to SDS-PAGE and immunoblotting using the indicated antibodies.
Figure Legend Snippet: HELZ interacts with the poly(A)-binding protein (PABP). GST pull-down using glutathione-sepharose beads was conducted by incubating crude HeLa cell lysates with recombinant GST, GST-Paip2 106–127 and GST-HELZ 1023–1199 ( A ), GST and the indicated GST-HELZ fragments ( D ), or crude HEK293 cell lysates with GST and GST-PABP 554–636 ( C ). Hypoxic HeLa cell lysates were incubated with GST or GST-HELZ 1023–1199 ( B ). Eluates were subjected to SDS-PAGE and immunoblotting using the indicated antibodies.

Techniques Used: Binding Assay, Recombinant, Incubation, SDS Page

2) Product Images from "Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap"

Article Title: Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2016.02119

Interaction of DDX3 with PAdV-3 and HAdV-5 pVIII. (A) Coomassie blue staining of purified protein. Purified GST.DDX3 protein was separated by 10% SDS-PAGE and stained with 0.25 Coomassie blue stain. (B) GST-pull down assay. Purified GSTor GST.DDX3 fusion protein immobilized on Glutathione-Sepharose 4B beads, incubated individually with in vitro translated [ 35 S] methionine labeled PAdV-3 pVIII or HAdV-5 pVIII, separated by 10% SDS-PAGE and detected by autoradiography. (C) Co-immunoprecipitation. Radio labeled in vitro transcribed and translated HAdV5 pVIII or PAdV-3 pVIII was incubated with in vitro transcribed and translated unlabeled DDX3 protein. Proteins were immunoprecipitated with either anti-DDX3 serum or rabbit pre immune sera, separated by 10% SDS-PAGE and auto radio-graphed. Immunoprecipitation (IP).
Figure Legend Snippet: Interaction of DDX3 with PAdV-3 and HAdV-5 pVIII. (A) Coomassie blue staining of purified protein. Purified GST.DDX3 protein was separated by 10% SDS-PAGE and stained with 0.25 Coomassie blue stain. (B) GST-pull down assay. Purified GSTor GST.DDX3 fusion protein immobilized on Glutathione-Sepharose 4B beads, incubated individually with in vitro translated [ 35 S] methionine labeled PAdV-3 pVIII or HAdV-5 pVIII, separated by 10% SDS-PAGE and detected by autoradiography. (C) Co-immunoprecipitation. Radio labeled in vitro transcribed and translated HAdV5 pVIII or PAdV-3 pVIII was incubated with in vitro transcribed and translated unlabeled DDX3 protein. Proteins were immunoprecipitated with either anti-DDX3 serum or rabbit pre immune sera, separated by 10% SDS-PAGE and auto radio-graphed. Immunoprecipitation (IP).

Techniques Used: Staining, Purification, SDS Page, Pull Down Assay, Incubation, In Vitro, Labeling, Autoradiography, Immunoprecipitation

Effect of pVIII on capped mRNA translation. (A). In vitro . The TNT ® T7 luciferase DNA (Promega) (i) was transcribed in vitro in the absence (uncapped) or presence (capped) of 40 mM Ribo m7GpppG cap analog (Promega) using RiboMAX RNA production system-T7 (Promega). The in vitro synthesized capped and uncapped luciferase mRNAs (ii) were translated in the supernatant collected after centrifugation of mixture of Flexi Rabbit Reticulo Lysate (Promega) incubated with Glutathione sepharose beads preloaded with GST.VIII or GST protein alone. The level of luciferase activity was measured using a luciferase kit (Promega) on a Luminometer (Turner Designs, Inc.). The results are shown as relative luciferase activity (iii). Error bars indicate SE of means for separate experiments. The relative luciferase intensity is determined based on GST compared to GST.pVIII. (B) In vivo . 293T cells were transfected with plasmid DNAs (2 μg of pcDNA3-RLuc-POLIRES-FLuc (i) and either 4 μg of pEY.pVIII or 4 μg of pEYFPN1). At 36 h post transfection, Firefly luciferase (FLuc) and Renilla reniformis luciferase (RLuc) activities were measured in a luminometer by using a dual luciferase assay kit (Promega) as per the company’s procedure. Expression of EYFP was used to normalize the transfection efficiency. The results are shown as relative luciferase activity (iii). The level of cytoplasmic RLuc-POLIRES-FLuc mRNA both in EY.pVIII and EYFP expressing plasmid transfected cells was quantified by RT-PCR (ii). Error bars indicate SE of means for three separate experiments. ∗ statistically significant.
Figure Legend Snippet: Effect of pVIII on capped mRNA translation. (A). In vitro . The TNT ® T7 luciferase DNA (Promega) (i) was transcribed in vitro in the absence (uncapped) or presence (capped) of 40 mM Ribo m7GpppG cap analog (Promega) using RiboMAX RNA production system-T7 (Promega). The in vitro synthesized capped and uncapped luciferase mRNAs (ii) were translated in the supernatant collected after centrifugation of mixture of Flexi Rabbit Reticulo Lysate (Promega) incubated with Glutathione sepharose beads preloaded with GST.VIII or GST protein alone. The level of luciferase activity was measured using a luciferase kit (Promega) on a Luminometer (Turner Designs, Inc.). The results are shown as relative luciferase activity (iii). Error bars indicate SE of means for separate experiments. The relative luciferase intensity is determined based on GST compared to GST.pVIII. (B) In vivo . 293T cells were transfected with plasmid DNAs (2 μg of pcDNA3-RLuc-POLIRES-FLuc (i) and either 4 μg of pEY.pVIII or 4 μg of pEYFPN1). At 36 h post transfection, Firefly luciferase (FLuc) and Renilla reniformis luciferase (RLuc) activities were measured in a luminometer by using a dual luciferase assay kit (Promega) as per the company’s procedure. Expression of EYFP was used to normalize the transfection efficiency. The results are shown as relative luciferase activity (iii). The level of cytoplasmic RLuc-POLIRES-FLuc mRNA both in EY.pVIII and EYFP expressing plasmid transfected cells was quantified by RT-PCR (ii). Error bars indicate SE of means for three separate experiments. ∗ statistically significant.

Techniques Used: In Vitro, Luciferase, Synthesized, Centrifugation, Incubation, Activity Assay, In Vivo, Transfection, Plasmid Preparation, Expressing, Reverse Transcription Polymerase Chain Reaction

m7GTP-sepharose binding assay. (A) The supernatant of the lysates of the cells collected at 36 h post BAdV-3 infection of MDBK cells (mock or BAdV-3) or transfection of 293T cells with plasmid DNAs (pEY.pVIII or pEYFPN1) were incubated with m7GTP sepharose cap affinity beads. After washing, the bound proteins were analyzed by Western blot using indicated protein specific antibodies and IRDye 800 conjugated goat anti-mouse IgG or Alexa Flour 680 goat anti-rabbit IgG as secondary antibody. The intensity of the bands of the Western blot in all cases was analyzed by Odyssey Software v2.1. The relative amount of proteins in BAdV-3 infected or pEY.VIII transfected cell lysates that are retained in the 7-methyl GTP resins as compared to mock infected or pEYFPN1 transfected cells, respectively (i.e., considering the amount of protein in mock infected or pEYFPN1 transfected cell lysates that are retained in the m7GTP resins as 100%) is plotted. Error bars indicate SE of means for three separate experiments. Proteins from the lysates of BAdV-3 infected or transfected cells were separated by 10% SDS-PAGE and probed in Western blot using anti-pVIII serum. (B) Proteins from the lysates of mock infected or BAdV-3 infected MDBK cells collected at 36 h post infection were separated by 10% SDS-PAGE and analyzed by Western blot using protein specific antibody and anti-rabbit IRDye 800 conjugated goat anti-mouse IgG (Li-COR biosciences) or Alexa Flour 680 goat anti-rabbit IgG as secondary antibody. β-actin was used as a loading control.
Figure Legend Snippet: m7GTP-sepharose binding assay. (A) The supernatant of the lysates of the cells collected at 36 h post BAdV-3 infection of MDBK cells (mock or BAdV-3) or transfection of 293T cells with plasmid DNAs (pEY.pVIII or pEYFPN1) were incubated with m7GTP sepharose cap affinity beads. After washing, the bound proteins were analyzed by Western blot using indicated protein specific antibodies and IRDye 800 conjugated goat anti-mouse IgG or Alexa Flour 680 goat anti-rabbit IgG as secondary antibody. The intensity of the bands of the Western blot in all cases was analyzed by Odyssey Software v2.1. The relative amount of proteins in BAdV-3 infected or pEY.VIII transfected cell lysates that are retained in the 7-methyl GTP resins as compared to mock infected or pEYFPN1 transfected cells, respectively (i.e., considering the amount of protein in mock infected or pEYFPN1 transfected cell lysates that are retained in the m7GTP resins as 100%) is plotted. Error bars indicate SE of means for three separate experiments. Proteins from the lysates of BAdV-3 infected or transfected cells were separated by 10% SDS-PAGE and probed in Western blot using anti-pVIII serum. (B) Proteins from the lysates of mock infected or BAdV-3 infected MDBK cells collected at 36 h post infection were separated by 10% SDS-PAGE and analyzed by Western blot using protein specific antibody and anti-rabbit IRDye 800 conjugated goat anti-mouse IgG (Li-COR biosciences) or Alexa Flour 680 goat anti-rabbit IgG as secondary antibody. β-actin was used as a loading control.

Techniques Used: Binding Assay, Infection, Transfection, Plasmid Preparation, Incubation, Western Blot, Software, SDS Page

Interaction of DDX3 with BAdV-3 pVIII. (A) Glutathione S-transferase (GST) pull down assay. Purified GST or GST.pVIII fusion protein immobilized on Glutathione-Sepharose 4B beads, incubated with in vitro translated [ 35 S] methionine labeled HA tagged DDX3 were separated by 10% SDS-PAGE and detected by autoradiography. (B,C) Co-immunoprecipitation in transfected cells. Proteins from the lysates of cells co-transfected with either pHA.DX3 and pEY.pVIII or pHA.DX3 and pEYFPN1 were immunoprecipitated with anti-pVIII serum (B) or anti-HA MAb (C) , separated by 10% SDS-PAGE and transferred to nitrocellulose membrane. The separated proteins were probed in Western blot using anti-HA MAb (B) or anti-pVIII serum (C) . (D) Co-immunoprecipitation in BAdV-3 infected cells. Proteins from the lysates of mock or BAdV-3 infected Madin-Darby Bovine Kidney (MDBK) cells were immunoprecipitated with anti-pVIII serum, separated by 10% SDS-PAGE, transferred to nitrocellulose membrane and probed in Western blot using anti-DDX3 MAb. Immunoprecipitation (IP). WB (Western blot). Ctl (Control) . (E–G) Confocal microscopy. MDBK cells mock infected (panels a and f) or infected with BAdV-3 (panels d and g1–g4) VERO cells untransfected (panel b) or transfected with indicated plasmid (panels c, e, and h1–h4) DNA, were fixed 36 h post-infection/transfection. The subcellular localization of DDX3 (panels a–c, g2, and h2) protein was visualized by indirect immunofluorescence (panels a–c, g2, h2) using anti-DDX3 MAb and fluorescein conjugated goat anti-mouse IgG-FITC (panels a and g2), anti-DDX3 MAb and Cy3 conjugated goat anti-mouse (pane b) secondary antibody, anti-HA MAb and Cy3 conjugated goat anti-mouse secondary antibody (panel c and h2). The subcellular localization of pVIII (panels d, e, f, g1, and h1) was visualized by direct fluorescence (panels e and h1) or indirect immunofluorescence using anti-pVIII serum and Cy3 conjugated goat anti-rabbit secondary antibody (panels d, f, and g1). Nuclei were stained with DAPI in each panel. A merge of the images is shown. Enlargement of panel g4 and h4 is shown, arrows in white shows few of the colocalization of pVIII and DDX3.
Figure Legend Snippet: Interaction of DDX3 with BAdV-3 pVIII. (A) Glutathione S-transferase (GST) pull down assay. Purified GST or GST.pVIII fusion protein immobilized on Glutathione-Sepharose 4B beads, incubated with in vitro translated [ 35 S] methionine labeled HA tagged DDX3 were separated by 10% SDS-PAGE and detected by autoradiography. (B,C) Co-immunoprecipitation in transfected cells. Proteins from the lysates of cells co-transfected with either pHA.DX3 and pEY.pVIII or pHA.DX3 and pEYFPN1 were immunoprecipitated with anti-pVIII serum (B) or anti-HA MAb (C) , separated by 10% SDS-PAGE and transferred to nitrocellulose membrane. The separated proteins were probed in Western blot using anti-HA MAb (B) or anti-pVIII serum (C) . (D) Co-immunoprecipitation in BAdV-3 infected cells. Proteins from the lysates of mock or BAdV-3 infected Madin-Darby Bovine Kidney (MDBK) cells were immunoprecipitated with anti-pVIII serum, separated by 10% SDS-PAGE, transferred to nitrocellulose membrane and probed in Western blot using anti-DDX3 MAb. Immunoprecipitation (IP). WB (Western blot). Ctl (Control) . (E–G) Confocal microscopy. MDBK cells mock infected (panels a and f) or infected with BAdV-3 (panels d and g1–g4) VERO cells untransfected (panel b) or transfected with indicated plasmid (panels c, e, and h1–h4) DNA, were fixed 36 h post-infection/transfection. The subcellular localization of DDX3 (panels a–c, g2, and h2) protein was visualized by indirect immunofluorescence (panels a–c, g2, h2) using anti-DDX3 MAb and fluorescein conjugated goat anti-mouse IgG-FITC (panels a and g2), anti-DDX3 MAb and Cy3 conjugated goat anti-mouse (pane b) secondary antibody, anti-HA MAb and Cy3 conjugated goat anti-mouse secondary antibody (panel c and h2). The subcellular localization of pVIII (panels d, e, f, g1, and h1) was visualized by direct fluorescence (panels e and h1) or indirect immunofluorescence using anti-pVIII serum and Cy3 conjugated goat anti-rabbit secondary antibody (panels d, f, and g1). Nuclei were stained with DAPI in each panel. A merge of the images is shown. Enlargement of panel g4 and h4 is shown, arrows in white shows few of the colocalization of pVIII and DDX3.

Techniques Used: Pull Down Assay, Purification, Incubation, In Vitro, Labeling, SDS Page, Autoradiography, Immunoprecipitation, Transfection, Western Blot, Infection, CTL Assay, Confocal Microscopy, Plasmid Preparation, Immunofluorescence, Fluorescence, Staining

3) Product Images from "Tyr728 in the Kinase Domain of the Murine Kinase Suppressor of RAS 1 Regulates Binding and Activation of the Mitogen-activated Protein Kinase Kinase *"

Article Title: Tyr728 in the Kinase Domain of the Murine Kinase Suppressor of RAS 1 Regulates Binding and Activation of the Mitogen-activated Protein Kinase Kinase *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M113.490235

LCK phosphorylates KSR1 on Tyr 728 . A , amino acid sequence of the murine KSR1 depicting phosphorylation sites identified by MS analysis. The known phosphorylation sites are highlighted in green , whereas the newly identified confident phosphorylation sites are highlighted in red . The conserved regulatory domains are shaded in gray. B and C , GST-tagged KSR1 WT and mutants Y673F ( B ) and Y728F ( C ) were coexpressed with active LCK in COS7 cells and precipitated by use of glutathione-Sepharose beads. The levels of tyrosine phosphorylation were detected by an anti-phosphotyrosine antibody. PD , pulldown; IB , immunoblot; α, anti.
Figure Legend Snippet: LCK phosphorylates KSR1 on Tyr 728 . A , amino acid sequence of the murine KSR1 depicting phosphorylation sites identified by MS analysis. The known phosphorylation sites are highlighted in green , whereas the newly identified confident phosphorylation sites are highlighted in red . The conserved regulatory domains are shaded in gray. B and C , GST-tagged KSR1 WT and mutants Y673F ( B ) and Y728F ( C ) were coexpressed with active LCK in COS7 cells and precipitated by use of glutathione-Sepharose beads. The levels of tyrosine phosphorylation were detected by an anti-phosphotyrosine antibody. PD , pulldown; IB , immunoblot; α, anti.

Techniques Used: Sequencing, Mass Spectrometry

Substitution of Arg 649 and Tyr 728 with different amino acids results in distinct outcomes for MEK binding and phosphorylation. A and B , GST-KSR1 WT and the indicated mutants were expressed in COS7 cells and precipitated by use of glutathione-Sepharose beads. The amounts and phosphorylation levels of (co)precipitated proteins were determined by use of appropriate antibodies. C , data from three independent experiments were quantified by optical densitometry. The bar diagram shows the relative amount of phosphorylated MEK bound to KSR1, where 1-fold represents phosphorylation level of MEK interacting with KSR1 WT. The data are presented as means ± S.D. of the respective measured parameters. ns (not significant), p ≥ 0.05; * (significant), p
Figure Legend Snippet: Substitution of Arg 649 and Tyr 728 with different amino acids results in distinct outcomes for MEK binding and phosphorylation. A and B , GST-KSR1 WT and the indicated mutants were expressed in COS7 cells and precipitated by use of glutathione-Sepharose beads. The amounts and phosphorylation levels of (co)precipitated proteins were determined by use of appropriate antibodies. C , data from three independent experiments were quantified by optical densitometry. The bar diagram shows the relative amount of phosphorylated MEK bound to KSR1, where 1-fold represents phosphorylation level of MEK interacting with KSR1 WT. The data are presented as means ± S.D. of the respective measured parameters. ns (not significant), p ≥ 0.05; * (significant), p

Techniques Used: Binding Assay

4) Product Images from "An adventitious interaction of filamin A with RhoGDI2(Tyr153Glu)"

Article Title: An adventitious interaction of filamin A with RhoGDI2(Tyr153Glu)

Journal: Biochemical and biophysical research communications

doi: 10.1016/j.bbrc.2015.12.044

Identification of RhoGDI2 as a potential FLNA binding partner (A) A scheme of how mechanical force induces conformational changes of FLNA molecule. N-terminal actin-binding domain (ABD). Immunoglobulin-like repeat 1 (R1). R19 and R23 are indicated in red. The right panel shows a bait construct pGBKT7 R19+23, which expresses the fusion protein of the GAL4-DNA-binding domain and FLNA repeats 19 and 23. (B) Interaction of RhoGDI2 (82–201aa) with FLNA19+23 by yeast two-hybrid assay. Photo images displayed yeast colony growth (DDO: double dropout) and β-galactosidase staining (X-gal). Expression of a bait or prey alone did not yield growth. Expression of FLNA19+23 with full-length RhoGDI2 resulted in growth on DDO plate but no color development or growth on X-gal plate. (C) GST-RhoGDI2 pulldown assay. Halo-tagged FLNA19–23 was pulled down with GST-RhoGDI2 immobilized on glutathione-Sepharose beads. Coomassie Brilliant Blue (CBB) stained gel shows purified proteins used.
Figure Legend Snippet: Identification of RhoGDI2 as a potential FLNA binding partner (A) A scheme of how mechanical force induces conformational changes of FLNA molecule. N-terminal actin-binding domain (ABD). Immunoglobulin-like repeat 1 (R1). R19 and R23 are indicated in red. The right panel shows a bait construct pGBKT7 R19+23, which expresses the fusion protein of the GAL4-DNA-binding domain and FLNA repeats 19 and 23. (B) Interaction of RhoGDI2 (82–201aa) with FLNA19+23 by yeast two-hybrid assay. Photo images displayed yeast colony growth (DDO: double dropout) and β-galactosidase staining (X-gal). Expression of a bait or prey alone did not yield growth. Expression of FLNA19+23 with full-length RhoGDI2 resulted in growth on DDO plate but no color development or growth on X-gal plate. (C) GST-RhoGDI2 pulldown assay. Halo-tagged FLNA19–23 was pulled down with GST-RhoGDI2 immobilized on glutathione-Sepharose beads. Coomassie Brilliant Blue (CBB) stained gel shows purified proteins used.

Techniques Used: Binding Assay, Construct, Y2H Assay, Staining, Expressing, Purification

5) Product Images from "Measles Virus Infection Inactivates Cellular Protein Phosphatase 5 with Consequent Suppression of Sp1 and c-Myc Activities"

Article Title: Measles Virus Infection Inactivates Cellular Protein Phosphatase 5 with Consequent Suppression of Sp1 and c-Myc Activities

Journal: Journal of Virology

doi: 10.1128/JVI.00825-15

Identification of Sp1-associated kinase in cells under unstimulated conditions. (A) Phosphorylation of GST-Sp1 by Sp1-assocated kinase activity. GST-Sp1 was bound to glutathione-Sepharose beads and incubated with lysates from mock- or MeV-infected 293SLAM cells. After the beads were washed, the presence of kinase activity that could phosphorylate Sp1 was assessed by an in vitro kinase assay containing [γ- 32 P]ATP. The reactions were analyzed by SDS–10% PAGE and autoradiography. Arrowhead indicates phosphorylated GST-Sp1. (B) List of kinases reported to phosphorylate Sp1 and their corresponding specific inhibitors. (C) 293SLAM cell lysates treated with each kinase inhibitor and then subjected to an in vitro kinase assay.
Figure Legend Snippet: Identification of Sp1-associated kinase in cells under unstimulated conditions. (A) Phosphorylation of GST-Sp1 by Sp1-assocated kinase activity. GST-Sp1 was bound to glutathione-Sepharose beads and incubated with lysates from mock- or MeV-infected 293SLAM cells. After the beads were washed, the presence of kinase activity that could phosphorylate Sp1 was assessed by an in vitro kinase assay containing [γ- 32 P]ATP. The reactions were analyzed by SDS–10% PAGE and autoradiography. Arrowhead indicates phosphorylated GST-Sp1. (B) List of kinases reported to phosphorylate Sp1 and their corresponding specific inhibitors. (C) 293SLAM cell lysates treated with each kinase inhibitor and then subjected to an in vitro kinase assay.

Techniques Used: Activity Assay, Incubation, Infection, In Vitro, Kinase Assay, Polyacrylamide Gel Electrophoresis, Autoradiography

6) Product Images from "Docking-dependent Ubiquitination of the Interferon Regulatory Factor-1 Tumor Suppressor Protein by the Ubiquitin Ligase CHIP *"

Article Title: Docking-dependent Ubiquitination of the Interferon Regulatory Factor-1 Tumor Suppressor Protein by the Ubiquitin Ligase CHIP *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.153122

Identification of CHIP as a novel IRF-1-binding protein. A , recombinant GST and GST-IRF-1 were immobilized on glutathione-Sepharose beads and incubated with A375 cell lysate (1 mg). Bound proteins and the input ( Lysate ; 2.5% loaded on gel) were analyzed
Figure Legend Snippet: Identification of CHIP as a novel IRF-1-binding protein. A , recombinant GST and GST-IRF-1 were immobilized on glutathione-Sepharose beads and incubated with A375 cell lysate (1 mg). Bound proteins and the input ( Lysate ; 2.5% loaded on gel) were analyzed

Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Recombinant, Incubation

7) Product Images from "Phosphorylation-enabled binding of Sgo1-PP2A to cohesin protects sororin and centromeric cohesion during mitosis"

Article Title: Phosphorylation-enabled binding of Sgo1-PP2A to cohesin protects sororin and centromeric cohesion during mitosis

Journal: Nature cell biology

doi: 10.1038/ncb2637

Sgo1 T346 phosphorylation promotes its binding to cohesin ( a ) Mitotic HeLa Tet-On cells stably expressing Myc-Sgo1 WT or T346A (TA) were lysed with or without Turbo Nuclease. The total cell lysates (Input) and IgG/α-Smc1 immunoprecipitates (IP) were blotted with the indicated antibodies. ( b ) HeLa Tet-On cells were either mock transfected or transfected with siSgo1, collected at 7 hr after a thymidine block (G2) or in mitosis (M), and lysed with the nuclease-containing buffer. The total cell lysates and α-Smc1 IP were blotted with the indicated antibodies. ( c ) Glutathione-Sepharose beads bound to GST, GST-Sgo1 WT, or T346A (TA) proteins treated with buffer or cyclin B–Cdk1 were incubated with mitotic HeLa Tet-On cell extracts. The proteins bound to beads were blotted with the indicated antibodies. ( d ) Lysates of mitotic HeLa Tet-On cells stably expressing StrepII-SA2 were incubated with Strep-Tactin beads. After washing, the beads were incubated with GST-Sgo1 WT or T346A (TA) that had been treated with buffer or cyclin B–Cdk1. The input Sgo1 proteins and proteins bound to beads were blotted with the indicated antibodies. ( e ) HeLa Tet-On cells stably expressing Myc-Sgo1 were mocked transfected or transfected with the indicated siRNAs, collected at mitosis, and lysed in the presence of nuclease. The total cell lysates (Input) and α-Smc1 IP were blotted with the indicated antibodies. IgG IP from mock transfected cells was used as a negative control. Note that the commercial Wapl antibody (Bethyl) failed to detect Wapl in α-Smc1 IPs. ( f ) GST or GST-Sgo1 pre-treated with buffer or cyclin B–Cdk1 were immobilized on glutathione-Sepharose beads. The beads were then incubated with lysates of Sf9 cells expressing recombinant human Scc1-His 6 and SA2. The Sf9 lysate (Input) and proteins bound to beads were blotted with the indicated antibodies.
Figure Legend Snippet: Sgo1 T346 phosphorylation promotes its binding to cohesin ( a ) Mitotic HeLa Tet-On cells stably expressing Myc-Sgo1 WT or T346A (TA) were lysed with or without Turbo Nuclease. The total cell lysates (Input) and IgG/α-Smc1 immunoprecipitates (IP) were blotted with the indicated antibodies. ( b ) HeLa Tet-On cells were either mock transfected or transfected with siSgo1, collected at 7 hr after a thymidine block (G2) or in mitosis (M), and lysed with the nuclease-containing buffer. The total cell lysates and α-Smc1 IP were blotted with the indicated antibodies. ( c ) Glutathione-Sepharose beads bound to GST, GST-Sgo1 WT, or T346A (TA) proteins treated with buffer or cyclin B–Cdk1 were incubated with mitotic HeLa Tet-On cell extracts. The proteins bound to beads were blotted with the indicated antibodies. ( d ) Lysates of mitotic HeLa Tet-On cells stably expressing StrepII-SA2 were incubated with Strep-Tactin beads. After washing, the beads were incubated with GST-Sgo1 WT or T346A (TA) that had been treated with buffer or cyclin B–Cdk1. The input Sgo1 proteins and proteins bound to beads were blotted with the indicated antibodies. ( e ) HeLa Tet-On cells stably expressing Myc-Sgo1 were mocked transfected or transfected with the indicated siRNAs, collected at mitosis, and lysed in the presence of nuclease. The total cell lysates (Input) and α-Smc1 IP were blotted with the indicated antibodies. IgG IP from mock transfected cells was used as a negative control. Note that the commercial Wapl antibody (Bethyl) failed to detect Wapl in α-Smc1 IPs. ( f ) GST or GST-Sgo1 pre-treated with buffer or cyclin B–Cdk1 were immobilized on glutathione-Sepharose beads. The beads were then incubated with lysates of Sf9 cells expressing recombinant human Scc1-His 6 and SA2. The Sf9 lysate (Input) and proteins bound to beads were blotted with the indicated antibodies.

Techniques Used: Binding Assay, Stable Transfection, Expressing, Transfection, Blocking Assay, Incubation, Negative Control, Recombinant

8) Product Images from "C1D and hMtr4p associate with the human exosome subunit PM/Scl-100 and are involved in pre-rRNA processing"

Article Title: C1D and hMtr4p associate with the human exosome subunit PM/Scl-100 and are involved in pre-rRNA processing

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkm082

C1D, MPP6 and PM/Scl-100 form a trimeric complex in vitro . GST-tagged C1D was incubated with either 35 S-labeled, in vitro translated C1D (lane 5), MPP6 (lane 6), PM/Scl-100 (lane 7) or MPP6 and PM/Scl-100 (lane 8). As a control, GST alone was incubated with all three labeled proteins (lane 4). After incubation, GST(-C1D)-containing complexes were precipitated with glutathione-Sepharose beads and analyzed by SDS-PAGE and autoradiography. In lanes 1–3, the in vitro translated C1D, MPP6 and PM/Scl-100 proteins were analyzed.
Figure Legend Snippet: C1D, MPP6 and PM/Scl-100 form a trimeric complex in vitro . GST-tagged C1D was incubated with either 35 S-labeled, in vitro translated C1D (lane 5), MPP6 (lane 6), PM/Scl-100 (lane 7) or MPP6 and PM/Scl-100 (lane 8). As a control, GST alone was incubated with all three labeled proteins (lane 4). After incubation, GST(-C1D)-containing complexes were precipitated with glutathione-Sepharose beads and analyzed by SDS-PAGE and autoradiography. In lanes 1–3, the in vitro translated C1D, MPP6 and PM/Scl-100 proteins were analyzed.

Techniques Used: In Vitro, Incubation, Labeling, SDS Page, Autoradiography

Association of C1D, MPP6 and hMtr4p with components of the exosome. ( A ) Co-immunoprecipitations were performed using anti-EGFP antibodies and extracts of HEp-2 cells transiently transfected with expression constructs for either EGFP alone (lane 2), EGFP-PM/Scl-75 (lane 3) or EGFP-C1D (lane 4). In the first lane, total extract from non-transfected HEp-2 cells was separated. The precipitated proteins were analyzed by western blotting, using anti-PM/Scl-positive patient serum R212 (upper part of the blot) or a monoclonal antibody to hRrp4p (lower part of the blot). Arrows indicate the positions of PM/Scl-100 and hRrp4p. The positions of molecular weight markers are indicated on the left. ( B ) Bacterially expressed, recombinant His-tagged PM/Scl-100 was immobilized using anti-PM/Scl-100 antibodies and incubated with either 35 S-labeled, in vitro translated GAPDH (lane 1), C1D (lane 4) or MPP6 (lane 7). Co-precipitated proteins were analyzed by SDS-PAGE and autoradiography. The positions of these proteins are indicated with arrows, and the positions of molecular weight markers are indicated on the left. Lanes 3, 6 and 9 show proteins co-precipitated with His-PM/Scl-100. In lanes 2, 5 and 8, material from control incubations, in which no recombinant His-PM/Scl-100 was added, was analyzed. ( C ) Glutathione-Sepharose beads were used to precipitate GST (lane 2), GST-C1D (lane 3) or GST-MPP6 (lane 4), which were incubated with 35 S-labeled, in vitro translated PM/Scl-100. After precipitation, bound PM/Scl-100 was analyzed by SDS-PAGE and autoradiography. In lane 1, 10% of the amount of labeled PM/Scl-100 used per incubation was loaded. On the left, the positions of molecular weight markers are indicated. ( D ) Similar experiments as described in ( C ), but now with 35 S-labeled, in vitro translated hMtr4p instead of PM/Scl-100.
Figure Legend Snippet: Association of C1D, MPP6 and hMtr4p with components of the exosome. ( A ) Co-immunoprecipitations were performed using anti-EGFP antibodies and extracts of HEp-2 cells transiently transfected with expression constructs for either EGFP alone (lane 2), EGFP-PM/Scl-75 (lane 3) or EGFP-C1D (lane 4). In the first lane, total extract from non-transfected HEp-2 cells was separated. The precipitated proteins were analyzed by western blotting, using anti-PM/Scl-positive patient serum R212 (upper part of the blot) or a monoclonal antibody to hRrp4p (lower part of the blot). Arrows indicate the positions of PM/Scl-100 and hRrp4p. The positions of molecular weight markers are indicated on the left. ( B ) Bacterially expressed, recombinant His-tagged PM/Scl-100 was immobilized using anti-PM/Scl-100 antibodies and incubated with either 35 S-labeled, in vitro translated GAPDH (lane 1), C1D (lane 4) or MPP6 (lane 7). Co-precipitated proteins were analyzed by SDS-PAGE and autoradiography. The positions of these proteins are indicated with arrows, and the positions of molecular weight markers are indicated on the left. Lanes 3, 6 and 9 show proteins co-precipitated with His-PM/Scl-100. In lanes 2, 5 and 8, material from control incubations, in which no recombinant His-PM/Scl-100 was added, was analyzed. ( C ) Glutathione-Sepharose beads were used to precipitate GST (lane 2), GST-C1D (lane 3) or GST-MPP6 (lane 4), which were incubated with 35 S-labeled, in vitro translated PM/Scl-100. After precipitation, bound PM/Scl-100 was analyzed by SDS-PAGE and autoradiography. In lane 1, 10% of the amount of labeled PM/Scl-100 used per incubation was loaded. On the left, the positions of molecular weight markers are indicated. ( D ) Similar experiments as described in ( C ), but now with 35 S-labeled, in vitro translated hMtr4p instead of PM/Scl-100.

Techniques Used: Transfection, Expressing, Construct, Western Blot, Molecular Weight, Recombinant, Incubation, Labeling, In Vitro, SDS Page, Autoradiography

9) Product Images from "Modulation of Interleukin-1 Transcriptional Response by the Interaction between VRK2 and the JIP1 Scaffold Protein"

Article Title: Modulation of Interleukin-1 Transcriptional Response by the Interaction between VRK2 and the JIP1 Scaffold Protein

Journal: PLoS ONE

doi: 10.1371/journal.pone.0001660

Mapping the region of JIP1 that interacts with VRK2 isoforms. (A–F). Cos1 cells were transfected with plasmids as indicated in the corresponding lane. The expression of the proteins was determined by western blot (bottom panel). The different lysates were pulled down with Glutathione-Sepharose to bring down the GST-JIP1 protein and associated molecules. The pull-down proteins were detected with antibodies that recognize the HA epitope in the VRK2 proteins. The constructs derived from VRK2 includes the two isoforms expressed from plasmids pCEFL-HA-VRK2A and pCEFL-HA-VRK2B as well as their catalytically inactive kinase-dead (KD) variants containing the K169E substitution. JIP1ΔJBD lacks the JNK binding domain (residues 127-282). (G). In vitro interaction between human VRK2 and JIP1 proteins. The human VRK2 protein was in vitro transcribed-translated and labeled with 35S methionine. This protein was used for a pulldown assay with bacterially expressed GST-JIP1 constructs. (F). Schematic representation of the GST-JIP1 proteins used in the pull-down assay and the interacting region with VRK2. (H). Binding of endogenous VRK2A to GST-JIP1. Cos1 cell were transfected with pCEFL-GST or pEBG-GST-JIP1, and the proteins in the pull-down were identified with a specific polyclonal antibody for VRK2 and GST. (I). Binding of endogenous JIP1 protein to GST-VRK2A. Cos1 cells were transfected with pCEFL-GST or PCEFL-GST-VRK2A. The JIP1 protein in the pull-down was detected with a specific polyclonal antibody.
Figure Legend Snippet: Mapping the region of JIP1 that interacts with VRK2 isoforms. (A–F). Cos1 cells were transfected with plasmids as indicated in the corresponding lane. The expression of the proteins was determined by western blot (bottom panel). The different lysates were pulled down with Glutathione-Sepharose to bring down the GST-JIP1 protein and associated molecules. The pull-down proteins were detected with antibodies that recognize the HA epitope in the VRK2 proteins. The constructs derived from VRK2 includes the two isoforms expressed from plasmids pCEFL-HA-VRK2A and pCEFL-HA-VRK2B as well as their catalytically inactive kinase-dead (KD) variants containing the K169E substitution. JIP1ΔJBD lacks the JNK binding domain (residues 127-282). (G). In vitro interaction between human VRK2 and JIP1 proteins. The human VRK2 protein was in vitro transcribed-translated and labeled with 35S methionine. This protein was used for a pulldown assay with bacterially expressed GST-JIP1 constructs. (F). Schematic representation of the GST-JIP1 proteins used in the pull-down assay and the interacting region with VRK2. (H). Binding of endogenous VRK2A to GST-JIP1. Cos1 cell were transfected with pCEFL-GST or pEBG-GST-JIP1, and the proteins in the pull-down were identified with a specific polyclonal antibody for VRK2 and GST. (I). Binding of endogenous JIP1 protein to GST-VRK2A. Cos1 cells were transfected with pCEFL-GST or PCEFL-GST-VRK2A. The JIP1 protein in the pull-down was detected with a specific polyclonal antibody.

Techniques Used: Transfection, Expressing, Western Blot, Construct, Derivative Assay, Binding Assay, In Vitro, Labeling, Pull Down Assay

10) Product Images from "ProLIF – quantitative integrin protein–protein interactions and synergistic membrane effects on proteoliposomes"

Article Title: ProLIF – quantitative integrin protein–protein interactions and synergistic membrane effects on proteoliposomes

Journal: Journal of Cell Science

doi: 10.1242/jcs.214270

ProLIF is a flow cytometry-based assay for detection of specific protein-lipid interactions. (A) Outline of ProLIF workflow. Step 1: Bio-Beads™ are added to lipids solubilised in Triton X-100 to remove the detergent and obtain liposomes. Step 2: liposomes are incubated with membrane-free cell extract containing the EGFP-tagged protein of interest. Step 3: Streptavidin–Sepharose (SA) beads are added in order to capture the liposomes via interaction with biotinylated lipids present in the liposome membrane. Step 4: SA beads are analysed by flow cytometry (FACS). Red dots and blue dots represent biotinylated lipids and PIs, respectively. Green fragments represent EGFP-tagged proteins from the cell lysate. (B) Biotinylated-lipid-containing liposomes were generated with and without encapsulated Cy5 dye, captured on SA beads in the presence or absence of increasing amounts of free biotin and analysed via FACS. The molar ratio between biotinylated lipids and soluble biotin added in each sample is indicated ( n =1). (C) Scatter plot and fluorescence histogram from SA beads alone incubated with cell lysate from EGFP-transfected cells and analysed by FACS. (D) Biotinylated-lipid-containing liposomes, with the indicated PI content, were incubated with cell lysates from EGFP alone- or BTK-PH–EGFP-transfected cells (equal EGFP concentrations) and then captured by SA beads and analysed by FACS. Shown are representative dot blots, and size gating in FACS, and histograms depicting EGFP fluorescence intensity (FL1) profiles (note that the axis labels are as in C). The red population in the scatter plot was gated for quantification. Data shown represent three individual experiments. (E) Binding of the BTK-PH–EGFP domain (from cell lysate as in D) to biotinylated-lipid-containing liposomes, with the indicated PI content, relative to control PI-free liposomes (data are normalised median fluorescence intensities shown as the mean±s.e.m.; n =5 independent experiments). (F) Binding of EGFP-tagged PLC-PH domain (from cell lysate) to biotinylated-lipid-containing liposomes, with the indicated PI content, relative to control PI-free liposomes (data are normalised median fluorescence intensities shown as the mean±s.e.m.; n =5 independent experiments). (G) Binding of tandem FYVE-EGFP domains (from cell lysate) to biotinylated-lipid-containing liposomes, with the indicated PI content, relative to PI-free liposomes (data are normalised median fluorescence intensities shown as the mean ±s.e.m.; n =6 independent experiments). ** P
Figure Legend Snippet: ProLIF is a flow cytometry-based assay for detection of specific protein-lipid interactions. (A) Outline of ProLIF workflow. Step 1: Bio-Beads™ are added to lipids solubilised in Triton X-100 to remove the detergent and obtain liposomes. Step 2: liposomes are incubated with membrane-free cell extract containing the EGFP-tagged protein of interest. Step 3: Streptavidin–Sepharose (SA) beads are added in order to capture the liposomes via interaction with biotinylated lipids present in the liposome membrane. Step 4: SA beads are analysed by flow cytometry (FACS). Red dots and blue dots represent biotinylated lipids and PIs, respectively. Green fragments represent EGFP-tagged proteins from the cell lysate. (B) Biotinylated-lipid-containing liposomes were generated with and without encapsulated Cy5 dye, captured on SA beads in the presence or absence of increasing amounts of free biotin and analysed via FACS. The molar ratio between biotinylated lipids and soluble biotin added in each sample is indicated ( n =1). (C) Scatter plot and fluorescence histogram from SA beads alone incubated with cell lysate from EGFP-transfected cells and analysed by FACS. (D) Biotinylated-lipid-containing liposomes, with the indicated PI content, were incubated with cell lysates from EGFP alone- or BTK-PH–EGFP-transfected cells (equal EGFP concentrations) and then captured by SA beads and analysed by FACS. Shown are representative dot blots, and size gating in FACS, and histograms depicting EGFP fluorescence intensity (FL1) profiles (note that the axis labels are as in C). The red population in the scatter plot was gated for quantification. Data shown represent three individual experiments. (E) Binding of the BTK-PH–EGFP domain (from cell lysate as in D) to biotinylated-lipid-containing liposomes, with the indicated PI content, relative to control PI-free liposomes (data are normalised median fluorescence intensities shown as the mean±s.e.m.; n =5 independent experiments). (F) Binding of EGFP-tagged PLC-PH domain (from cell lysate) to biotinylated-lipid-containing liposomes, with the indicated PI content, relative to control PI-free liposomes (data are normalised median fluorescence intensities shown as the mean±s.e.m.; n =5 independent experiments). (G) Binding of tandem FYVE-EGFP domains (from cell lysate) to biotinylated-lipid-containing liposomes, with the indicated PI content, relative to PI-free liposomes (data are normalised median fluorescence intensities shown as the mean ±s.e.m.; n =6 independent experiments). ** P

Techniques Used: Flow Cytometry, Cytometry, Incubation, FACS, Generated, Fluorescence, Transfection, Binding Assay, Planar Chromatography

11) Product Images from "Regulation of Bone Morphogenetic Protein Signaling by ADP-ribosylation *"

Article Title: Regulation of Bone Morphogenetic Protein Signaling by ADP-ribosylation *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M116.729699

PARP1 ADP-ribosylates, whereas PARG de-ADP-ribosylates Smad1 and Smad5. A , in vitro ADP-ribosylation assay of Smad1, Smad5, Smad4, and Smad3. GST-Smad proteins were incubated with 32 P-β-NAD + and recombinant PARP1. After glutathione-agarose pulldown, ADP-ribosylated GST-Smad1/5/4/3 were imaged by autoradiography. The radioactive protein bands of PARP1 and GST-Smads are marked. The lower panel shows GST-Smad proteins stained with Coomassie Brilliant Blue after SDS-PAGE. M , molecular size marker. A representative autoradiogram of four assays is shown. Molecular size markers in kDa are also marked. B , in vitro de-PARylation of GST-Smad1 and GST-Smad5. PARG or vehicle were incubated with equal amounts of GST-Smad1/5, 32 P-β-NAD + , and recombinant PARP1 for 30 min at 37 °C. ADP-ribosylated proteins were imaged by autoradiography. The radioactive protein bands of PARP1 and GST-Smads are marked. The lower panel shows total GST proteins stained with Coomassie Brilliant Blue. M , molecular size marker. A representative autoradiogram of five assays is shown. Molecular size markers in kDa are also marked. C , immunoblot of endogenous PARP1 from HEK293T cell extracts bound to the indicated GST-Smad1 MH1 domain mutants. TCL shows the levels of endogenous PARP1. Total GST-Smad1 mutant proteins used for immunoblotting of endogenous PARP1 are stained with Coomassie Brilliant Blue in the middle panel . The Smad1 sequence motif that was mutated ( red letters ) and that represents a genuine ADP-ribosylation target sequence is shown in the bottom panel . A representative immunoblot of three repeats is shown. Molecular size markers in kDa are also marked. D , in vitro ADP-ribosylation assay of GST-Smad1-MH1 domain mutants. Control GST, beads, WT-Smad1-MH1 domain, and three mutants (as shown in C ) were incubated with 32 P-β-NAD + and recombinant PARP1. ADP-ribosylated proteins were imaged via autoradiography. The radioactive protein bands of PARP1 and GST-Smad1-MH1 are marked. Total GST proteins were checked by Coomassie Brilliant Blue staining. Lane 1/3 WT indicates a reaction where one-third of the GST-Smad1-MH1 protein was used compared with the WT lanes. A representative autoradiogram of two assays is shown. Molecular size markers in kDa are also marked. E , immunoblot of recombinant PARP1 (20 ng) bound to the indicated GST-Smad1 MH1 domain mutants. The experiment is a repeat of the ribosylation assay of Fig. 8 D , except that only cold β-NAD + was used during incubation, followed by pulldown and immunoblotting. On the side, increasing amounts of recombinant PARP1 along with TCL from HEK293T cells show the levels of recombinant PARP1 used in the assay relative to endogenous PARP1. Total GST-Smad1 mutant proteins checked by Coomassie Brilliant Blue staining, used for immunoblotting of recombinant PARP1. A representative immunoblot of two repeats is shown. Molecular size markers in kDa are also marked. F , molecular model adapted to a detail from the crystal structure of two Smad3 MH1 domains bound to the Smad-binding DNA element (PDB code 1mhd ). Shown is a ribbon diagram of the whole Smad3 MH1 domain with colored amino acids and the acceptor glutamate ( red ) and lysine ( blue ) residues drawn as stick and ball structures on the bottom side of the surface of the regulatory α-helix of one Smad3 MH1 subunit ( white arrow ). The β-hairpin that contacts DNA is also indicated ( white arrow ). WB , Western blotting.
Figure Legend Snippet: PARP1 ADP-ribosylates, whereas PARG de-ADP-ribosylates Smad1 and Smad5. A , in vitro ADP-ribosylation assay of Smad1, Smad5, Smad4, and Smad3. GST-Smad proteins were incubated with 32 P-β-NAD + and recombinant PARP1. After glutathione-agarose pulldown, ADP-ribosylated GST-Smad1/5/4/3 were imaged by autoradiography. The radioactive protein bands of PARP1 and GST-Smads are marked. The lower panel shows GST-Smad proteins stained with Coomassie Brilliant Blue after SDS-PAGE. M , molecular size marker. A representative autoradiogram of four assays is shown. Molecular size markers in kDa are also marked. B , in vitro de-PARylation of GST-Smad1 and GST-Smad5. PARG or vehicle were incubated with equal amounts of GST-Smad1/5, 32 P-β-NAD + , and recombinant PARP1 for 30 min at 37 °C. ADP-ribosylated proteins were imaged by autoradiography. The radioactive protein bands of PARP1 and GST-Smads are marked. The lower panel shows total GST proteins stained with Coomassie Brilliant Blue. M , molecular size marker. A representative autoradiogram of five assays is shown. Molecular size markers in kDa are also marked. C , immunoblot of endogenous PARP1 from HEK293T cell extracts bound to the indicated GST-Smad1 MH1 domain mutants. TCL shows the levels of endogenous PARP1. Total GST-Smad1 mutant proteins used for immunoblotting of endogenous PARP1 are stained with Coomassie Brilliant Blue in the middle panel . The Smad1 sequence motif that was mutated ( red letters ) and that represents a genuine ADP-ribosylation target sequence is shown in the bottom panel . A representative immunoblot of three repeats is shown. Molecular size markers in kDa are also marked. D , in vitro ADP-ribosylation assay of GST-Smad1-MH1 domain mutants. Control GST, beads, WT-Smad1-MH1 domain, and three mutants (as shown in C ) were incubated with 32 P-β-NAD + and recombinant PARP1. ADP-ribosylated proteins were imaged via autoradiography. The radioactive protein bands of PARP1 and GST-Smad1-MH1 are marked. Total GST proteins were checked by Coomassie Brilliant Blue staining. Lane 1/3 WT indicates a reaction where one-third of the GST-Smad1-MH1 protein was used compared with the WT lanes. A representative autoradiogram of two assays is shown. Molecular size markers in kDa are also marked. E , immunoblot of recombinant PARP1 (20 ng) bound to the indicated GST-Smad1 MH1 domain mutants. The experiment is a repeat of the ribosylation assay of Fig. 8 D , except that only cold β-NAD + was used during incubation, followed by pulldown and immunoblotting. On the side, increasing amounts of recombinant PARP1 along with TCL from HEK293T cells show the levels of recombinant PARP1 used in the assay relative to endogenous PARP1. Total GST-Smad1 mutant proteins checked by Coomassie Brilliant Blue staining, used for immunoblotting of recombinant PARP1. A representative immunoblot of two repeats is shown. Molecular size markers in kDa are also marked. F , molecular model adapted to a detail from the crystal structure of two Smad3 MH1 domains bound to the Smad-binding DNA element (PDB code 1mhd ). Shown is a ribbon diagram of the whole Smad3 MH1 domain with colored amino acids and the acceptor glutamate ( red ) and lysine ( blue ) residues drawn as stick and ball structures on the bottom side of the surface of the regulatory α-helix of one Smad3 MH1 subunit ( white arrow ). The β-hairpin that contacts DNA is also indicated ( white arrow ). WB , Western blotting.

Techniques Used: In Vitro, Incubation, Recombinant, Autoradiography, Staining, SDS Page, Marker, Mutagenesis, Sequencing, Binding Assay, Western Blot

12) Product Images from "A proteomic approach to identify endosomal cargoes controlling cancer invasiveness"

Article Title: A proteomic approach to identify endosomal cargoes controlling cancer invasiveness

Journal: Journal of Cell Science

doi: 10.1242/jcs.190835

Rab17 is a key regulator and interacting protein of the endosomal v-SNARE Vamp8. (A) Kaplan–Meier plot showing the influence of Rab17 expression on overall survival from breast cancer. The red and black lines represent patients with Rab17 expression above and below the median, respectively. n =1778 patients (low Rab17); n =1776 patients (high Rab17). Log rank test, P =1.2×10 −11 . (B) MDA-MB-231 cells that had been SILAC-labelled with heavy- and light-isotope amino acids were transfected with non-targeting siRNA (si-Con) or an siRNA targeting Rab17 (si-Rab17), respectively [forward (Fw) experiment]. For the reverse (Rev) experiment, these labelling conditions were swapped. Scatter plot indicates the SILAC ratio between siRab17 and siCon cells (si-Rab17/si-Con; log 2 scale) for each protein obtained for the forward versus the reverse experiments for the whole-cell proteome. Blue dotted lines indicate the regions encompassing significantly affected proteins (significance B statistical test, FDR of 5%, Perseus software). (C–E) MDA-MB-231 cells were transfected with siRNAs targeting Rab17 (SMARTPool or individual oligonucleotide Rab17#1), Vamp8 [SMARTPool (SP)], ERK2 or non-targeting controls (si-Con#1 and si-Con#2). Vamp8, Vamp7, Vamp3 and ERK1 and ERK2 expression levels were then determined by western blotting. In E, Rab17 expression was determined by using quantitative PCR (qPCR) relative to that in control (con) (graph). Data are mean±s.e.m. (F) MDA-MB-231 cells were transfected with GFP or GFP–Rab17. GFP-tagged proteins were isolated using GFP-Trap_A agarose beads (Chromotek). Proteins eluted from the beads were resolved by SDS-PAGE, stained with Coomassie Blue (Expedeon) and in-gel digested for MS analysis. Statistical testing of differences between means (Welch’s t -test) was performed to show the most differentially expressed proteins across three independent experimental replicates. The red dotted line indicates the significance threshold. Blue dots denote the 95 significant interacting proteins identified. (G) MDA-MB-231 cells were transfected with GFP, GFP–Rab17 or GFP–Rab24. GFP-tagged proteins were isolated as described in A, and immunoprecipitates were analysed by western blotting with antibodies recognising GFP, Vamp8 or Vamp7. IP, immunoprecipitation. (H) Zoom of plot in Fig. 1 F showing the identity (gene name) of late-endosome- and lysosome-associated proteins (red dots) in the Rab17 interactome. 43% of the members of the Rab17 interactome belong to the lysosome and late-endosome category, and have been annotated according to Chapel et al. (2013) .
Figure Legend Snippet: Rab17 is a key regulator and interacting protein of the endosomal v-SNARE Vamp8. (A) Kaplan–Meier plot showing the influence of Rab17 expression on overall survival from breast cancer. The red and black lines represent patients with Rab17 expression above and below the median, respectively. n =1778 patients (low Rab17); n =1776 patients (high Rab17). Log rank test, P =1.2×10 −11 . (B) MDA-MB-231 cells that had been SILAC-labelled with heavy- and light-isotope amino acids were transfected with non-targeting siRNA (si-Con) or an siRNA targeting Rab17 (si-Rab17), respectively [forward (Fw) experiment]. For the reverse (Rev) experiment, these labelling conditions were swapped. Scatter plot indicates the SILAC ratio between siRab17 and siCon cells (si-Rab17/si-Con; log 2 scale) for each protein obtained for the forward versus the reverse experiments for the whole-cell proteome. Blue dotted lines indicate the regions encompassing significantly affected proteins (significance B statistical test, FDR of 5%, Perseus software). (C–E) MDA-MB-231 cells were transfected with siRNAs targeting Rab17 (SMARTPool or individual oligonucleotide Rab17#1), Vamp8 [SMARTPool (SP)], ERK2 or non-targeting controls (si-Con#1 and si-Con#2). Vamp8, Vamp7, Vamp3 and ERK1 and ERK2 expression levels were then determined by western blotting. In E, Rab17 expression was determined by using quantitative PCR (qPCR) relative to that in control (con) (graph). Data are mean±s.e.m. (F) MDA-MB-231 cells were transfected with GFP or GFP–Rab17. GFP-tagged proteins were isolated using GFP-Trap_A agarose beads (Chromotek). Proteins eluted from the beads were resolved by SDS-PAGE, stained with Coomassie Blue (Expedeon) and in-gel digested for MS analysis. Statistical testing of differences between means (Welch’s t -test) was performed to show the most differentially expressed proteins across three independent experimental replicates. The red dotted line indicates the significance threshold. Blue dots denote the 95 significant interacting proteins identified. (G) MDA-MB-231 cells were transfected with GFP, GFP–Rab17 or GFP–Rab24. GFP-tagged proteins were isolated as described in A, and immunoprecipitates were analysed by western blotting with antibodies recognising GFP, Vamp8 or Vamp7. IP, immunoprecipitation. (H) Zoom of plot in Fig. 1 F showing the identity (gene name) of late-endosome- and lysosome-associated proteins (red dots) in the Rab17 interactome. 43% of the members of the Rab17 interactome belong to the lysosome and late-endosome category, and have been annotated according to Chapel et al. (2013) .

Techniques Used: Expressing, Multiple Displacement Amplification, Transfection, Software, Western Blot, Real-time Polymerase Chain Reaction, Isolation, SDS Page, Staining, Mass Spectrometry, Immunoprecipitation

13) Product Images from "The N-Terminal Region of the Human Autophagy Protein ATG16L1 Contains a Domain That Folds into a Helical Structure Consistent with Formation of a Coiled-Coil"

Article Title: The N-Terminal Region of the Human Autophagy Protein ATG16L1 Contains a Domain That Folds into a Helical Structure Consistent with Formation of a Coiled-Coil

Journal: PLoS ONE

doi: 10.1371/journal.pone.0076237

Purification of recombinant human ATG16L1 coiled-coil. (A) GST-CCD1-FLAG-6His expression produces a truncated product (orange arrowhead). (B) Glutathione sepharose purification of GST-CCD2-FLAG-6His. 1 – total cell lysate, 2 – soluble extract, 3 – unbound flow through, 4–7 successive elution fractions. (C) CCD2-FLAG-6His following removal of the GST tag by TEV cleavage (lane 1). (D) Elution fractions of CCD2-FLAG-6His after anion exchange. The position of the truncated protein is marked by an orange arrowhead. (E) CCD3-FLAG-6His (lane 1) is highly pure and shows no evidence of truncation following glutathione sepharose affinity purification, TEV cleavage and hydrophobic interaction chromatography. In all panels the black arrowhead marks the bands representing the expected size of the ATG16L1 construct; M denotes PageRuler™ Plus Prestained protein standards (Thermo Scientific).
Figure Legend Snippet: Purification of recombinant human ATG16L1 coiled-coil. (A) GST-CCD1-FLAG-6His expression produces a truncated product (orange arrowhead). (B) Glutathione sepharose purification of GST-CCD2-FLAG-6His. 1 – total cell lysate, 2 – soluble extract, 3 – unbound flow through, 4–7 successive elution fractions. (C) CCD2-FLAG-6His following removal of the GST tag by TEV cleavage (lane 1). (D) Elution fractions of CCD2-FLAG-6His after anion exchange. The position of the truncated protein is marked by an orange arrowhead. (E) CCD3-FLAG-6His (lane 1) is highly pure and shows no evidence of truncation following glutathione sepharose affinity purification, TEV cleavage and hydrophobic interaction chromatography. In all panels the black arrowhead marks the bands representing the expected size of the ATG16L1 construct; M denotes PageRuler™ Plus Prestained protein standards (Thermo Scientific).

Techniques Used: Purification, Recombinant, Expressing, Flow Cytometry, Affinity Purification, Hydrophobic Interaction Chromatography, Construct

14) Product Images from "PRAME Is a Golgi-Targeted Protein That Associates with the Elongin BC Complex and Is Upregulated by Interferon-Gamma and Bacterial PAMPs"

Article Title: PRAME Is a Golgi-Targeted Protein That Associates with the Elongin BC Complex and Is Upregulated by Interferon-Gamma and Bacterial PAMPs

Journal: PLoS ONE

doi: 10.1371/journal.pone.0058052

PRAME associates with the Elongin BC complex. ( A ) SDS-PAGE and silver staining showing affinity capture of proteins from HL60 whole cell extracts (WCE) by immobilised GST or GST-PRAME proteins. GST and GST-PRAME proteins are indicated. Putative PRAME-specific bands are indicated and bands of approximately 12 kDa and 17 kDa were excised for mass spectrometry analysis. ( B ) Co-immunoprecipitation of PRAME with Elongin complex components. Whole cell extracts of HEK293 cells transfected with PRAME-FLAG-6xHis (or empty vector control) applied to anti-FLAG sepharose beads as described in Materials and Methods. After extensive washing, co-purified PRAME and E3 ubiquitin ligase complex components were detected by western blotting using specific antibodies as indicated. ( C ) GST-pulldown experiment showing binding of ELB and ELC proteins in HL60 whole cell extracts to GST or GST-PRAME proteins. The top panel is a Coomassie-stained gel showing the input whole cell extract, and the purified GST and GST-PRAME proteins. The lower panels are western blots revealing PRAME, ELB and ELC proteins bound to GST proteins. ( D ) GST-pulldown experiments revealing interactions of 35 [S]-labelled in vitro translated human ELC (hELC), C.elegans ELC (wELC), C.elegans ELC (L47D-L49D-Y88D-Y91D) (wELC mutant) and C.elegans ELB proteins with GST or GST-PRAME. ( E ) Yeast two hybrid assays of LexA-PRAME interactions with GAL4 AD-fused human ELC (hELC) or C.elegans proteins (wELB, wELC, wELC mutant). Western blots of the HA-tagged elongin fusion proteins are also shown. Reporter activity is expressed as β-galactosidase activity normalised to amount of protein in the extracts. ( F ) Immunofluorescence staining showing subcellular localisation of endogenous ELC, ELB and CUL2 proteins in HL60 cells. ( G ) Immunofluorescence staining showing colocalisation of endogenous ELC and PRAME proteins in HL60 cells following treatment with LPS/IFNγ for 4 hours.
Figure Legend Snippet: PRAME associates with the Elongin BC complex. ( A ) SDS-PAGE and silver staining showing affinity capture of proteins from HL60 whole cell extracts (WCE) by immobilised GST or GST-PRAME proteins. GST and GST-PRAME proteins are indicated. Putative PRAME-specific bands are indicated and bands of approximately 12 kDa and 17 kDa were excised for mass spectrometry analysis. ( B ) Co-immunoprecipitation of PRAME with Elongin complex components. Whole cell extracts of HEK293 cells transfected with PRAME-FLAG-6xHis (or empty vector control) applied to anti-FLAG sepharose beads as described in Materials and Methods. After extensive washing, co-purified PRAME and E3 ubiquitin ligase complex components were detected by western blotting using specific antibodies as indicated. ( C ) GST-pulldown experiment showing binding of ELB and ELC proteins in HL60 whole cell extracts to GST or GST-PRAME proteins. The top panel is a Coomassie-stained gel showing the input whole cell extract, and the purified GST and GST-PRAME proteins. The lower panels are western blots revealing PRAME, ELB and ELC proteins bound to GST proteins. ( D ) GST-pulldown experiments revealing interactions of 35 [S]-labelled in vitro translated human ELC (hELC), C.elegans ELC (wELC), C.elegans ELC (L47D-L49D-Y88D-Y91D) (wELC mutant) and C.elegans ELB proteins with GST or GST-PRAME. ( E ) Yeast two hybrid assays of LexA-PRAME interactions with GAL4 AD-fused human ELC (hELC) or C.elegans proteins (wELB, wELC, wELC mutant). Western blots of the HA-tagged elongin fusion proteins are also shown. Reporter activity is expressed as β-galactosidase activity normalised to amount of protein in the extracts. ( F ) Immunofluorescence staining showing subcellular localisation of endogenous ELC, ELB and CUL2 proteins in HL60 cells. ( G ) Immunofluorescence staining showing colocalisation of endogenous ELC and PRAME proteins in HL60 cells following treatment with LPS/IFNγ for 4 hours.

Techniques Used: SDS Page, Silver Staining, Mass Spectrometry, Immunoprecipitation, Transfection, Plasmid Preparation, Purification, Western Blot, Binding Assay, Staining, In Vitro, Mutagenesis, Activity Assay, Immunofluorescence

15) Product Images from "Tumor suppressor protein DAB2IP participates in chromosomal stability maintenance through activating spindle assembly checkpoint and stabilizing kinetochore-microtubule attachments"

Article Title: Tumor suppressor protein DAB2IP participates in chromosomal stability maintenance through activating spindle assembly checkpoint and stabilizing kinetochore-microtubule attachments

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw746

DAB2IP interacts with Plk1 and promotes Plk1 activity. ( A ) Paired PC3 and ( B ) C4-2 cells were treated with nocodazole (50 ng/ml) for 16 h. Mitotic cells and untreated cells were analyzed by immunoblotting with anti- phospho-Plk1 (Thr210), anti-Plk1 and anti-Actin antibodies. ( C ) Mitotic and untreated C4-2 Neo and C4-2 D2 cells were analyzed for Plk1 activity by the Plk1 kinase assay using GST-Cdc25C as the substrate. Phosphorylation of GST-Cdc25C was visualized by autoradiography. Immunoblot analysis of Plk1 showed similar levels of Plk1 in the kinase assay. ( D ) C4-2 D2 cells were synchronized with nocodazole (50 ng/ml) for 16 h and mitotic cells were collected by shake off. Mitotic cell lysates were immunoprecipitated with anti-DAB2IP, anti-Plk1 antibodies or rabbit IgG followed by immunoblot analysis to detect DAB2IP and Plk1 levels. ( E ) Top: Schema of the truncated domains of DAB2IP. Bottom: Various cDNA constructs of DAB2IP truncations were co-transfected with HA-Plk1 into 293T cells and immunoprecipitated using anti-Flag antibody; HA signal was examined by Western blotting. ( F ) Direct protein–protein interaction between DAB2IP and Plk1. GST fusion proteins carrying C-terminal of DAB2IP was incubated with His-tagged full-length Plk1 or Plk1-PBD domain followed by glutathione agarose pull-down. The bound Plk1 or PBD domain was detected by anti-His antibody.
Figure Legend Snippet: DAB2IP interacts with Plk1 and promotes Plk1 activity. ( A ) Paired PC3 and ( B ) C4-2 cells were treated with nocodazole (50 ng/ml) for 16 h. Mitotic cells and untreated cells were analyzed by immunoblotting with anti- phospho-Plk1 (Thr210), anti-Plk1 and anti-Actin antibodies. ( C ) Mitotic and untreated C4-2 Neo and C4-2 D2 cells were analyzed for Plk1 activity by the Plk1 kinase assay using GST-Cdc25C as the substrate. Phosphorylation of GST-Cdc25C was visualized by autoradiography. Immunoblot analysis of Plk1 showed similar levels of Plk1 in the kinase assay. ( D ) C4-2 D2 cells were synchronized with nocodazole (50 ng/ml) for 16 h and mitotic cells were collected by shake off. Mitotic cell lysates were immunoprecipitated with anti-DAB2IP, anti-Plk1 antibodies or rabbit IgG followed by immunoblot analysis to detect DAB2IP and Plk1 levels. ( E ) Top: Schema of the truncated domains of DAB2IP. Bottom: Various cDNA constructs of DAB2IP truncations were co-transfected with HA-Plk1 into 293T cells and immunoprecipitated using anti-Flag antibody; HA signal was examined by Western blotting. ( F ) Direct protein–protein interaction between DAB2IP and Plk1. GST fusion proteins carrying C-terminal of DAB2IP was incubated with His-tagged full-length Plk1 or Plk1-PBD domain followed by glutathione agarose pull-down. The bound Plk1 or PBD domain was detected by anti-His antibody.

Techniques Used: Activity Assay, Kinase Assay, Autoradiography, Immunoprecipitation, Construct, Transfection, Western Blot, Incubation

16) Product Images from "Functional Interaction between Chfr and Kif22 Controls Genomic Stability *Functional Interaction between Chfr and Kif22 Controls Genomic Stability * S⃞"

Article Title: Functional Interaction between Chfr and Kif22 Controls Genomic Stability *Functional Interaction between Chfr and Kif22 Controls Genomic Stability * S⃞

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M900333200

Chfr interacts with Kif22. A , 293T cells stably expressing triple-tagged Chfr ( SFB-Chfr ) were used in tandem affinity purification, and the associated proteins in the Chfr complex were separated by SDS-PAGE and visualized by Coomassie staining. The presence of Kif22 in the Chfr complex identified by mass spectrometry analysis was indicated. MW , molecular mass standards. B and C , immunoprecipitation ( IP ) using control IgG or Kif22 ( B ) or Kif11 antibody ( C ) was performed using 293T extracts, and the association of endogenous Chfr was evaluated by immunoblotting. D , 293T cells were transfected with plasmids encoding FLAG-tagged Kif22 or Myc-tagged Chfr alone or in combination. The immunoprecipitation was performed using anti-FLAG antibodies, and the associated Chfr was identified by Western blotting ( WB ) using anti-Myc antibody. E , reverse co-immunoprecipitation was performed using anti-Myc antibody, and the presence of Kif22 in Chfr immunocomplex was examined by Western blotting using anti-FLAG antibody. F , either control GST or GST-Chfr fusion proteins immobilized on agarose beads were incubated with extracts prepared from 293T cells expressing FLAG-tagged Kif22, and the interaction of Kif22 with Chfr was assessed by immunoblotting. G , either control GST or GST-Chfr fusion proteins immobilized on agarose beads were incubated with bacterially expressed recombinant MBP-Kif22, and the interaction of Kif22 with Chfr was assessed by immunoblotting with anti-MBP antibody. H , T24 cells were allowed to grow to confluency for 96 h and then trypsinized and released into fresh medium. Samples were taken at the indicated time points and analyzed by fluorescence-activated cell sorter and Western blotting.
Figure Legend Snippet: Chfr interacts with Kif22. A , 293T cells stably expressing triple-tagged Chfr ( SFB-Chfr ) were used in tandem affinity purification, and the associated proteins in the Chfr complex were separated by SDS-PAGE and visualized by Coomassie staining. The presence of Kif22 in the Chfr complex identified by mass spectrometry analysis was indicated. MW , molecular mass standards. B and C , immunoprecipitation ( IP ) using control IgG or Kif22 ( B ) or Kif11 antibody ( C ) was performed using 293T extracts, and the association of endogenous Chfr was evaluated by immunoblotting. D , 293T cells were transfected with plasmids encoding FLAG-tagged Kif22 or Myc-tagged Chfr alone or in combination. The immunoprecipitation was performed using anti-FLAG antibodies, and the associated Chfr was identified by Western blotting ( WB ) using anti-Myc antibody. E , reverse co-immunoprecipitation was performed using anti-Myc antibody, and the presence of Kif22 in Chfr immunocomplex was examined by Western blotting using anti-FLAG antibody. F , either control GST or GST-Chfr fusion proteins immobilized on agarose beads were incubated with extracts prepared from 293T cells expressing FLAG-tagged Kif22, and the interaction of Kif22 with Chfr was assessed by immunoblotting. G , either control GST or GST-Chfr fusion proteins immobilized on agarose beads were incubated with bacterially expressed recombinant MBP-Kif22, and the interaction of Kif22 with Chfr was assessed by immunoblotting with anti-MBP antibody. H , T24 cells were allowed to grow to confluency for 96 h and then trypsinized and released into fresh medium. Samples were taken at the indicated time points and analyzed by fluorescence-activated cell sorter and Western blotting.

Techniques Used: Stable Transfection, Expressing, Affinity Purification, SDS Page, Staining, Mass Spectrometry, Immunoprecipitation, Transfection, Western Blot, Incubation, Recombinant, Fluorescence

17) Product Images from "The NS Segment of an H5N1 Highly Pathogenic Avian Influenza Virus (HPAIV) Is Sufficient To Alter Replication Efficiency, Cell Tropism, and Host Range of an H7N1 HPAIV ▿The NS Segment of an H5N1 Highly Pathogenic Avian Influenza Virus (HPAIV) Is Sufficient To Alter Replication Efficiency, Cell Tropism, and Host Range of an H7N1 HPAIV ▿ †"

Article Title: The NS Segment of an H5N1 Highly Pathogenic Avian Influenza Virus (HPAIV) Is Sufficient To Alter Replication Efficiency, Cell Tropism, and Host Range of an H7N1 HPAIV ▿The NS Segment of an H5N1 Highly Pathogenic Avian Influenza Virus (HPAIV) Is Sufficient To Alter Replication Efficiency, Cell Tropism, and Host Range of an H7N1 HPAIV ▿ †

Journal: Journal of Virology

doi: 10.1128/JVI.01668-09

The NS1 proteins from FPV and GD have similar F2F3-binding capacities. In a GST pulldown assay, in vitro translated and 35 S-labeled NS1 proteins of FPV, GD, or PR8 were mixed with F2F3-GST or H 2 O as a control and precipitated using glutathione-Sepharose
Figure Legend Snippet: The NS1 proteins from FPV and GD have similar F2F3-binding capacities. In a GST pulldown assay, in vitro translated and 35 S-labeled NS1 proteins of FPV, GD, or PR8 were mixed with F2F3-GST or H 2 O as a control and precipitated using glutathione-Sepharose

Techniques Used: Binding Assay, GST Pulldown Assay, In Vitro, Labeling

18) Product Images from "The BEACH-containing protein WDR81 coordinates p62 and LC3C to promote aggrephagy"

Article Title: The BEACH-containing protein WDR81 coordinates p62 and LC3C to promote aggrephagy

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201608039

WDR81 interacts with LC3C through two LIRs to facilitate aggrephagy. (A and B) Co-IP of GFP-tagged WDR81 fragments with mCh-LC3C. Individual GFP-WDR81 fragments were coexpressed with mCh-LC3C in HEK293 cells. IPs were performed with mCherry antibody, and precipitated proteins were detected with antibodies to GFP and mCherry. (C) Alignment of canonical and noncanonical LIRs in WDR81 with those in p62, NBR1, FUNDC1, or NDP52. Conserved residues are shown in red. (D) Co-IP of mCh-LC3C with wild-type (WT), LIR, or CLIR mutants of GFP-WDR81(341–650) in HEK293 cells. WDR81 mutations are shown in the box. IPs were performed with mCherry antibody and precipitated proteins were detected with antibodies to GFP and mCherry. (E) Co-IP of mCh-LC3C with WT or the indicated LIR mutants of Flag-WDR81 full-length protein in HEK293 cells. IPs were performed as in A. (F) Pull-down of Flag-WDR81 by GST-LC3C. Purified recombinant GST and GST-LC3C immobilized on glutathione-Sepharose beads were incubated overnight at 4°C with lysates of HEK293 cells expressing Flag-WDR81 or the indicated Flag-WDR81 mutants. After extensive washing, bound proteins were subjected to Western blot with Flag antibody. (G and H) KO-81 HeLa cells were transfected with Flag-WDR81 or the indicated Flag-WDR81 mutants. 48 h later, cells were subjected to immunostaining (G) or immunoblotting (H) with p62 antibody. Quantification (mean ± SEM) of p62 in (G) is shown in the right panel. ***, P
Figure Legend Snippet: WDR81 interacts with LC3C through two LIRs to facilitate aggrephagy. (A and B) Co-IP of GFP-tagged WDR81 fragments with mCh-LC3C. Individual GFP-WDR81 fragments were coexpressed with mCh-LC3C in HEK293 cells. IPs were performed with mCherry antibody, and precipitated proteins were detected with antibodies to GFP and mCherry. (C) Alignment of canonical and noncanonical LIRs in WDR81 with those in p62, NBR1, FUNDC1, or NDP52. Conserved residues are shown in red. (D) Co-IP of mCh-LC3C with wild-type (WT), LIR, or CLIR mutants of GFP-WDR81(341–650) in HEK293 cells. WDR81 mutations are shown in the box. IPs were performed with mCherry antibody and precipitated proteins were detected with antibodies to GFP and mCherry. (E) Co-IP of mCh-LC3C with WT or the indicated LIR mutants of Flag-WDR81 full-length protein in HEK293 cells. IPs were performed as in A. (F) Pull-down of Flag-WDR81 by GST-LC3C. Purified recombinant GST and GST-LC3C immobilized on glutathione-Sepharose beads were incubated overnight at 4°C with lysates of HEK293 cells expressing Flag-WDR81 or the indicated Flag-WDR81 mutants. After extensive washing, bound proteins were subjected to Western blot with Flag antibody. (G and H) KO-81 HeLa cells were transfected with Flag-WDR81 or the indicated Flag-WDR81 mutants. 48 h later, cells were subjected to immunostaining (G) or immunoblotting (H) with p62 antibody. Quantification (mean ± SEM) of p62 in (G) is shown in the right panel. ***, P

Techniques Used: Co-Immunoprecipitation Assay, Purification, Recombinant, Incubation, Expressing, Western Blot, Transfection, Immunostaining

WDR81 interacts with p62. (A) Immunostaining of p62 and WDR81 in wild-type (WT) and ATG5 −/− MEF cells. Insets show a magnified view (1.8×) of the boxed area in the merged images. (B) Images of BFP-WDR81 and GFP-p62 coexpressed in HeLa cells (top row); and images of overexpressed GFP-p62 and immunostained endogenous WDR81 (bottom). Dashed lines indicate the cell outline. (C) Colocalization of BFP-WDR81 with GFP-p62 (green arrows) or mCh-WDR91 (red arrows) in HeLa cells. (D) Colocalization of GFP-tagged WDR81 truncations with mCherry-p62 (mCh-p62) in HeLa cells. Schematic representations of the WDR81 truncations are shown in the top panel; the colocalizations are shown in the bottom panel. (E–G) Co-IP of HA-p62 with Flag-WDR81 (E), of HA-p62 with Flag-WDR81(1–650) (F), and of HA-p62 with Flag-WDR81(WD40) (G) in HEK293 cells. IPs were performed using Flag antibody, and precipitated proteins were detected with the indicated antibodies. (H) Interaction of GST-p62 with Flag-WDR81. Purified GST and GST-p62 (left) immobilized on glutathione-Sepharose beads were incubated for 12–16 h at 4°C with lysates of HEK293 cells expressing Flag-WDR81, Flag-WDR81(1–650), or Flag-WDR81(WD40). After extensive washing, bound proteins were subjected to immunoblotting (IB) with antibodies to Flag (right). (I) In vitro interaction of GST-p62 with 35 S-labeled WDR81. (J) Colocalization of GFP-WDR81(1–650) with mCh-tagged p62 truncations in HeLa cells. Schematic representations of the p62 truncations are shown in the top panel and protein colocalizations are shown in the bottom panel. Bars, 10 µm. H, leucine-rich nuclear export signal; L, LC3-interaction region; PB1, Phox and Bem1p domain; UBA, ubiquitin-associated domain; ZZ, ZZ-type zinc-finger domain. (K) In vitro interaction of GST-p62, GST-p62(PB1) with 35 S-labeled WDR81(1–650). (L) Co-IP of mCh-tagged p62 truncations with Flag-WDR81(1–650) in HEK293 cells. IPs were performed using Flag antibody, and precipitated proteins were detected with antibodies to Flag and mCherry. Bars: (main images) 10 µm; (A, insets) 5 µm.
Figure Legend Snippet: WDR81 interacts with p62. (A) Immunostaining of p62 and WDR81 in wild-type (WT) and ATG5 −/− MEF cells. Insets show a magnified view (1.8×) of the boxed area in the merged images. (B) Images of BFP-WDR81 and GFP-p62 coexpressed in HeLa cells (top row); and images of overexpressed GFP-p62 and immunostained endogenous WDR81 (bottom). Dashed lines indicate the cell outline. (C) Colocalization of BFP-WDR81 with GFP-p62 (green arrows) or mCh-WDR91 (red arrows) in HeLa cells. (D) Colocalization of GFP-tagged WDR81 truncations with mCherry-p62 (mCh-p62) in HeLa cells. Schematic representations of the WDR81 truncations are shown in the top panel; the colocalizations are shown in the bottom panel. (E–G) Co-IP of HA-p62 with Flag-WDR81 (E), of HA-p62 with Flag-WDR81(1–650) (F), and of HA-p62 with Flag-WDR81(WD40) (G) in HEK293 cells. IPs were performed using Flag antibody, and precipitated proteins were detected with the indicated antibodies. (H) Interaction of GST-p62 with Flag-WDR81. Purified GST and GST-p62 (left) immobilized on glutathione-Sepharose beads were incubated for 12–16 h at 4°C with lysates of HEK293 cells expressing Flag-WDR81, Flag-WDR81(1–650), or Flag-WDR81(WD40). After extensive washing, bound proteins were subjected to immunoblotting (IB) with antibodies to Flag (right). (I) In vitro interaction of GST-p62 with 35 S-labeled WDR81. (J) Colocalization of GFP-WDR81(1–650) with mCh-tagged p62 truncations in HeLa cells. Schematic representations of the p62 truncations are shown in the top panel and protein colocalizations are shown in the bottom panel. Bars, 10 µm. H, leucine-rich nuclear export signal; L, LC3-interaction region; PB1, Phox and Bem1p domain; UBA, ubiquitin-associated domain; ZZ, ZZ-type zinc-finger domain. (K) In vitro interaction of GST-p62, GST-p62(PB1) with 35 S-labeled WDR81(1–650). (L) Co-IP of mCh-tagged p62 truncations with Flag-WDR81(1–650) in HEK293 cells. IPs were performed using Flag antibody, and precipitated proteins were detected with antibodies to Flag and mCherry. Bars: (main images) 10 µm; (A, insets) 5 µm.

Techniques Used: Immunostaining, Co-Immunoprecipitation Assay, Purification, Incubation, Expressing, In Vitro, Labeling

19) Product Images from "Herpes Simplex Virus ICP27 Protein Directly Interacts with the Nuclear Pore Complex through Nup62, Inhibiting Host Nucleocytoplasmic Transport Pathways *"

Article Title: Herpes Simplex Virus ICP27 Protein Directly Interacts with the Nuclear Pore Complex through Nup62, Inhibiting Host Nucleocytoplasmic Transport Pathways *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.331777

Interaction of GST-Nup62 and ICP27 does not depend on RNA or virus infection. A , Nup62 fused to GST and purified from bacteria in the absence of any other mammalian proteins. Nup62-GST was incubated with WT HSV-1-, Δ27-, and mock-infected cell extracts or with extracts from uninfected cells expressing ICP27 from a plasmid behind the CMV promoter. Upper panel , Western blotting with Abs against ICP27. Lower panel , Western blotting with GAPDH Abs as a loading control for lysates. Input and pull-downs with GST alone were added on the ends for controls. B , GST-Nup62 was incubated with WT HSV-1-infected lysates either with or without RNase I treatment. Again, GST alone and input were used as controls. C , Rae1, another transport receptor, does not pull down ICP27. Bacterially purified GST-Rae1 (67 kDa) was incubated with lysates from WT HSV-1-, Δ27 null virus-, a mutant HSV-1 strain (M15) carrying a C-terminal point mutant (P465L/G466E) in ICP27-, or mock-infected cells. Upper panel , proteins co-purifying on glutathione-Sepharose beads were analyzed by Western blotting with Ab against ICP27. GST-Rae1 did not pull down ICP27 from any of the added cell extracts. A doublet band running lower in lanes 2–5 and marked with an asterisk is a band from the GST-Rae1 bacterial protein preparation cross-reacting nonspecifically with anti-ICP27 Ab. D , bacterially expressed GST-Rae1 protein from the fusion protein preparation used in binding assays shown in Fig. 4 C was analyzed on a Coomassie-stained gel. The expected full-length protein position is shown with an asterisk , whereas the double asterisks indicate cleavage products of the full-length protein. E , GST-Rae1 and GST-TAP pull-down known partner protein Nup98. Bacterially purified fusion protein GST-Rae1 (67 kDa) was incubated in the presence of RNase I with HSV-1 Δ27 null virus-infected or mock-infected cell lysates. Another fusion protein, GST-TAP, earlier shown to interact with Nup98, and GST alone were also incubated with HSV-1 WT virus-infected cell lysates. Proteins co-purifying on glutathione-Sepharose beads shown as bound samples and unbound leftover samples were analyzed by Western blotting with Ab against Nup98. Mock-infected and Δ27 null virus-infected cell lysates were loaded as input. Bound samples show that GST-Rae1 pulled down Nup98 from Δ27 null virus-infected or mock-infected cell lysates, and GST-TAP also pulled down Nup98 from WT virus-infected lysates. GST alone showed a weak band as background in bound samples; however, the majority of Nup98 is not pulled down and is present in unbound samples with GST alone, whereas not much is left as unbound with mock.
Figure Legend Snippet: Interaction of GST-Nup62 and ICP27 does not depend on RNA or virus infection. A , Nup62 fused to GST and purified from bacteria in the absence of any other mammalian proteins. Nup62-GST was incubated with WT HSV-1-, Δ27-, and mock-infected cell extracts or with extracts from uninfected cells expressing ICP27 from a plasmid behind the CMV promoter. Upper panel , Western blotting with Abs against ICP27. Lower panel , Western blotting with GAPDH Abs as a loading control for lysates. Input and pull-downs with GST alone were added on the ends for controls. B , GST-Nup62 was incubated with WT HSV-1-infected lysates either with or without RNase I treatment. Again, GST alone and input were used as controls. C , Rae1, another transport receptor, does not pull down ICP27. Bacterially purified GST-Rae1 (67 kDa) was incubated with lysates from WT HSV-1-, Δ27 null virus-, a mutant HSV-1 strain (M15) carrying a C-terminal point mutant (P465L/G466E) in ICP27-, or mock-infected cells. Upper panel , proteins co-purifying on glutathione-Sepharose beads were analyzed by Western blotting with Ab against ICP27. GST-Rae1 did not pull down ICP27 from any of the added cell extracts. A doublet band running lower in lanes 2–5 and marked with an asterisk is a band from the GST-Rae1 bacterial protein preparation cross-reacting nonspecifically with anti-ICP27 Ab. D , bacterially expressed GST-Rae1 protein from the fusion protein preparation used in binding assays shown in Fig. 4 C was analyzed on a Coomassie-stained gel. The expected full-length protein position is shown with an asterisk , whereas the double asterisks indicate cleavage products of the full-length protein. E , GST-Rae1 and GST-TAP pull-down known partner protein Nup98. Bacterially purified fusion protein GST-Rae1 (67 kDa) was incubated in the presence of RNase I with HSV-1 Δ27 null virus-infected or mock-infected cell lysates. Another fusion protein, GST-TAP, earlier shown to interact with Nup98, and GST alone were also incubated with HSV-1 WT virus-infected cell lysates. Proteins co-purifying on glutathione-Sepharose beads shown as bound samples and unbound leftover samples were analyzed by Western blotting with Ab against Nup98. Mock-infected and Δ27 null virus-infected cell lysates were loaded as input. Bound samples show that GST-Rae1 pulled down Nup98 from Δ27 null virus-infected or mock-infected cell lysates, and GST-TAP also pulled down Nup98 from WT virus-infected lysates. GST alone showed a weak band as background in bound samples; however, the majority of Nup98 is not pulled down and is present in unbound samples with GST alone, whereas not much is left as unbound with mock.

Techniques Used: Infection, Purification, Incubation, Expressing, Plasmid Preparation, Western Blot, Mutagenesis, Binding Assay, Staining

The Nup62-ICP27 interaction is direct, and TAP also binds ICP27 under similar conditions. A , GST-TAP pulls down ICP27 from WT HSV-1-infected cell extracts but not from extracts of cells infected with a viral ICP27 C-terminal point mutant (M15; upper panel ), whereas in the same binding reactions, GST-TAP pulls down Nup62 from WT HSV-1-infected cell extracts and from extracts of cells infected with a viral ICP27 C-terminal point mutant (M15) or with ICP27 null mutant Δ27 virus and from mock-infected cell extracts ( upper panel ). Bacterially expressed GST or GST-TAP was incubated with extracts from WT-, Δ27-, or M15-infected HSV-1 strains. Proteins co-purifying on glutathione-Sepharose beads were analyzed by Western blot with ICP27 or Nup62 Abs. B , ICP27 and TAP co-immunoprecipitate from HeLa cell extracts in the presence of RNase I. Co-immunoprecipitation was carried out by mixing anti-ICP27 antibodies 1113 and 1119 with HSV-1 WT and ICP27 mutant M15 and null Δ27 viruses and mock-infected HeLa cell extracts. Complexes formed were separated by SDS-PAGE followed by Western blotting with anti-TAP and ICP27 Abs. ICP27 co-precipitated TAP. Upper panel , co-immunoprecipitation of TAP in WT-infected HeLa cell extracts in the presence of RNase I by ICP27 but no co-immunoprecipitation with M15-, Δ27-, and mock-infected cells. Input , mock-infected HeLa cell extract. The lower band in the input lane could be due to another cellular protein cross-reacting with this TAP Ab, but the correct size upper band specific to TAP is clearly seen. The band marked with an asterisk is the heavy chain of IgGs used for immunoprecipitations. ICP27 immunoprecipitated itself. The lower panel shows immunoprecipitation of ICP27 in WT- and M15-infected HeLa cell extracts in the presence of RNase I by its Ab but no immunoprecipitation in Δ27- and mock-infected cell extracts. C , the purity of various fusion proteins used for in vitro binding assays. Coomassie-stained 10% SDS-PAGE of bacterially expressed proteins used in Figs. 4 – 7 . The single asterisk indicates the molecular weight of the expected protein product, whereas the double asterisks indicate cleavage products of the full-length protein. Prestained molecular weight protein markers are loaded on the leftmost lane with GST alone. D , bacterially purified His-tagged ICP27 was incubated with bacterially purified GST-Nup62, GST, or GST-TAP proteins on glutathione-Sepharose beads. Protein complexes formed were eluted by reduced glutathione and separated by SDS-PAGE and Western blotted with anti-His Ab. His-ICP27 interacted with GST-Nup62 and GST-TAP but not GST alone. E , an unrelated nonspecific His-tagged fusion protein (His-mRFP) does not interact with GST-tagged TAP or Nup62 fusion proteins. Bacterially purified His-tagged mRFP fusion protein was incubated with bacterially purified GST-TAP, GST-Nup62, or GST alone on glutathione-Sepharose beads in the presence of RNase I. Protein complexes eluted by reduced glutathione and separated by SDS-PAGE are shown on a Coomassie-stained gel. His-mRFP (corresponding to the band in input) does not interact with GST-TAP, GST-Nup62, and GST alone in bound samples run on gel ( upper panel ), but His-mRFP is present in all unbound pull-down samples run on Coomassie ( lower panel ). Purified His-mRFP alone protein added to the pull-down reactions was loaded as input. Molecular weight protein marker sizes are given in kDa on the left .
Figure Legend Snippet: The Nup62-ICP27 interaction is direct, and TAP also binds ICP27 under similar conditions. A , GST-TAP pulls down ICP27 from WT HSV-1-infected cell extracts but not from extracts of cells infected with a viral ICP27 C-terminal point mutant (M15; upper panel ), whereas in the same binding reactions, GST-TAP pulls down Nup62 from WT HSV-1-infected cell extracts and from extracts of cells infected with a viral ICP27 C-terminal point mutant (M15) or with ICP27 null mutant Δ27 virus and from mock-infected cell extracts ( upper panel ). Bacterially expressed GST or GST-TAP was incubated with extracts from WT-, Δ27-, or M15-infected HSV-1 strains. Proteins co-purifying on glutathione-Sepharose beads were analyzed by Western blot with ICP27 or Nup62 Abs. B , ICP27 and TAP co-immunoprecipitate from HeLa cell extracts in the presence of RNase I. Co-immunoprecipitation was carried out by mixing anti-ICP27 antibodies 1113 and 1119 with HSV-1 WT and ICP27 mutant M15 and null Δ27 viruses and mock-infected HeLa cell extracts. Complexes formed were separated by SDS-PAGE followed by Western blotting with anti-TAP and ICP27 Abs. ICP27 co-precipitated TAP. Upper panel , co-immunoprecipitation of TAP in WT-infected HeLa cell extracts in the presence of RNase I by ICP27 but no co-immunoprecipitation with M15-, Δ27-, and mock-infected cells. Input , mock-infected HeLa cell extract. The lower band in the input lane could be due to another cellular protein cross-reacting with this TAP Ab, but the correct size upper band specific to TAP is clearly seen. The band marked with an asterisk is the heavy chain of IgGs used for immunoprecipitations. ICP27 immunoprecipitated itself. The lower panel shows immunoprecipitation of ICP27 in WT- and M15-infected HeLa cell extracts in the presence of RNase I by its Ab but no immunoprecipitation in Δ27- and mock-infected cell extracts. C , the purity of various fusion proteins used for in vitro binding assays. Coomassie-stained 10% SDS-PAGE of bacterially expressed proteins used in Figs. 4 – 7 . The single asterisk indicates the molecular weight of the expected protein product, whereas the double asterisks indicate cleavage products of the full-length protein. Prestained molecular weight protein markers are loaded on the leftmost lane with GST alone. D , bacterially purified His-tagged ICP27 was incubated with bacterially purified GST-Nup62, GST, or GST-TAP proteins on glutathione-Sepharose beads. Protein complexes formed were eluted by reduced glutathione and separated by SDS-PAGE and Western blotted with anti-His Ab. His-ICP27 interacted with GST-Nup62 and GST-TAP but not GST alone. E , an unrelated nonspecific His-tagged fusion protein (His-mRFP) does not interact with GST-tagged TAP or Nup62 fusion proteins. Bacterially purified His-tagged mRFP fusion protein was incubated with bacterially purified GST-TAP, GST-Nup62, or GST alone on glutathione-Sepharose beads in the presence of RNase I. Protein complexes eluted by reduced glutathione and separated by SDS-PAGE are shown on a Coomassie-stained gel. His-mRFP (corresponding to the band in input) does not interact with GST-TAP, GST-Nup62, and GST alone in bound samples run on gel ( upper panel ), but His-mRFP is present in all unbound pull-down samples run on Coomassie ( lower panel ). Purified His-mRFP alone protein added to the pull-down reactions was loaded as input. Molecular weight protein marker sizes are given in kDa on the left .

Techniques Used: Infection, Mutagenesis, Binding Assay, Incubation, Western Blot, Immunoprecipitation, SDS Page, In Vitro, Staining, Molecular Weight, Purification, Marker

Mapping of Nup62 interaction sites on ICP27. A, schematic of ICP27, N-terminal deletion mutants, and C-terminal point mutants. The NES, NLS, and the RGG viral RNA binding site are marked along with the three hnRNP K homology domains ( KH1 to - 3 ). In the third conserved domain are the Sm and zinc finger regions that are disrupted by the M15 (P465L/G466E) and M16 point mutations (C488L) ( 57 ), respectively, and are represented with double and single asterisks , respectively. B , GST-Nup62 was incubated with lysates from cells infected with herpesviruses carrying the listed ICP27 mutations. The material that bound to GST-Nup62 and eluted from glutathione-Sepharose beads is shown in the upper panels , whereas the unbound material is shown in the lower panels to confirm that the viral proteins were expressed. All co-precipitated proteins are visualized with the ICP27 monoclonal Abs, either 1113 or 1119 because 1113 Ab is raised against a region of ICP27 that is missing in mutant d3–4 and 1119 Ab is raised against a region that is missing in dleu mutant. Standard GST alone and input controls were employed.
Figure Legend Snippet: Mapping of Nup62 interaction sites on ICP27. A, schematic of ICP27, N-terminal deletion mutants, and C-terminal point mutants. The NES, NLS, and the RGG viral RNA binding site are marked along with the three hnRNP K homology domains ( KH1 to - 3 ). In the third conserved domain are the Sm and zinc finger regions that are disrupted by the M15 (P465L/G466E) and M16 point mutations (C488L) ( 57 ), respectively, and are represented with double and single asterisks , respectively. B , GST-Nup62 was incubated with lysates from cells infected with herpesviruses carrying the listed ICP27 mutations. The material that bound to GST-Nup62 and eluted from glutathione-Sepharose beads is shown in the upper panels , whereas the unbound material is shown in the lower panels to confirm that the viral proteins were expressed. All co-precipitated proteins are visualized with the ICP27 monoclonal Abs, either 1113 or 1119 because 1113 Ab is raised against a region of ICP27 that is missing in mutant d3–4 and 1119 Ab is raised against a region that is missing in dleu mutant. Standard GST alone and input controls were employed.

Techniques Used: RNA Binding Assay, Incubation, Infection, Mutagenesis

Nup62 interacts with ORF57, an ICP27 homologue from KSHV. A , mock-infected HeLa extracts were incubated with either GST-ICP27, GST-ORF57 (a homologue of ICP27 from KSHV), or GST alone. Complexes isolated on glutathione-Sepharose beads were eluted after washing, separated on 10% SDS-PAGE, transferred to nitrocellulose membranes, and incubated with Abs against Nup62. Both ICP27 homologs pulled down Nup62. 33% of the HeLa cell extracts used for binding were loaded as input, and one-half of the pull-down reaction samples eluted from the beads were loaded on the gel. The lower band marked with an asterisk in lane 2 is a band from the GST-ICP27 protein preparation cross-reacting nonspecifically with Nup62 Ab. B , control protein GAPDH did not bind to ICP27 and ORF57 fusion proteins. Pulled down reactions of ICP27, ORF57 fusion proteins, and GST alone with HeLa lysates were blotted with anti-GAPDH Ab, and this showed GAPDH present only in the input and not pulled down in bound samples. C , Coomassie-stained 10% SDS-PAGE of bacterially expressed proteins GST-ICP27, GST-ORF57, and GST alone present on the beads used in the binding reactions here and in Fig. 5 A . The single asterisk indicates the molecular weight of the expected protein product, whereas the double asterisks indicate cleavage products of the full-length protein.
Figure Legend Snippet: Nup62 interacts with ORF57, an ICP27 homologue from KSHV. A , mock-infected HeLa extracts were incubated with either GST-ICP27, GST-ORF57 (a homologue of ICP27 from KSHV), or GST alone. Complexes isolated on glutathione-Sepharose beads were eluted after washing, separated on 10% SDS-PAGE, transferred to nitrocellulose membranes, and incubated with Abs against Nup62. Both ICP27 homologs pulled down Nup62. 33% of the HeLa cell extracts used for binding were loaded as input, and one-half of the pull-down reaction samples eluted from the beads were loaded on the gel. The lower band marked with an asterisk in lane 2 is a band from the GST-ICP27 protein preparation cross-reacting nonspecifically with Nup62 Ab. B , control protein GAPDH did not bind to ICP27 and ORF57 fusion proteins. Pulled down reactions of ICP27, ORF57 fusion proteins, and GST alone with HeLa lysates were blotted with anti-GAPDH Ab, and this showed GAPDH present only in the input and not pulled down in bound samples. C , Coomassie-stained 10% SDS-PAGE of bacterially expressed proteins GST-ICP27, GST-ORF57, and GST alone present on the beads used in the binding reactions here and in Fig. 5 A . The single asterisk indicates the molecular weight of the expected protein product, whereas the double asterisks indicate cleavage products of the full-length protein.

Techniques Used: Infection, Incubation, Isolation, SDS Page, Binding Assay, Staining, Molecular Weight

20) Product Images from "Several Phenylalanine-Glycine Motives in the Nucleoporin Nup214 Are Essential for Binding of the Nuclear Export Receptor CRM1 *"

Article Title: Several Phenylalanine-Glycine Motives in the Nucleoporin Nup214 Are Essential for Binding of the Nuclear Export Receptor CRM1 *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.433243

FG motives are involved in Nup214 binding to CRM1. A , schematic view of His- or GST-tagged Nup214 fragments and mutants. Dots indicate the approximate location of FGs. B , GST or GST-Ran loaded with GDP or GTP was immobilized on glutathione-Sepharose beads
Figure Legend Snippet: FG motives are involved in Nup214 binding to CRM1. A , schematic view of His- or GST-tagged Nup214 fragments and mutants. Dots indicate the approximate location of FGs. B , GST or GST-Ran loaded with GDP or GTP was immobilized on glutathione-Sepharose beads

Techniques Used: Binding Assay

21) Product Images from "Hypoxia-inducible Factor Prolyl-4-hydroxylase PHD2 Protein Abundance Depends on Integral Membrane Anchoring of FKBP38 *"

Article Title: Hypoxia-inducible Factor Prolyl-4-hydroxylase PHD2 Protein Abundance Depends on Integral Membrane Anchoring of FKBP38 *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.032631

FKBP38 regulates proteasomal activity. A and B , recombinant GST or GST-FKBP38 proteins were incubated with recombinant S2 or S4 and PHD2 proteins, and protein complexes were pulled down with glutathione-Sepharose beads, separated by SDS-PAGE, and visualized
Figure Legend Snippet: FKBP38 regulates proteasomal activity. A and B , recombinant GST or GST-FKBP38 proteins were incubated with recombinant S2 or S4 and PHD2 proteins, and protein complexes were pulled down with glutathione-Sepharose beads, separated by SDS-PAGE, and visualized

Techniques Used: Activity Assay, Recombinant, Incubation, SDS Page

Mapping the interaction domain of PHD2. A , schematic representation of the PHD2 domain architecture and the PHD2 constructs used. B , IVTT 35 S-labeled PHD2 variants were incubated with GST-FKBP38 or GST alone. Protein complexes were pulled down with glutathione-Sepharose
Figure Legend Snippet: Mapping the interaction domain of PHD2. A , schematic representation of the PHD2 domain architecture and the PHD2 constructs used. B , IVTT 35 S-labeled PHD2 variants were incubated with GST-FKBP38 or GST alone. Protein complexes were pulled down with glutathione-Sepharose

Techniques Used: Construct, Labeling, Incubation

22) Product Images from "Hypoxia-inducible Factor Prolyl-4-hydroxylase PHD2 Protein Abundance Depends on Integral Membrane Anchoring of FKBP38 *"

Article Title: Hypoxia-inducible Factor Prolyl-4-hydroxylase PHD2 Protein Abundance Depends on Integral Membrane Anchoring of FKBP38 *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.032631

FKBP38 regulates proteasomal activity. A and B , recombinant GST or GST-FKBP38 proteins were incubated with recombinant S2 or S4 and PHD2 proteins, and protein complexes were pulled down with glutathione-Sepharose beads, separated by SDS-PAGE, and visualized
Figure Legend Snippet: FKBP38 regulates proteasomal activity. A and B , recombinant GST or GST-FKBP38 proteins were incubated with recombinant S2 or S4 and PHD2 proteins, and protein complexes were pulled down with glutathione-Sepharose beads, separated by SDS-PAGE, and visualized

Techniques Used: Activity Assay, Recombinant, Incubation, SDS Page

Mapping the interaction domain of PHD2. A , schematic representation of the PHD2 domain architecture and the PHD2 constructs used. B , IVTT 35 S-labeled PHD2 variants were incubated with GST-FKBP38 or GST alone. Protein complexes were pulled down with glutathione-Sepharose
Figure Legend Snippet: Mapping the interaction domain of PHD2. A , schematic representation of the PHD2 domain architecture and the PHD2 constructs used. B , IVTT 35 S-labeled PHD2 variants were incubated with GST-FKBP38 or GST alone. Protein complexes were pulled down with glutathione-Sepharose

Techniques Used: Construct, Labeling, Incubation

23) Product Images from "PNUTS functions as a proto-oncogene by sequestering PTEN"

Article Title: PNUTS functions as a proto-oncogene by sequestering PTEN

Journal: Cancer research

doi: 10.1158/0008-5472.CAN-12-1394

PNUTS is a novel PTEN associated protein (a) Co-immunoprecipitation of endogenous PTEN and PNUTS was carried out using extracts prepared from HEK 293T cells. The presence of PNUTS in PTEN complex and vice versa was evaluated by immunoblotting with the indicated antibodies. (b) Lysates from bacteria expressing MBP-PTEN were pulled down with GST alone or GST-PNUTS containing sepharose beads and immunoblotted with anti-MBP antibody. (c) Schematic representation of N-terminal SFB-tagged PTEN (FL), along with its various deletion mutants (D1-D5). (d) SFB-tagged PTEN Full Length and domain deletions were expressed in HEK 293T cells along with Full Length MYC-PNUTS, the cell lysates were pulled down with Streptavidin beads and the interaction of PNUTS was detected with anti-Myc antibody. The expression of PTEN and PNUTS was checked by immunoblotting with anti-Flag and anti-Myc antibodies respectively. (e) Schematic representation of N-terminal SFB-tagged PNUTS (FL), along with its deletion mutants (D1-D7). (f) SFB-tagged PNUTS constructs and Myc-PTEN were co-expressed in HEK293T cells, the cell lysates were pulled down with streptavidin beads and the interaction of PTEN was detected by immunoblotting with anti-Myc antibodies.
Figure Legend Snippet: PNUTS is a novel PTEN associated protein (a) Co-immunoprecipitation of endogenous PTEN and PNUTS was carried out using extracts prepared from HEK 293T cells. The presence of PNUTS in PTEN complex and vice versa was evaluated by immunoblotting with the indicated antibodies. (b) Lysates from bacteria expressing MBP-PTEN were pulled down with GST alone or GST-PNUTS containing sepharose beads and immunoblotted with anti-MBP antibody. (c) Schematic representation of N-terminal SFB-tagged PTEN (FL), along with its various deletion mutants (D1-D5). (d) SFB-tagged PTEN Full Length and domain deletions were expressed in HEK 293T cells along with Full Length MYC-PNUTS, the cell lysates were pulled down with Streptavidin beads and the interaction of PNUTS was detected with anti-Myc antibody. The expression of PTEN and PNUTS was checked by immunoblotting with anti-Flag and anti-Myc antibodies respectively. (e) Schematic representation of N-terminal SFB-tagged PNUTS (FL), along with its deletion mutants (D1-D7). (f) SFB-tagged PNUTS constructs and Myc-PTEN were co-expressed in HEK293T cells, the cell lysates were pulled down with streptavidin beads and the interaction of PTEN was detected by immunoblotting with anti-Myc antibodies.

Techniques Used: Immunoprecipitation, Expressing, Construct

24) Product Images from "Nuclear import of cutaneous beta genus HPV8 E7 oncoprotein is mediated by hydrophobic interactions between its zinc-binding domain and FG nucleoporins"

Article Title: Nuclear import of cutaneous beta genus HPV8 E7 oncoprotein is mediated by hydrophobic interactions between its zinc-binding domain and FG nucleoporins

Journal: Virology

doi: 10.1016/j.virol.2013.11.020

A. HPV8 E7 interacts via its zinc-binding domain with the FG domain of Nup62 and mutations of hydrophobic residues disrupt its interaction. Hela cells were transfected with EGFP-8E7 (lane 1), EGFP-8E7 LRLFV65AAAAA (lane 2), EGFP-8E7 R66A (lane 3), EGFP-8cE7 (lane 4), EGFP-8cE7 LRLFV65AAAAA (lane 5), EGFP-8cE7 R66A (lane 6) and EGFP (lane 7) and cell lysates were prepared 24 h post-transfection and probed with a GFP antibody. HeLa cells lysate was also probed for Kap β2 (lane 8). GST-Nup62N (lanes 9-15) and GST (lanes 17 and 23) immobilized on glutathione-Sepharose were incubated with the cell lysates and the bound proteins were eluted and analyzed by immunobloting with a GFP antibody (lanes 9 and 17, EGFP-8E7; lanes 10 and 18, EGFP-8E7 LRLFV65AAAAA ; lanes 11 and 19, EGFP-8E7 R66A ; lanes 12 and 20, EGFP-8cE7; lanes 13 and 21, EGFP-8cE7 LRLFV65AAAAA ; lanes 14 and 22, EGFP-8cE7 R66A ; lanes 15 and 23, EGFP). Binding of Kap β2 to GST-Nup62N and GST was also analyzed (lanes 16 and 24). B. HPV8 E7 binds to Nup153 and mutations of hydrophobic residues within its zinc-binding domain inhibit this interaction. A. Hela cells were transfected with EGFP-8E7 (lane 1), EGFP-8E7 LRLFV65AAAAA (lane 2), EGFP-8E7 R66A (lane 3), EGFP-8cE7 (lane 4), EGFP-8cE7 LRLFV65AAAAA (lane 5), EGFP-8cE7 R66A (lane 6) and EGFP (lane 7) and cell lysates were prepared 24 h post transfection and probed with GFP antibody. HeLa cells lysate was also probed for Kap β2 (lane 8). GST-Nup153 (lanes 9 to 15) and GST (lanes 17 to 23) immobilized on glutathione-Sepharose were incubated with the cell lysates and the bound proteins were eluted and analyzed by immunobloting with a GFP antibody (lanes 9 and 17, EGFP-8E7; lanes 10 and 18, EGFP-8E7 LRLFV65AAAAA ; lanes 11 and 19, EGFP-8E7 R66A ; lanes 12 and 20, EGFP-8cE7; lanes 13 and 21, EGFP-8cE7 LRLFV65AAAAA ; lanes 14 and 22, EGFP-8cE7 R66A ; lanes 15 and 23, EGFP). Binding of Kap β2 to GST-Nup153 and GST was also analyzed (lanes 16 and 24).
Figure Legend Snippet: A. HPV8 E7 interacts via its zinc-binding domain with the FG domain of Nup62 and mutations of hydrophobic residues disrupt its interaction. Hela cells were transfected with EGFP-8E7 (lane 1), EGFP-8E7 LRLFV65AAAAA (lane 2), EGFP-8E7 R66A (lane 3), EGFP-8cE7 (lane 4), EGFP-8cE7 LRLFV65AAAAA (lane 5), EGFP-8cE7 R66A (lane 6) and EGFP (lane 7) and cell lysates were prepared 24 h post-transfection and probed with a GFP antibody. HeLa cells lysate was also probed for Kap β2 (lane 8). GST-Nup62N (lanes 9-15) and GST (lanes 17 and 23) immobilized on glutathione-Sepharose were incubated with the cell lysates and the bound proteins were eluted and analyzed by immunobloting with a GFP antibody (lanes 9 and 17, EGFP-8E7; lanes 10 and 18, EGFP-8E7 LRLFV65AAAAA ; lanes 11 and 19, EGFP-8E7 R66A ; lanes 12 and 20, EGFP-8cE7; lanes 13 and 21, EGFP-8cE7 LRLFV65AAAAA ; lanes 14 and 22, EGFP-8cE7 R66A ; lanes 15 and 23, EGFP). Binding of Kap β2 to GST-Nup62N and GST was also analyzed (lanes 16 and 24). B. HPV8 E7 binds to Nup153 and mutations of hydrophobic residues within its zinc-binding domain inhibit this interaction. A. Hela cells were transfected with EGFP-8E7 (lane 1), EGFP-8E7 LRLFV65AAAAA (lane 2), EGFP-8E7 R66A (lane 3), EGFP-8cE7 (lane 4), EGFP-8cE7 LRLFV65AAAAA (lane 5), EGFP-8cE7 R66A (lane 6) and EGFP (lane 7) and cell lysates were prepared 24 h post transfection and probed with GFP antibody. HeLa cells lysate was also probed for Kap β2 (lane 8). GST-Nup153 (lanes 9 to 15) and GST (lanes 17 to 23) immobilized on glutathione-Sepharose were incubated with the cell lysates and the bound proteins were eluted and analyzed by immunobloting with a GFP antibody (lanes 9 and 17, EGFP-8E7; lanes 10 and 18, EGFP-8E7 LRLFV65AAAAA ; lanes 11 and 19, EGFP-8E7 R66A ; lanes 12 and 20, EGFP-8cE7; lanes 13 and 21, EGFP-8cE7 LRLFV65AAAAA ; lanes 14 and 22, EGFP-8cE7 R66A ; lanes 15 and 23, EGFP). Binding of Kap β2 to GST-Nup153 and GST was also analyzed (lanes 16 and 24).

Techniques Used: Binding Assay, Transfection, Incubation, Western Blot

25) Product Images from "NADP+ is an endogenous PARP inhibitor in DNA damage response and tumor suppression"

Article Title: NADP+ is an endogenous PARP inhibitor in DNA damage response and tumor suppression

Journal: Nature Communications

doi: 10.1038/s41467-019-08530-5

NADP + suppresses poly(ADP-ribose) polymerase (PARP) activity in vitro. a PARPs can bind to NADP + . HIS-PARP1, HIS-PARP2, and GST-PARP10 were immobilized on Ni Sepharose and glutathione S -transferase (GST) beads respectively, followed by incubating with [ 32 P]NADP + . [ 32 P]NADP + was heat-released from Ni Sepharose or GST beads and examined by thin-layer chromatography. As the negative controls (NC), Ni Sepharose or GST beads without recombinant PARP proteins was incubated with [ 32 P]NADP + . b , c NADP + suppresses PARP1’s activity in vitro. In vitro PAPR1-mediated PARylation assay was performed using different ratio of NADP + /NAD + . Auto-PARylation of PARP1 was examined by western blotting ( b ). [ 32 P]NAD + was used as the donor in PARP1-mediated in vitro PARylation assay. The auto-PARylation of PARP1 was examined by autoradiography. Coomassie staining of His-PARP1 was shown as the loading control ( c ). d NADP + suppresses PARP2 in vitro. Auto-PARylation of PARP2 was examined by western blotting with the indicated antibody. e NADP + suppresses PARP10 in vitro. Auto-MARylation level of PARP10 was examined by autoradiography
Figure Legend Snippet: NADP + suppresses poly(ADP-ribose) polymerase (PARP) activity in vitro. a PARPs can bind to NADP + . HIS-PARP1, HIS-PARP2, and GST-PARP10 were immobilized on Ni Sepharose and glutathione S -transferase (GST) beads respectively, followed by incubating with [ 32 P]NADP + . [ 32 P]NADP + was heat-released from Ni Sepharose or GST beads and examined by thin-layer chromatography. As the negative controls (NC), Ni Sepharose or GST beads without recombinant PARP proteins was incubated with [ 32 P]NADP + . b , c NADP + suppresses PARP1’s activity in vitro. In vitro PAPR1-mediated PARylation assay was performed using different ratio of NADP + /NAD + . Auto-PARylation of PARP1 was examined by western blotting ( b ). [ 32 P]NAD + was used as the donor in PARP1-mediated in vitro PARylation assay. The auto-PARylation of PARP1 was examined by autoradiography. Coomassie staining of His-PARP1 was shown as the loading control ( c ). d NADP + suppresses PARP2 in vitro. Auto-PARylation of PARP2 was examined by western blotting with the indicated antibody. e NADP + suppresses PARP10 in vitro. Auto-MARylation level of PARP10 was examined by autoradiography

Techniques Used: Activity Assay, In Vitro, Thin Layer Chromatography, Recombinant, Incubation, Western Blot, Autoradiography, Staining

26) Product Images from "Neuronal Ceroid Lipofuscinoses Are Connected at Molecular Level: Interaction of CLN5 Protein with CLN2 and CLN3"

Article Title: Neuronal Ceroid Lipofuscinoses Are Connected at Molecular Level: Interaction of CLN5 Protein with CLN2 and CLN3

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E02-01-0031

Interaction analyses of CLN-proteins. (A) COS-1 cells were transfected by either WT or mutant CLN5 cDNA constructs, and cell lysates were immunoprecipitated with CLN2-specific antibody. CLN2 was expressed endogenously. Results were obtained by Western blotting with CLN5-specific antibody. (B) COS-1 cells were transfected with CLN3 cDNA construct and with either WT or mutant CLN5 cDNA constructs. Cell lysates were immunoprecipitated with CLN5-specific antibody. Results were obtained by Western blotting with a CLN3-specific antibody. (C) Radiolabeled CLN2 or CLN3, produced by in vitro translation, was coupled with GST or GST-CLN5 produced in E. coli and pulled down with Glutathione-Sepharose. Results were obtained by SDS-PAGE and fluorography. Coupled proteins are indicated above, molecular weights of the marker bands are shown on the left, and the CLN-specific bands on the right. Posit., crude COS-1 cell lysate (A) expressing endogenous CLN2 (B) transfected with CLN5 cDNA.
Figure Legend Snippet: Interaction analyses of CLN-proteins. (A) COS-1 cells were transfected by either WT or mutant CLN5 cDNA constructs, and cell lysates were immunoprecipitated with CLN2-specific antibody. CLN2 was expressed endogenously. Results were obtained by Western blotting with CLN5-specific antibody. (B) COS-1 cells were transfected with CLN3 cDNA construct and with either WT or mutant CLN5 cDNA constructs. Cell lysates were immunoprecipitated with CLN5-specific antibody. Results were obtained by Western blotting with a CLN3-specific antibody. (C) Radiolabeled CLN2 or CLN3, produced by in vitro translation, was coupled with GST or GST-CLN5 produced in E. coli and pulled down with Glutathione-Sepharose. Results were obtained by SDS-PAGE and fluorography. Coupled proteins are indicated above, molecular weights of the marker bands are shown on the left, and the CLN-specific bands on the right. Posit., crude COS-1 cell lysate (A) expressing endogenous CLN2 (B) transfected with CLN5 cDNA.

Techniques Used: Transfection, Mutagenesis, Construct, Immunoprecipitation, Western Blot, Produced, In Vitro, SDS Page, Marker, Expressing

27) Product Images from "HCMV Encoded Glycoprotein M (UL100) Interacts with Rab11 Effector Protein FIP4"

Article Title: HCMV Encoded Glycoprotein M (UL100) Interacts with Rab11 Effector Protein FIP4

Journal: Traffic (Copenhagen, Denmark)

doi: 10.1111/j.1600-0854.2009.00967.x

FIP4 interact with the gM-CT (A) Pull down assay in which glutathione sepharose beads containing gM-CT constructs (GST-gM-CT, GST-gM-ac1, GST-gM-ac2) or purified GST alone were incubated with the HK293 cell lysate expressing FIP4- myc . After extensive washing protein samples were boiled in sample buffer, resolved by SDS-PAGE and assayed by western analysis. Western blots were probed with anti-myc (9E10) mab and detected with the HRP (top panel). The lower panel shows the Coomassie blue stained gels of the GST purified input of proteins used in pull down assay. (B) Pull down assay of FIP3 or FIP4 by cytoplasmic tail of gM. Glutathione sepharose beads containing gM-CT constructs or GST alone were incubated with HK293 cell lysates expressing FIP3- myc or FIP4- myc. After extensive washing, beads were boiled and eluted proteins resolved by SDS-PAGE followed by western blot analysis. Western blots were probed with anti-myc mab and detected with the HRP. (C) Fluorescence Resonance Energy Transfer (FRET) indicating strong FIP4 and gM interaction in HCMV infected cells. For FRET assays, HFF cells were electroporated with FIP4 or FIP3 myc- tagged constructs and plated on the coverslips. 24 hours later, the cells were infected with HCMV. The cells were fixed in 4% PFA and stained with IMP anti-gM monoclonal antibodies (specific for the C-terminal tail of gM) or anti- myc antibody followed by labeling with secondary antibody conjugated with FITC or TxRed. A non-bleaching region was selected as an internal control of FRET analysis detail description of the assay is provided in Materials and Methods.
Figure Legend Snippet: FIP4 interact with the gM-CT (A) Pull down assay in which glutathione sepharose beads containing gM-CT constructs (GST-gM-CT, GST-gM-ac1, GST-gM-ac2) or purified GST alone were incubated with the HK293 cell lysate expressing FIP4- myc . After extensive washing protein samples were boiled in sample buffer, resolved by SDS-PAGE and assayed by western analysis. Western blots were probed with anti-myc (9E10) mab and detected with the HRP (top panel). The lower panel shows the Coomassie blue stained gels of the GST purified input of proteins used in pull down assay. (B) Pull down assay of FIP3 or FIP4 by cytoplasmic tail of gM. Glutathione sepharose beads containing gM-CT constructs or GST alone were incubated with HK293 cell lysates expressing FIP3- myc or FIP4- myc. After extensive washing, beads were boiled and eluted proteins resolved by SDS-PAGE followed by western blot analysis. Western blots were probed with anti-myc mab and detected with the HRP. (C) Fluorescence Resonance Energy Transfer (FRET) indicating strong FIP4 and gM interaction in HCMV infected cells. For FRET assays, HFF cells were electroporated with FIP4 or FIP3 myc- tagged constructs and plated on the coverslips. 24 hours later, the cells were infected with HCMV. The cells were fixed in 4% PFA and stained with IMP anti-gM monoclonal antibodies (specific for the C-terminal tail of gM) or anti- myc antibody followed by labeling with secondary antibody conjugated with FITC or TxRed. A non-bleaching region was selected as an internal control of FRET analysis detail description of the assay is provided in Materials and Methods.

Techniques Used: Pull Down Assay, Construct, Purification, Incubation, Expressing, SDS Page, Western Blot, Staining, Fluorescence, Förster Resonance Energy Transfer, Infection, Labeling

FIP4 bound to the gM-CT recruits Rab11 but fails to bind Arf5 or Arf6. HK293 cells were transfected with FIP4- myc alone or co-transfected with FIP4-myc and either with HA-Arf5, HA-Arf6, GFP-Rab11, and Arf1-HA as a control. Two days post transfection cells were lysed and analyzed by immunoprecipitation with myc antibody-tagged magnetic beads as described in the Materials and Methods or incubated with sepharose beads containing purified GST-gM-CT or GST alone. After immunoprecipitation and extensive washing, precipitated protein were resolved by SDS-PAGE, transferred to membranes and probed with appropriate antibody. The asterisk indicates HA-Arf6 band which was visualized only after prolonged exposure of the membrane that had been probed with anti-HA and developed with HRP.
Figure Legend Snippet: FIP4 bound to the gM-CT recruits Rab11 but fails to bind Arf5 or Arf6. HK293 cells were transfected with FIP4- myc alone or co-transfected with FIP4-myc and either with HA-Arf5, HA-Arf6, GFP-Rab11, and Arf1-HA as a control. Two days post transfection cells were lysed and analyzed by immunoprecipitation with myc antibody-tagged magnetic beads as described in the Materials and Methods or incubated with sepharose beads containing purified GST-gM-CT or GST alone. After immunoprecipitation and extensive washing, precipitated protein were resolved by SDS-PAGE, transferred to membranes and probed with appropriate antibody. The asterisk indicates HA-Arf6 band which was visualized only after prolonged exposure of the membrane that had been probed with anti-HA and developed with HRP.

Techniques Used: Transfection, Immunoprecipitation, Magnetic Beads, Incubation, Purification, SDS Page

28) Product Images from "CD4 and Major Histocompatibility Complex Class I Downregulation by the Human Immunodeficiency Virus Type 1 Nef Protein in Pediatric AIDS Progression"

Article Title: CD4 and Major Histocompatibility Complex Class I Downregulation by the Human Immunodeficiency Virus Type 1 Nef Protein in Pediatric AIDS Progression

Journal: Journal of Virology

doi: 10.1128/JVI.77.21.11536-11545.2003

Binding of AP-1 to GST-Nef fusion proteins. GST alone, GST-Nef wild-type (NL4-3), or GST fused to the indicated Nef proteins derived from patients was immobilized on Sepharose beads and incubated with Jurkat cell lysates. Bound proteins were eluted, separated by SDS-PAGE, immunoblotted with anti-AP-1 γ subunit (top panels) or anti-GST antibody (lower panels), and quantified by densitometry. As a control, 20 μg of Jurkat cell lysate was loaded on the same gel. Relative AP-1 binding activity was calculated as the amount of AP-1 γ subunit normalized for the amount of GST fusion protein and expressed as a percentage of the value measured for GST-wild-type Nef. Data representative of one of three independent experiments are shown. CD4 and MHC-I downregulation activities relative to wild-type Nef are indicated.
Figure Legend Snippet: Binding of AP-1 to GST-Nef fusion proteins. GST alone, GST-Nef wild-type (NL4-3), or GST fused to the indicated Nef proteins derived from patients was immobilized on Sepharose beads and incubated with Jurkat cell lysates. Bound proteins were eluted, separated by SDS-PAGE, immunoblotted with anti-AP-1 γ subunit (top panels) or anti-GST antibody (lower panels), and quantified by densitometry. As a control, 20 μg of Jurkat cell lysate was loaded on the same gel. Relative AP-1 binding activity was calculated as the amount of AP-1 γ subunit normalized for the amount of GST fusion protein and expressed as a percentage of the value measured for GST-wild-type Nef. Data representative of one of three independent experiments are shown. CD4 and MHC-I downregulation activities relative to wild-type Nef are indicated.

Techniques Used: Binding Assay, Derivative Assay, Incubation, SDS Page, Activity Assay

29) Product Images from "Paxillin phosphorylation at Ser273 localizes a GIT1-PIX-PAK complex and regulates adhesion and protrusion dynamics"

Article Title: Paxillin phosphorylation at Ser273 localizes a GIT1-PIX-PAK complex and regulates adhesion and protrusion dynamics

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200509075

S273-paxillin phosphorylation by PAK regulates paxillin–GIT1 binding. (a) CHO-K1 lysates treated (right) and untreated (left) with 5 nM CalyculinA (CalA) were probed (top) using a phospho–S273-paxillin–specific antibody. Total paxillin levels were assayed with an anti-paxillin antibody (bottom). A single band corresponding to the molecular mass of paxillin (∼68 kD) was detected in treated lysates. (b) Kinase assay was performed with FLAG-WT-paxillin and either KD- or CA-myc-PAK synthesized in vitro, and S273-paxillin phosphorylation was assessed with a phospho–S273-paxillin antibody. Bottom blots show equal loading by probing with anti-FLAG and anti-myc antibodies, respectively. Phospho–S273-paxillin levels increased eightfold with CA-PAK compared with KD-PAK. (c) Paxillin was immunoprecipitated using a GFP antibody from CHO-K1 lysates expressing paxillin-GFP and either KD- or CA-myc-PAK, and S273-paxillin phosphorylation levels were assayed using a phospho–S273-paxillin antibody. The lower two panels show equal levels of paxillin–GFP and myc-PAK, and the GFP blot shows equal loading in the lysates. S273-paxillin phosphorylation increased eightfold with CA-PAK as compared with KD-PAK. (d) A GFP antibody was used to immunoprecipitate paxillin from CHO-K1 lysates expressing GFP control or WT-, S273A-, or S273D-paxillin-GFP and FLAG-GIT1. GIT1 binding was probed using an anti-FLAG antibody. The bottom two panels show equivalent expression of S273-paxillin mutants and FLAG-GIT1 in the lysates. GIT1 binding to S273D-paxillin increased threefold, whereas it was reduced twofold with S273A-paxillin, when compared with WT-paxillin. (e) Paxillin was immunoprecipitated from CHO-K1 lysates expressing GFP control or WT-, S273A-, or S273D-paxillin-GFP and myc-FAK using a GFP antibody, and FAK binding was assessed with an anti-myc antibody. The bottom two panels show equivalent expression of S273-paxillin mutants and myc-FAK in the lysates. S273-paxillin phosphorylation only marginally affected FAK binding. (f) GIT1 was immunoprecipitated from in vitro mixtures of FLAG-GIT1, untagged WT-paxillin, and either KD- or CA-PAK using anti-FLAG M2-conjugated agarose, and phospho–S273-paxillin binding was probed using a phospho–S273-paxillin antibody. The middle blot shows equal levels of FLAG-GIT1 using an anti-FLAG antibody. (bottom) Equal loading of the lysates using anti-myc and anti-paxillin antibodies, respectively. Phospho–S273-paxillin–GIT1 binding increased sevenfold with CA-PAK compared with KD-PAK. (g) Anti-FLAG M2-conjugated agarose was used to immunoprecipitate GIT1 from in vitro mixtures of FLAG-GIT1, untagged WT-paxillin, and CA-PAK preincubated with 500-fold molar excess of phospho– or nonphospho–S273-paxillin peptide, and phospho–S273-paxillin binding was assessed with a phospho–S273-paxillin antibody. Very low levels of phospho–S273-paxillin–GIT1 binding was detected with the competitive phosphopeptide (left), whereas a robust signal was observed with the noncompetitive peptide (right), confirming that the PAK-mediated increase in phospho–S273-paxillin–GIT1 binding is specific to S273-paxillin phosphorylation.
Figure Legend Snippet: S273-paxillin phosphorylation by PAK regulates paxillin–GIT1 binding. (a) CHO-K1 lysates treated (right) and untreated (left) with 5 nM CalyculinA (CalA) were probed (top) using a phospho–S273-paxillin–specific antibody. Total paxillin levels were assayed with an anti-paxillin antibody (bottom). A single band corresponding to the molecular mass of paxillin (∼68 kD) was detected in treated lysates. (b) Kinase assay was performed with FLAG-WT-paxillin and either KD- or CA-myc-PAK synthesized in vitro, and S273-paxillin phosphorylation was assessed with a phospho–S273-paxillin antibody. Bottom blots show equal loading by probing with anti-FLAG and anti-myc antibodies, respectively. Phospho–S273-paxillin levels increased eightfold with CA-PAK compared with KD-PAK. (c) Paxillin was immunoprecipitated using a GFP antibody from CHO-K1 lysates expressing paxillin-GFP and either KD- or CA-myc-PAK, and S273-paxillin phosphorylation levels were assayed using a phospho–S273-paxillin antibody. The lower two panels show equal levels of paxillin–GFP and myc-PAK, and the GFP blot shows equal loading in the lysates. S273-paxillin phosphorylation increased eightfold with CA-PAK as compared with KD-PAK. (d) A GFP antibody was used to immunoprecipitate paxillin from CHO-K1 lysates expressing GFP control or WT-, S273A-, or S273D-paxillin-GFP and FLAG-GIT1. GIT1 binding was probed using an anti-FLAG antibody. The bottom two panels show equivalent expression of S273-paxillin mutants and FLAG-GIT1 in the lysates. GIT1 binding to S273D-paxillin increased threefold, whereas it was reduced twofold with S273A-paxillin, when compared with WT-paxillin. (e) Paxillin was immunoprecipitated from CHO-K1 lysates expressing GFP control or WT-, S273A-, or S273D-paxillin-GFP and myc-FAK using a GFP antibody, and FAK binding was assessed with an anti-myc antibody. The bottom two panels show equivalent expression of S273-paxillin mutants and myc-FAK in the lysates. S273-paxillin phosphorylation only marginally affected FAK binding. (f) GIT1 was immunoprecipitated from in vitro mixtures of FLAG-GIT1, untagged WT-paxillin, and either KD- or CA-PAK using anti-FLAG M2-conjugated agarose, and phospho–S273-paxillin binding was probed using a phospho–S273-paxillin antibody. The middle blot shows equal levels of FLAG-GIT1 using an anti-FLAG antibody. (bottom) Equal loading of the lysates using anti-myc and anti-paxillin antibodies, respectively. Phospho–S273-paxillin–GIT1 binding increased sevenfold with CA-PAK compared with KD-PAK. (g) Anti-FLAG M2-conjugated agarose was used to immunoprecipitate GIT1 from in vitro mixtures of FLAG-GIT1, untagged WT-paxillin, and CA-PAK preincubated with 500-fold molar excess of phospho– or nonphospho–S273-paxillin peptide, and phospho–S273-paxillin binding was assessed with a phospho–S273-paxillin antibody. Very low levels of phospho–S273-paxillin–GIT1 binding was detected with the competitive phosphopeptide (left), whereas a robust signal was observed with the noncompetitive peptide (right), confirming that the PAK-mediated increase in phospho–S273-paxillin–GIT1 binding is specific to S273-paxillin phosphorylation.

Techniques Used: Binding Assay, Kinase Assay, Synthesized, In Vitro, Immunoprecipitation, Expressing

30) Product Images from "?-Catenin Binds to the Activation Function 2 Region of the Androgen Receptor and Modulates the Effects of the N-Terminal Domain and TIF2 on Ligand-Dependent Transcription"

Article Title: ?-Catenin Binds to the Activation Function 2 Region of the Androgen Receptor and Modulates the Effects of the N-Terminal Domain and TIF2 on Ligand-Dependent Transcription

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.23.5.1674-1687.2003

(A) Structural arrangement of VP16/β-catenin constructs in this experiment, demonstrating the location of armadillo repeats. (B) CV-1 cells in a mammalian two-hybrid assay were cotransfected with 10 ng of GAL4/AR LBD, 100 ng of pFR-LUC, 10 ng of Renilla null luciferase reporter, and 50 ng of VP16/β-catenin(S33A), VP16/β-cateninΔN, or VP16/β-cateninΔC(S33A). (C) CV-1 cells in a transactivation assay were cotransfected with 10 ng of pCMVhAR507-919, 100 ng of MMTV-LUC, 50 ng of VP16/β-catenin(S33A), VP16/β-cateninΔN, VP16/β-cateninΔC(S33A), or equimolar amounts of VP16 empty vector. (D) In vitro interaction of the androgen receptor LBD with β-catenin. [ 35 S]AR LBD was synthesized in vitro and incubated in the presence or absence of 1 μM R1881, with glutathione-Sepharose beads bound with GST-β-catenin, GST-β-catenin(1-257), or GST-β-catenin(418-781). Control beads contained GST alone. Beads were washed, and bound 35 S-labeled proteins were eluted and analyzed by SDS-PAGE and autoradiography. The input lane contained 20% of the total radioactivity added for each reaction.
Figure Legend Snippet: (A) Structural arrangement of VP16/β-catenin constructs in this experiment, demonstrating the location of armadillo repeats. (B) CV-1 cells in a mammalian two-hybrid assay were cotransfected with 10 ng of GAL4/AR LBD, 100 ng of pFR-LUC, 10 ng of Renilla null luciferase reporter, and 50 ng of VP16/β-catenin(S33A), VP16/β-cateninΔN, or VP16/β-cateninΔC(S33A). (C) CV-1 cells in a transactivation assay were cotransfected with 10 ng of pCMVhAR507-919, 100 ng of MMTV-LUC, 50 ng of VP16/β-catenin(S33A), VP16/β-cateninΔN, VP16/β-cateninΔC(S33A), or equimolar amounts of VP16 empty vector. (D) In vitro interaction of the androgen receptor LBD with β-catenin. [ 35 S]AR LBD was synthesized in vitro and incubated in the presence or absence of 1 μM R1881, with glutathione-Sepharose beads bound with GST-β-catenin, GST-β-catenin(1-257), or GST-β-catenin(418-781). Control beads contained GST alone. Beads were washed, and bound 35 S-labeled proteins were eluted and analyzed by SDS-PAGE and autoradiography. The input lane contained 20% of the total radioactivity added for each reaction.

Techniques Used: Construct, Two Hybrid Assay, Luciferase, Transactivation Assay, Plasmid Preparation, In Vitro, Synthesized, Incubation, Labeling, SDS Page, Autoradiography, Radioactivity

31) Product Images from "The K-bZIP Protein from Kaposi's Sarcoma-Associated Herpesvirus Interacts with p53 and Represses Its Transcriptional Activity"

Article Title: The K-bZIP Protein from Kaposi's Sarcoma-Associated Herpesvirus Interacts with p53 and Represses Its Transcriptional Activity

Journal: Journal of Virology

doi:

K-bZIP interacts with p53 in vitro. Equal amounts of GST, GST-p53, and GST-KbZIP were incubated with 35 S-labeled in vitro-translated K-bZIP (A) or luciferase (Luc) (B), and an aliquot [Input (10%)] from each binding reaction was precipitated with glutathione-Sepharose beads. The bead-bound proteins were eluted and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). K-bZIP and Luc (both indicated by an arrow) were visualized by autoradiography. GST-KbZIP was used as a positive control for K-bZIP binding ability.
Figure Legend Snippet: K-bZIP interacts with p53 in vitro. Equal amounts of GST, GST-p53, and GST-KbZIP were incubated with 35 S-labeled in vitro-translated K-bZIP (A) or luciferase (Luc) (B), and an aliquot [Input (10%)] from each binding reaction was precipitated with glutathione-Sepharose beads. The bead-bound proteins were eluted and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). K-bZIP and Luc (both indicated by an arrow) were visualized by autoradiography. GST-KbZIP was used as a positive control for K-bZIP binding ability.

Techniques Used: In Vitro, Incubation, Labeling, Luciferase, Binding Assay, Polyacrylamide Gel Electrophoresis, SDS Page, Autoradiography, Positive Control

32) Product Images from "Using Affinity Chromatography to Investigate Novel Protein-Protein Interactions in an Undergraduate Cell and Molecular Biology Lab Course"

Article Title: Using Affinity Chromatography to Investigate Novel Protein-Protein Interactions in an Undergraduate Cell and Molecular Biology Lab Course

Journal: CBE Life Sciences Education

doi: 10.1187/cbe.09-03-0019

Students did not identify a subdomain of GST-Nup1-C that associates with Mex67-GFP. (A) Students expressed GST-Nup1 fusion proteins in E. coli and purified the recombinant GST fusion proteins using glutathione-Sepharose beads. For each sample, a lane
Figure Legend Snippet: Students did not identify a subdomain of GST-Nup1-C that associates with Mex67-GFP. (A) Students expressed GST-Nup1 fusion proteins in E. coli and purified the recombinant GST fusion proteins using glutathione-Sepharose beads. For each sample, a lane

Techniques Used: Purification, Recombinant

33) Product Images from "Coilin displays differential affinity for specific RNAs in vivo and is linked to telomerase RNA biogenesis"

Article Title: Coilin displays differential affinity for specific RNAs in vivo and is linked to telomerase RNA biogenesis

Journal: Journal of molecular biology

doi: 10.1016/j.jmb.2012.12.014

(A) Diagrams of seven GST-tagged coilin constructs showing precipitation behavior of equal total protein, 160 μg, following incubation with or without DNase and RNase, with “-” being no visible precipitation and “++++” the most relative precipitate; SA = self-association, FL = full length. (B) Top panel , Coomassie stained SDS-PAGE gel of equal total partially purified GST-tagged proteins untreated; Bottom panel , equal volume of soluble protein following incubation with DNase and RNase; * indicates full length GST-tagged protein of interest for each construct; % Soluble value indicates the percentage of soluble protein remaining after nuclease treatment, relative to untreated soluble protein, as calculated by relative desitometric analysis of protein bands in top and bottom panels. (C) Equal amounts of total GST-tagged protein loaded into an agarose gel and stained with ethidium bromide to visualize relative amounts of co-purified nucleic acid. (D) Representative image of visible precipitate which forms upon nuclease treatment, seen with RNase treatment or the combination of DNase and RNase.
Figure Legend Snippet: (A) Diagrams of seven GST-tagged coilin constructs showing precipitation behavior of equal total protein, 160 μg, following incubation with or without DNase and RNase, with “-” being no visible precipitation and “++++” the most relative precipitate; SA = self-association, FL = full length. (B) Top panel , Coomassie stained SDS-PAGE gel of equal total partially purified GST-tagged proteins untreated; Bottom panel , equal volume of soluble protein following incubation with DNase and RNase; * indicates full length GST-tagged protein of interest for each construct; % Soluble value indicates the percentage of soluble protein remaining after nuclease treatment, relative to untreated soluble protein, as calculated by relative desitometric analysis of protein bands in top and bottom panels. (C) Equal amounts of total GST-tagged protein loaded into an agarose gel and stained with ethidium bromide to visualize relative amounts of co-purified nucleic acid. (D) Representative image of visible precipitate which forms upon nuclease treatment, seen with RNase treatment or the combination of DNase and RNase.

Techniques Used: Construct, Incubation, Staining, SDS Page, Purification, Agarose Gel Electrophoresis

(A) Coomassie stained SDS-PAGE gels containing fully purified nucleic acid-free GST-tagged proteins; ladder to the right of each separate panel denotes marker locations of 175, 80, 58, 46, 30 and 25 kDa for each individual gel. (B) Agarose gels containing RNase assay results visualized by ethidium bromide; 500 ng RNA per lane; numbers beneath lanes indicate amount of protein in μg; the 28S rRNA band is indicated, with both the 32S and 18S also visible. (C) Line graph showing densitometric analysis of the 28S rRNA bands from images shown in B; percent band density of the control (no protein) band is plotted for each protein and amount.
Figure Legend Snippet: (A) Coomassie stained SDS-PAGE gels containing fully purified nucleic acid-free GST-tagged proteins; ladder to the right of each separate panel denotes marker locations of 175, 80, 58, 46, 30 and 25 kDa for each individual gel. (B) Agarose gels containing RNase assay results visualized by ethidium bromide; 500 ng RNA per lane; numbers beneath lanes indicate amount of protein in μg; the 28S rRNA band is indicated, with both the 32S and 18S also visible. (C) Line graph showing densitometric analysis of the 28S rRNA bands from images shown in B; percent band density of the control (no protein) band is plotted for each protein and amount.

Techniques Used: Staining, SDS Page, Purification, Marker

34) Product Images from "Regulation of CHMP4/ESCRT-III Function in Human Immunodeficiency Virus Type 1 Budding by CC2D1A"

Article Title: Regulation of CHMP4/ESCRT-III Function in Human Immunodeficiency Virus Type 1 Budding by CC2D1A

Journal: Journal of Virology

doi: 10.1128/JVI.06539-11

DM14 domain 1-dependent CHMP4B binding and inhibition of HIV-1 budding by an N-terminal CC2D1A fragment. (A) DM14 domain 1 is required for CHMP4B binding by an N-terminal CC2D1A fragment. The schematically illustrated GST fusion proteins were expressed in 293T cells together with CHMP4B-FLAG, and proteins precipitated from the cell lysates by glutathione-Sepharose beads were analyzed by Western blotting with anti-FLAG antibody or by Coomassie staining to detect the GST-DM14 fusion proteins. (B) Effects of the N-terminal CC2D1A fragments on the rescue of HIV-1 ΔPTAPP by ALIX.
Figure Legend Snippet: DM14 domain 1-dependent CHMP4B binding and inhibition of HIV-1 budding by an N-terminal CC2D1A fragment. (A) DM14 domain 1 is required for CHMP4B binding by an N-terminal CC2D1A fragment. The schematically illustrated GST fusion proteins were expressed in 293T cells together with CHMP4B-FLAG, and proteins precipitated from the cell lysates by glutathione-Sepharose beads were analyzed by Western blotting with anti-FLAG antibody or by Coomassie staining to detect the GST-DM14 fusion proteins. (B) Effects of the N-terminal CC2D1A fragments on the rescue of HIV-1 ΔPTAPP by ALIX.

Techniques Used: Binding Assay, Inhibition, Western Blot, Staining

CHMP4B binding by isolated DM14 domains and their effects on the function of ALIX in HIV-1 budding. (A) Ability of GST-DM14 domain fusion proteins to pull down CHMP4B. The schematically illustrated GST fusion proteins were expressed in 293T cells together with CHMP4B-HA, and proteins precipitated from the cell lysates by glutathione-Sepharose beads were analyzed by Western blotting with an anti-HA antibody or by Coomassie staining to detect the GST-DM14 fusion proteins. (B) Effects of the GST-DM14 domain fusion proteins on the rescue of HIV-1 ΔPTAPP by ALIX. 293T cells were cotransfected with ΔPTAPP HIV-1 proviral DNA, a vector expressing HA-ALIX, and vectors expressing GST or the indicated GST-DM14 domain fusion proteins. Virion pellets and the cell lysates were analyzed by Western blotting with anti-CA and anti-HA antibodies to detect Gag, Gag cleavage products, and ALIX.
Figure Legend Snippet: CHMP4B binding by isolated DM14 domains and their effects on the function of ALIX in HIV-1 budding. (A) Ability of GST-DM14 domain fusion proteins to pull down CHMP4B. The schematically illustrated GST fusion proteins were expressed in 293T cells together with CHMP4B-HA, and proteins precipitated from the cell lysates by glutathione-Sepharose beads were analyzed by Western blotting with an anti-HA antibody or by Coomassie staining to detect the GST-DM14 fusion proteins. (B) Effects of the GST-DM14 domain fusion proteins on the rescue of HIV-1 ΔPTAPP by ALIX. 293T cells were cotransfected with ΔPTAPP HIV-1 proviral DNA, a vector expressing HA-ALIX, and vectors expressing GST or the indicated GST-DM14 domain fusion proteins. Virion pellets and the cell lysates were analyzed by Western blotting with anti-CA and anti-HA antibodies to detect Gag, Gag cleavage products, and ALIX.

Techniques Used: Binding Assay, Isolation, Western Blot, Staining, Plasmid Preparation, Expressing

35) Product Images from "The Arabidopsis SOS2 protein kinase physically interacts with and is activated by the calcium-binding protein SOS3"

Article Title: The Arabidopsis SOS2 protein kinase physically interacts with and is activated by the calcium-binding protein SOS3

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

doi:

SOS2 interacts with SOS3 in vitro . ( A ) Interaction between SOS3 and SOS2 in a pull-down assay. [ 3 H]leucine-labeled SOS3 was incubated with glutathione-Sepharose immobilized GST, GST fusion proteins of SOS2, or RB. Proteins bound to the Sepharose beads were pelleted, washed thoroughly, electrophoresed, and detected by fluorography. ( B ) Interaction between SOS3 and SOS2 in a gel blot overlay assay. GST, partially purified GST-RB, GST-SOS2, GST-SOS2(K40N), GST-SOS2(G197E), and protein size markers (M) were separated by SDS/PAGE. Two identical gels were run; one was stained with Coomassie blue ( Left ), and the other was electroblotted and probed with [ 32 P]labeled SOS3 ( Right ).
Figure Legend Snippet: SOS2 interacts with SOS3 in vitro . ( A ) Interaction between SOS3 and SOS2 in a pull-down assay. [ 3 H]leucine-labeled SOS3 was incubated with glutathione-Sepharose immobilized GST, GST fusion proteins of SOS2, or RB. Proteins bound to the Sepharose beads were pelleted, washed thoroughly, electrophoresed, and detected by fluorography. ( B ) Interaction between SOS3 and SOS2 in a gel blot overlay assay. GST, partially purified GST-RB, GST-SOS2, GST-SOS2(K40N), GST-SOS2(G197E), and protein size markers (M) were separated by SDS/PAGE. Two identical gels were run; one was stained with Coomassie blue ( Left ), and the other was electroblotted and probed with [ 32 P]labeled SOS3 ( Right ).

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

36) Product Images from "Peroxisomal Targeting Signal Receptor Pex5p Interacts with Cargoes and Import Machinery Components in a Spatiotemporally Differentiated Manner: Conserved Pex5p WXXXF/Y Motifs Are Critical for Matrix Protein Import"

Article Title: Peroxisomal Targeting Signal Receptor Pex5p Interacts with Cargoes and Import Machinery Components in a Spatiotemporally Differentiated Manner: Conserved Pex5p WXXXF/Y Motifs Are Critical for Matrix Protein Import

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.22.6.1639-1655.2002

Functional domain mapping of Pex5pL. (A) Schematic representation of Chinese hamster ClPEX5L product constructs. The truncated products of ClPEX5L (the longer isoform of ClPEX5 ) were expressed in E. coli as fusion proteins placed at the C terminus of GST. Numbers, amino acid residues of ClPex5pL; vertical bars, highly conserved pentapeptide WXXXF/Y motifs; hatched bars, Pex5pL-specific 37-amino-acid sequence. (B) Interaction of Chinese hamster Pex5pL variants with other peroxins and PTS1. Binding assays were performed using recombinant fusions of GST to Pex5pL and its variants. GST fusion proteins (5 μg each) were incubated with lysates of 207P7 cells (10 6 ) expressing a high level of Pex7p. Purified Pex13p (0.1 μg) and His 6 -GFP-SKL (5 μg) were separately incubated with glutathione-Sepharose beads conjugated to GST fusion proteins (5 μg each). After a thorough washing, proteins bound to Sepharose beads were analyzed by SDS-PAGE on 12% gels and immunoblotting using antibodies specific for Pex7p, Pex13p, Pex14p, and GFP. (C) Heterologous expression and purification of recombinant PEX5L proteins. GST-Pex5pL variants were expressed in E. coli and purified as described in Materials and Methods. GST fusion proteins (1 μg each) were analyzed by SDS-PAGE and stained with Coomassie blue. (D) GST-Pex5pL(190-233) and GST-Pex5pL(190-233)S214F were likewise incubated with the lysates of 207P7 cells (10 6 ). Pex7p in bound fractions was probed with an anti-Pex7p antibody (top). GST fusion proteins were stained with Coomassie blue (bottom).
Figure Legend Snippet: Functional domain mapping of Pex5pL. (A) Schematic representation of Chinese hamster ClPEX5L product constructs. The truncated products of ClPEX5L (the longer isoform of ClPEX5 ) were expressed in E. coli as fusion proteins placed at the C terminus of GST. Numbers, amino acid residues of ClPex5pL; vertical bars, highly conserved pentapeptide WXXXF/Y motifs; hatched bars, Pex5pL-specific 37-amino-acid sequence. (B) Interaction of Chinese hamster Pex5pL variants with other peroxins and PTS1. Binding assays were performed using recombinant fusions of GST to Pex5pL and its variants. GST fusion proteins (5 μg each) were incubated with lysates of 207P7 cells (10 6 ) expressing a high level of Pex7p. Purified Pex13p (0.1 μg) and His 6 -GFP-SKL (5 μg) were separately incubated with glutathione-Sepharose beads conjugated to GST fusion proteins (5 μg each). After a thorough washing, proteins bound to Sepharose beads were analyzed by SDS-PAGE on 12% gels and immunoblotting using antibodies specific for Pex7p, Pex13p, Pex14p, and GFP. (C) Heterologous expression and purification of recombinant PEX5L proteins. GST-Pex5pL variants were expressed in E. coli and purified as described in Materials and Methods. GST fusion proteins (1 μg each) were analyzed by SDS-PAGE and stained with Coomassie blue. (D) GST-Pex5pL(190-233) and GST-Pex5pL(190-233)S214F were likewise incubated with the lysates of 207P7 cells (10 6 ). Pex7p in bound fractions was probed with an anti-Pex7p antibody (top). GST fusion proteins were stained with Coomassie blue (bottom).

Techniques Used: Functional Assay, Construct, Sequencing, Binding Assay, Recombinant, Incubation, Expressing, Purification, SDS Page, Staining

Interaction of Pex14p and Pex13p with cargo-loaded or unloaded Pex5p. (A) In vitro binding assays were performed using fusion proteins GST-Pex14p (2 μg), GST-Pex13p (2 μg), Pex5pL (2 μg), and His 6 -GFP-SKL (4 μg) (top) or recombinant catalase (4 μg) (bottom). GST pull-down assays were likewise done in the absence of cargoes (top, lanes 6 and 7). Components added to the assay mixtures, including GST in place of GST fusion proteins, are indicated at the top. Pex5pL, His 6 -GFP-SKL, and catalase in fractions bound to GST-Pex14p- and GST-Pex13p-linked Sepharose were detected by immunoblotting using antibodies specific for the respective proteins. (B) Formation of a hetero-oligomeric complex comprising Pex14p, Pex13p, Pex5p, and PTS1 cargo protein. Binding assays were done as for panel A using GST-Pex14p (2 μg), purified recombinant proteins, Pex13p (0.1 μg), Pex5pS (2 μg), Pex5pL (2 μg), and His 6 -GFP-SKL (4 μg). One-tenth aliquots of the input, Pex5pS, Pex13p, and His 6 -GFP-SKL were loaded in lane 10; Pex5pL was in lane 11. Components added to the assay mixtures are indicated at the top. Pex5p, Pex13p, and His 6 -GFP-SKL in fractions bound to GST-Pex14p were detected as for panel A.
Figure Legend Snippet: Interaction of Pex14p and Pex13p with cargo-loaded or unloaded Pex5p. (A) In vitro binding assays were performed using fusion proteins GST-Pex14p (2 μg), GST-Pex13p (2 μg), Pex5pL (2 μg), and His 6 -GFP-SKL (4 μg) (top) or recombinant catalase (4 μg) (bottom). GST pull-down assays were likewise done in the absence of cargoes (top, lanes 6 and 7). Components added to the assay mixtures, including GST in place of GST fusion proteins, are indicated at the top. Pex5pL, His 6 -GFP-SKL, and catalase in fractions bound to GST-Pex14p- and GST-Pex13p-linked Sepharose were detected by immunoblotting using antibodies specific for the respective proteins. (B) Formation of a hetero-oligomeric complex comprising Pex14p, Pex13p, Pex5p, and PTS1 cargo protein. Binding assays were done as for panel A using GST-Pex14p (2 μg), purified recombinant proteins, Pex13p (0.1 μg), Pex5pS (2 μg), Pex5pL (2 μg), and His 6 -GFP-SKL (4 μg). One-tenth aliquots of the input, Pex5pS, Pex13p, and His 6 -GFP-SKL were loaded in lane 10; Pex5pL was in lane 11. Components added to the assay mixtures are indicated at the top. Pex5p, Pex13p, and His 6 -GFP-SKL in fractions bound to GST-Pex14p were detected as for panel A.

Techniques Used: In Vitro, Binding Assay, Recombinant, Protein Binding, Purification

37) Product Images from "She4p/Dim1p Interacts with the Motor Domain of Unconventional Myosins in the Budding Yeast, Saccharomyces cerevisiae"

Article Title: She4p/Dim1p Interacts with the Motor Domain of Unconventional Myosins in the Budding Yeast, Saccharomyces cerevisiae

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E02-09-0616

She4p interacts with Myo5p in a temperature-dependent manner. Myo5p-TAP from YKT323 lysate was adsorbed to IgG Sepharose. GST-She4p was purified from bacterial lysate. IgG Sepharose with or without Myo5p-TAP was incubated with GST-She4p at 4 or 30°C. The IgG Sepharose was collected, and bound proteins were analyzed by SDS-PAGE and SYPRO orange staining. The result shown is a representative of four experiments.
Figure Legend Snippet: She4p interacts with Myo5p in a temperature-dependent manner. Myo5p-TAP from YKT323 lysate was adsorbed to IgG Sepharose. GST-She4p was purified from bacterial lysate. IgG Sepharose with or without Myo5p-TAP was incubated with GST-She4p at 4 or 30°C. The IgG Sepharose was collected, and bound proteins were analyzed by SDS-PAGE and SYPRO orange staining. The result shown is a representative of four experiments.

Techniques Used: Purification, Incubation, SDS Page, Staining

38) Product Images from "Novel regulation of mitotic exit by the Cdc42 effectors Gic1 and Gic2"

Article Title: Novel regulation of mitotic exit by the Cdc42 effectors Gic1 and Gic2

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200309080

Gic1 interacts with MEN components. (A) Gic1 interacts with Cdc42, Bfa1, Bub2, and Cdc14 in the yeast two-hybrid system. Yeast strains containing the indicated two-hybrid plasmids were overlaid with top agar containing X-Gal and were incubated for 3 h at 30°. (B) Gic1 binds directly to Bfa1, Bub2, Tem1, and Cdc14. Purified GST, GST-Gic1, and GST-Sec22 (20 nM) were bound to Sepharose beads and incubated with the indicated MBP fusion proteins (20 nM). Eluted proteins were analyzed by immunoblotting using antibodies against MBP and GST. The bottom panel shows GST-Gic1, GST-Sec22, and GST eluted from the Sepharose beads of the MBP-Bfa1 experiment (visualized with the anti-GST antibodies). Similar blots were obtained for the other binding experiments using MBP-Bub2, MBP-Tem1, and MBP-Cdc14.
Figure Legend Snippet: Gic1 interacts with MEN components. (A) Gic1 interacts with Cdc42, Bfa1, Bub2, and Cdc14 in the yeast two-hybrid system. Yeast strains containing the indicated two-hybrid plasmids were overlaid with top agar containing X-Gal and were incubated for 3 h at 30°. (B) Gic1 binds directly to Bfa1, Bub2, Tem1, and Cdc14. Purified GST, GST-Gic1, and GST-Sec22 (20 nM) were bound to Sepharose beads and incubated with the indicated MBP fusion proteins (20 nM). Eluted proteins were analyzed by immunoblotting using antibodies against MBP and GST. The bottom panel shows GST-Gic1, GST-Sec22, and GST eluted from the Sepharose beads of the MBP-Bfa1 experiment (visualized with the anti-GST antibodies). Similar blots were obtained for the other binding experiments using MBP-Bub2, MBP-Tem1, and MBP-Cdc14.

Techniques Used: Incubation, Purification, Binding Assay

39) Product Images from "SM protein Munc18-2 facilitates transition of Syntaxin 11-mediated lipid mixing to complete fusion for T-lymphocyte cytotoxicity"

Article Title: SM protein Munc18-2 facilitates transition of Syntaxin 11-mediated lipid mixing to complete fusion for T-lymphocyte cytotoxicity

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

doi: 10.1073/pnas.1617981114

VAMP8 or VAMP2 bind to a recombinant STX11/GST–SNAP23 complex. ( A ) Equivalent amounts of recombinant GST (GST alone) or GST–SNAP23 were bound to glutathione-Sepharose beads, and increasing concentrations (0.5, 1.0, or 1.5 μg) of
Figure Legend Snippet: VAMP8 or VAMP2 bind to a recombinant STX11/GST–SNAP23 complex. ( A ) Equivalent amounts of recombinant GST (GST alone) or GST–SNAP23 were bound to glutathione-Sepharose beads, and increasing concentrations (0.5, 1.0, or 1.5 μg) of

Techniques Used: Recombinant

40) Product Images from "Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap"

Article Title: Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2016.02119

Interaction of DDX3 with PAdV-3 and HAdV-5 pVIII. (A) Coomassie blue staining of purified protein. Purified GST.DDX3 protein was separated by 10% SDS-PAGE and stained with 0.25 Coomassie blue stain. (B) GST-pull down assay. Purified GSTor GST.DDX3 fusion protein immobilized on Glutathione-Sepharose 4B beads, incubated individually with in vitro translated [ 35 S] methionine labeled PAdV-3 pVIII or HAdV-5 pVIII, separated by 10% SDS-PAGE and detected by autoradiography. (C) Co-immunoprecipitation. Radio labeled in vitro transcribed and translated HAdV5 pVIII or PAdV-3 pVIII was incubated with in vitro transcribed and translated unlabeled DDX3 protein. Proteins were immunoprecipitated with either anti-DDX3 serum or rabbit pre immune sera, separated by 10% SDS-PAGE and auto radio-graphed. Immunoprecipitation (IP).
Figure Legend Snippet: Interaction of DDX3 with PAdV-3 and HAdV-5 pVIII. (A) Coomassie blue staining of purified protein. Purified GST.DDX3 protein was separated by 10% SDS-PAGE and stained with 0.25 Coomassie blue stain. (B) GST-pull down assay. Purified GSTor GST.DDX3 fusion protein immobilized on Glutathione-Sepharose 4B beads, incubated individually with in vitro translated [ 35 S] methionine labeled PAdV-3 pVIII or HAdV-5 pVIII, separated by 10% SDS-PAGE and detected by autoradiography. (C) Co-immunoprecipitation. Radio labeled in vitro transcribed and translated HAdV5 pVIII or PAdV-3 pVIII was incubated with in vitro transcribed and translated unlabeled DDX3 protein. Proteins were immunoprecipitated with either anti-DDX3 serum or rabbit pre immune sera, separated by 10% SDS-PAGE and auto radio-graphed. Immunoprecipitation (IP).

Techniques Used: Staining, Purification, SDS Page, Pull Down Assay, Incubation, In Vitro, Labeling, Autoradiography, Immunoprecipitation

Effect of pVIII on capped mRNA translation. (A). In vitro . The TNT ® T7 luciferase DNA (Promega) (i) was transcribed in vitro in the absence (uncapped) or presence (capped) of 40 mM Ribo m7GpppG cap analog (Promega) using RiboMAX RNA production system-T7 (Promega). The in vitro synthesized capped and uncapped luciferase mRNAs (ii) were translated in the supernatant collected after centrifugation of mixture of Flexi Rabbit Reticulo Lysate (Promega) incubated with Glutathione sepharose beads preloaded with GST.VIII or GST protein alone. The level of luciferase activity was measured using a luciferase kit (Promega) on a Luminometer (Turner Designs, Inc.). The results are shown as relative luciferase activity (iii). Error bars indicate SE of means for separate experiments. The relative luciferase intensity is determined based on GST compared to GST.pVIII. (B) In vivo . 293T cells were transfected with plasmid DNAs (2 μg of pcDNA3-RLuc-POLIRES-FLuc (i) and either 4 μg of pEY.pVIII or 4 μg of pEYFPN1). At 36 h post transfection, Firefly luciferase (FLuc) and Renilla reniformis luciferase (RLuc) activities were measured in a luminometer by using a dual luciferase assay kit (Promega) as per the company’s procedure. Expression of EYFP was used to normalize the transfection efficiency. The results are shown as relative luciferase activity (iii). The level of cytoplasmic RLuc-POLIRES-FLuc mRNA both in EY.pVIII and EYFP expressing plasmid transfected cells was quantified by RT-PCR (ii). Error bars indicate SE of means for three separate experiments. ∗ statistically significant.
Figure Legend Snippet: Effect of pVIII on capped mRNA translation. (A). In vitro . The TNT ® T7 luciferase DNA (Promega) (i) was transcribed in vitro in the absence (uncapped) or presence (capped) of 40 mM Ribo m7GpppG cap analog (Promega) using RiboMAX RNA production system-T7 (Promega). The in vitro synthesized capped and uncapped luciferase mRNAs (ii) were translated in the supernatant collected after centrifugation of mixture of Flexi Rabbit Reticulo Lysate (Promega) incubated with Glutathione sepharose beads preloaded with GST.VIII or GST protein alone. The level of luciferase activity was measured using a luciferase kit (Promega) on a Luminometer (Turner Designs, Inc.). The results are shown as relative luciferase activity (iii). Error bars indicate SE of means for separate experiments. The relative luciferase intensity is determined based on GST compared to GST.pVIII. (B) In vivo . 293T cells were transfected with plasmid DNAs (2 μg of pcDNA3-RLuc-POLIRES-FLuc (i) and either 4 μg of pEY.pVIII or 4 μg of pEYFPN1). At 36 h post transfection, Firefly luciferase (FLuc) and Renilla reniformis luciferase (RLuc) activities were measured in a luminometer by using a dual luciferase assay kit (Promega) as per the company’s procedure. Expression of EYFP was used to normalize the transfection efficiency. The results are shown as relative luciferase activity (iii). The level of cytoplasmic RLuc-POLIRES-FLuc mRNA both in EY.pVIII and EYFP expressing plasmid transfected cells was quantified by RT-PCR (ii). Error bars indicate SE of means for three separate experiments. ∗ statistically significant.

Techniques Used: In Vitro, Luciferase, Synthesized, Centrifugation, Incubation, Activity Assay, In Vivo, Transfection, Plasmid Preparation, Expressing, Reverse Transcription Polymerase Chain Reaction

m7GTP-sepharose binding assay. (A) The supernatant of the lysates of the cells collected at 36 h post BAdV-3 infection of MDBK cells (mock or BAdV-3) or transfection of 293T cells with plasmid DNAs (pEY.pVIII or pEYFPN1) were incubated with m7GTP sepharose cap affinity beads. After washing, the bound proteins were analyzed by Western blot using indicated protein specific antibodies and IRDye 800 conjugated goat anti-mouse IgG or Alexa Flour 680 goat anti-rabbit IgG as secondary antibody. The intensity of the bands of the Western blot in all cases was analyzed by Odyssey Software v2.1. The relative amount of proteins in BAdV-3 infected or pEY.VIII transfected cell lysates that are retained in the 7-methyl GTP resins as compared to mock infected or pEYFPN1 transfected cells, respectively (i.e., considering the amount of protein in mock infected or pEYFPN1 transfected cell lysates that are retained in the m7GTP resins as 100%) is plotted. Error bars indicate SE of means for three separate experiments. Proteins from the lysates of BAdV-3 infected or transfected cells were separated by 10% SDS-PAGE and probed in Western blot using anti-pVIII serum. (B) Proteins from the lysates of mock infected or BAdV-3 infected MDBK cells collected at 36 h post infection were separated by 10% SDS-PAGE and analyzed by Western blot using protein specific antibody and anti-rabbit IRDye 800 conjugated goat anti-mouse IgG (Li-COR biosciences) or Alexa Flour 680 goat anti-rabbit IgG as secondary antibody. β-actin was used as a loading control.
Figure Legend Snippet: m7GTP-sepharose binding assay. (A) The supernatant of the lysates of the cells collected at 36 h post BAdV-3 infection of MDBK cells (mock or BAdV-3) or transfection of 293T cells with plasmid DNAs (pEY.pVIII or pEYFPN1) were incubated with m7GTP sepharose cap affinity beads. After washing, the bound proteins were analyzed by Western blot using indicated protein specific antibodies and IRDye 800 conjugated goat anti-mouse IgG or Alexa Flour 680 goat anti-rabbit IgG as secondary antibody. The intensity of the bands of the Western blot in all cases was analyzed by Odyssey Software v2.1. The relative amount of proteins in BAdV-3 infected or pEY.VIII transfected cell lysates that are retained in the 7-methyl GTP resins as compared to mock infected or pEYFPN1 transfected cells, respectively (i.e., considering the amount of protein in mock infected or pEYFPN1 transfected cell lysates that are retained in the m7GTP resins as 100%) is plotted. Error bars indicate SE of means for three separate experiments. Proteins from the lysates of BAdV-3 infected or transfected cells were separated by 10% SDS-PAGE and probed in Western blot using anti-pVIII serum. (B) Proteins from the lysates of mock infected or BAdV-3 infected MDBK cells collected at 36 h post infection were separated by 10% SDS-PAGE and analyzed by Western blot using protein specific antibody and anti-rabbit IRDye 800 conjugated goat anti-mouse IgG (Li-COR biosciences) or Alexa Flour 680 goat anti-rabbit IgG as secondary antibody. β-actin was used as a loading control.

Techniques Used: Binding Assay, Infection, Transfection, Plasmid Preparation, Incubation, Western Blot, Software, SDS Page

Interaction of DDX3 with BAdV-3 pVIII. (A) Glutathione S-transferase (GST) pull down assay. Purified GST or GST.pVIII fusion protein immobilized on Glutathione-Sepharose 4B beads, incubated with in vitro translated [ 35 S] methionine labeled HA tagged DDX3 were separated by 10% SDS-PAGE and detected by autoradiography. (B,C) Co-immunoprecipitation in transfected cells. Proteins from the lysates of cells co-transfected with either pHA.DX3 and pEY.pVIII or pHA.DX3 and pEYFPN1 were immunoprecipitated with anti-pVIII serum (B) or anti-HA MAb (C) , separated by 10% SDS-PAGE and transferred to nitrocellulose membrane. The separated proteins were probed in Western blot using anti-HA MAb (B) or anti-pVIII serum (C) . (D) Co-immunoprecipitation in BAdV-3 infected cells. Proteins from the lysates of mock or BAdV-3 infected Madin-Darby Bovine Kidney (MDBK) cells were immunoprecipitated with anti-pVIII serum, separated by 10% SDS-PAGE, transferred to nitrocellulose membrane and probed in Western blot using anti-DDX3 MAb. Immunoprecipitation (IP). WB (Western blot). Ctl (Control) . (E–G) Confocal microscopy. MDBK cells mock infected (panels a and f) or infected with BAdV-3 (panels d and g1–g4) VERO cells untransfected (panel b) or transfected with indicated plasmid (panels c, e, and h1–h4) DNA, were fixed 36 h post-infection/transfection. The subcellular localization of DDX3 (panels a–c, g2, and h2) protein was visualized by indirect immunofluorescence (panels a–c, g2, h2) using anti-DDX3 MAb and fluorescein conjugated goat anti-mouse IgG-FITC (panels a and g2), anti-DDX3 MAb and Cy3 conjugated goat anti-mouse (pane b) secondary antibody, anti-HA MAb and Cy3 conjugated goat anti-mouse secondary antibody (panel c and h2). The subcellular localization of pVIII (panels d, e, f, g1, and h1) was visualized by direct fluorescence (panels e and h1) or indirect immunofluorescence using anti-pVIII serum and Cy3 conjugated goat anti-rabbit secondary antibody (panels d, f, and g1). Nuclei were stained with DAPI in each panel. A merge of the images is shown. Enlargement of panel g4 and h4 is shown, arrows in white shows few of the colocalization of pVIII and DDX3.
Figure Legend Snippet: Interaction of DDX3 with BAdV-3 pVIII. (A) Glutathione S-transferase (GST) pull down assay. Purified GST or GST.pVIII fusion protein immobilized on Glutathione-Sepharose 4B beads, incubated with in vitro translated [ 35 S] methionine labeled HA tagged DDX3 were separated by 10% SDS-PAGE and detected by autoradiography. (B,C) Co-immunoprecipitation in transfected cells. Proteins from the lysates of cells co-transfected with either pHA.DX3 and pEY.pVIII or pHA.DX3 and pEYFPN1 were immunoprecipitated with anti-pVIII serum (B) or anti-HA MAb (C) , separated by 10% SDS-PAGE and transferred to nitrocellulose membrane. The separated proteins were probed in Western blot using anti-HA MAb (B) or anti-pVIII serum (C) . (D) Co-immunoprecipitation in BAdV-3 infected cells. Proteins from the lysates of mock or BAdV-3 infected Madin-Darby Bovine Kidney (MDBK) cells were immunoprecipitated with anti-pVIII serum, separated by 10% SDS-PAGE, transferred to nitrocellulose membrane and probed in Western blot using anti-DDX3 MAb. Immunoprecipitation (IP). WB (Western blot). Ctl (Control) . (E–G) Confocal microscopy. MDBK cells mock infected (panels a and f) or infected with BAdV-3 (panels d and g1–g4) VERO cells untransfected (panel b) or transfected with indicated plasmid (panels c, e, and h1–h4) DNA, were fixed 36 h post-infection/transfection. The subcellular localization of DDX3 (panels a–c, g2, and h2) protein was visualized by indirect immunofluorescence (panels a–c, g2, h2) using anti-DDX3 MAb and fluorescein conjugated goat anti-mouse IgG-FITC (panels a and g2), anti-DDX3 MAb and Cy3 conjugated goat anti-mouse (pane b) secondary antibody, anti-HA MAb and Cy3 conjugated goat anti-mouse secondary antibody (panel c and h2). The subcellular localization of pVIII (panels d, e, f, g1, and h1) was visualized by direct fluorescence (panels e and h1) or indirect immunofluorescence using anti-pVIII serum and Cy3 conjugated goat anti-rabbit secondary antibody (panels d, f, and g1). Nuclei were stained with DAPI in each panel. A merge of the images is shown. Enlargement of panel g4 and h4 is shown, arrows in white shows few of the colocalization of pVIII and DDX3.

Techniques Used: Pull Down Assay, Purification, Incubation, In Vitro, Labeling, SDS Page, Autoradiography, Immunoprecipitation, Transfection, Western Blot, Infection, CTL Assay, Confocal Microscopy, Plasmid Preparation, Immunofluorescence, Fluorescence, Staining

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Expressing:

Article Title: TP53INP2 contributes to autophagosome formation by promoting LC3-ATG7 interaction
Article Snippet: .. For the GST-LC3B[G120] affinity-isolation assay, HEK293 cells expressing TP53INP2[NLSΔ], TP53INP2[Δ1-28],[NLSΔ] TP53INP2W35,I38A [NLSΔ], TP53INP2[Δ67-111],[NLSΔ] or TP53INP2[Δ112-144],[NLSΔ] were lysed and the cell lysate was incubated with purified GST or GST-LC3B[G120] proteins at 4°C for 4 h. Then the glutathione-sepharose 4B beads were added to the mixture followed by incubation at 4°C for 2 h. Immunocomplexes were washed and used for western blot. .. Protein bands were detected with Coomassie Brilliant Blue and quantified using the ImageJ software.

Western Blot:

Article Title: Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap
Article Snippet: .. pVIII Protein Interacts with eIFs Indirectly via Its Interaction with DDX3 To determine if the interaction of pVIII and DDX3 also affects the level of eIFs, the pellet and supernatant fractions of the rabbit-reticulo Lysates incubated with glutathione sepharose beads preloaded with purified GST.pVIII or GST were analyzed by Western blot using protein specific antibodies. ..

Article Title: TP53INP2 contributes to autophagosome formation by promoting LC3-ATG7 interaction
Article Snippet: .. For the GST-LC3B[G120] affinity-isolation assay, HEK293 cells expressing TP53INP2[NLSΔ], TP53INP2[Δ1-28],[NLSΔ] TP53INP2W35,I38A [NLSΔ], TP53INP2[Δ67-111],[NLSΔ] or TP53INP2[Δ112-144],[NLSΔ] were lysed and the cell lysate was incubated with purified GST or GST-LC3B[G120] proteins at 4°C for 4 h. Then the glutathione-sepharose 4B beads were added to the mixture followed by incubation at 4°C for 2 h. Immunocomplexes were washed and used for western blot. .. Protein bands were detected with Coomassie Brilliant Blue and quantified using the ImageJ software.

Recombinant:

Article Title: The Putative RNA Helicase HELZ Promotes Cell Proliferation, Translation Initiation and Ribosomal Protein S6 Phosphorylation
Article Snippet: .. Crude HeLa cell lysates were incubated with recombinant GST, the indicated GST-HELZ fragments, GST-HIF-1α530–826 as well as GST-Paip2106–127 and GST pull–down was conducted using glutathione–sepharose beads. .. Eluates were subjected to SDS–PAGE and immunoblotting. (TIF) Click here for additional data file.

Chromatin Immunoprecipitation:

Article Title: Docking-dependent Ubiquitination of the Interferon Regulatory Factor-1 Tumor Suppressor Protein by the Ubiquitin Ligase CHIP *
Article Snippet: .. When A375 cell lysate was passed through a column prepared by immobilizing GST-IRF-1 on glutathione-Sepharose beads, endogenous CHIP bound specifically to GST-IRF-1 and not to a GST alone control column ( A ). .. Additionally, CHIP was co-immunoprecipitated with IRF-1 from A375 cells in which both proteins were overexpressed ( B ).

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  • 94
    GE Healthcare glutathione sepharose 4b
    α-syn Aptamers Were Selected through SELEX (A) Schematic illustration of the method used for α-syn aptamer selection. GST-tagged α-syn was immobilized on <t>glutathione-sepharose</t> beads. The ssDNA library was incubated with the target beads for binding. Unbound oligonucleotides were washed away, and the bound ones were released by heating at 95°C. The selected binders were amplified by PCR with biotinylated primers. ssDNAs were subsequently purified from the PCR product using streptavidin-coated magnetic beads, resulting in an enriched DNA pool, which was used in the next SELEX round. After the last round, the selected ssDNAs were sequenced by deep sequencing. (B) The aptamer candidates. After deep sequencing, the two sequences with most frequently appearing were selected as the aptamer candidates. (C) Aptamer binding specificity assay by dot blotting. Five microgram samples (α-syn, GST, Aβ 42 , BSA, and three domains of α-syn) were respectively immobilized onto the nitrocellulose membrane for binding of each aptamer.
    Glutathione Sepharose 4b, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 94/100, based on 713 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/glutathione sepharose 4b/product/GE Healthcare
    Average 94 stars, based on 713 article reviews
    Price from $9.99 to $1999.99
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    94
    GE Healthcare glutathione sepharose 4b beads
    Beta-1 adrenergic receptor (β1AR) binds directly to golgin-160 (1–393) . Representative gels for the purification of golgin-160 (1–393) and its binding to β1AR are shown. ( A ) The NEB IMPACT system was used to create a purified, untagged golgin-160 (1–393) following cleavage of the intein tag. DTT-induced cleavage caused enrichment of an approximately 60 kDa protein, which was specifically eluted off of the chitin column. This protein band could be detected using immunoblotting with an antibody to the N-terminus of golgin-160. Input, protein added to the chitin column; Cleaved, protein on the chitin column after addition of DTT but before elution; Eluate, protein released from the column after cleavage; *, golgin-160 (1–393) ; **, GST fusion proteins; ( B ) The purified, untagged golgin-160 head domain was incubated with purified GST or GST-β1AR L3 pre-bound to <t>glutathione-Sepharose</t> 4B beads. The beads were washed and bound golgin-160 (1–393) was detected by Coomassie blue staining after SDS-PAGE. Note that the samples in panel A were run on a 4%–12% gradient gel, whereas those in B were run on a 10% gel.
    Glutathione Sepharose 4b Beads, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 94/100, based on 719 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/glutathione sepharose 4b beads/product/GE Healthcare
    Average 94 stars, based on 719 article reviews
    Price from $9.99 to $1999.99
    glutathione sepharose 4b beads - by Bioz Stars, 2020-08
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    α-syn Aptamers Were Selected through SELEX (A) Schematic illustration of the method used for α-syn aptamer selection. GST-tagged α-syn was immobilized on glutathione-sepharose beads. The ssDNA library was incubated with the target beads for binding. Unbound oligonucleotides were washed away, and the bound ones were released by heating at 95°C. The selected binders were amplified by PCR with biotinylated primers. ssDNAs were subsequently purified from the PCR product using streptavidin-coated magnetic beads, resulting in an enriched DNA pool, which was used in the next SELEX round. After the last round, the selected ssDNAs were sequenced by deep sequencing. (B) The aptamer candidates. After deep sequencing, the two sequences with most frequently appearing were selected as the aptamer candidates. (C) Aptamer binding specificity assay by dot blotting. Five microgram samples (α-syn, GST, Aβ 42 , BSA, and three domains of α-syn) were respectively immobilized onto the nitrocellulose membrane for binding of each aptamer.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Novel DNA Aptamers for Parkinson’s Disease Treatment Inhibit α-Synuclein Aggregation and Facilitate its Degradation

    doi: 10.1016/j.omtn.2018.02.011

    Figure Lengend Snippet: α-syn Aptamers Were Selected through SELEX (A) Schematic illustration of the method used for α-syn aptamer selection. GST-tagged α-syn was immobilized on glutathione-sepharose beads. The ssDNA library was incubated with the target beads for binding. Unbound oligonucleotides were washed away, and the bound ones were released by heating at 95°C. The selected binders were amplified by PCR with biotinylated primers. ssDNAs were subsequently purified from the PCR product using streptavidin-coated magnetic beads, resulting in an enriched DNA pool, which was used in the next SELEX round. After the last round, the selected ssDNAs were sequenced by deep sequencing. (B) The aptamer candidates. After deep sequencing, the two sequences with most frequently appearing were selected as the aptamer candidates. (C) Aptamer binding specificity assay by dot blotting. Five microgram samples (α-syn, GST, Aβ 42 , BSA, and three domains of α-syn) were respectively immobilized onto the nitrocellulose membrane for binding of each aptamer.

    Article Snippet: Then the fusion protein GST-α-syn was purified on glutathione-sepharose 4B according to the manufacturer’s instructions (GE Healthcare, Boston, MA).

    Techniques: Selection, Incubation, Binding Assay, Amplification, Polymerase Chain Reaction, Purification, Magnetic Beads, Sequencing

    Beta-1 adrenergic receptor (β1AR) binds directly to golgin-160 (1–393) . Representative gels for the purification of golgin-160 (1–393) and its binding to β1AR are shown. ( A ) The NEB IMPACT system was used to create a purified, untagged golgin-160 (1–393) following cleavage of the intein tag. DTT-induced cleavage caused enrichment of an approximately 60 kDa protein, which was specifically eluted off of the chitin column. This protein band could be detected using immunoblotting with an antibody to the N-terminus of golgin-160. Input, protein added to the chitin column; Cleaved, protein on the chitin column after addition of DTT but before elution; Eluate, protein released from the column after cleavage; *, golgin-160 (1–393) ; **, GST fusion proteins; ( B ) The purified, untagged golgin-160 head domain was incubated with purified GST or GST-β1AR L3 pre-bound to glutathione-Sepharose 4B beads. The beads were washed and bound golgin-160 (1–393) was detected by Coomassie blue staining after SDS-PAGE. Note that the samples in panel A were run on a 4%–12% gradient gel, whereas those in B were run on a 10% gel.

    Journal: International Journal of Molecular Sciences

    Article Title: Three Basic Residues of Intracellular Loop 3 of the Beta-1 Adrenergic Receptor Are Required for Golgin-160-Dependent Trafficking

    doi: 10.3390/ijms15022929

    Figure Lengend Snippet: Beta-1 adrenergic receptor (β1AR) binds directly to golgin-160 (1–393) . Representative gels for the purification of golgin-160 (1–393) and its binding to β1AR are shown. ( A ) The NEB IMPACT system was used to create a purified, untagged golgin-160 (1–393) following cleavage of the intein tag. DTT-induced cleavage caused enrichment of an approximately 60 kDa protein, which was specifically eluted off of the chitin column. This protein band could be detected using immunoblotting with an antibody to the N-terminus of golgin-160. Input, protein added to the chitin column; Cleaved, protein on the chitin column after addition of DTT but before elution; Eluate, protein released from the column after cleavage; *, golgin-160 (1–393) ; **, GST fusion proteins; ( B ) The purified, untagged golgin-160 head domain was incubated with purified GST or GST-β1AR L3 pre-bound to glutathione-Sepharose 4B beads. The beads were washed and bound golgin-160 (1–393) was detected by Coomassie blue staining after SDS-PAGE. Note that the samples in panel A were run on a 4%–12% gradient gel, whereas those in B were run on a 10% gel.

    Article Snippet: The soluble fraction of the lysed cells was incubated 2 h at 4 °C with 10 μg GST alone or GST-tagged golgin-160(1–393) that had been pre-conjugated to glutathione-Sepharose 4B beads.

    Techniques: Purification, Binding Assay, Incubation, Staining, SDS Page

    HELZ interacts with the poly(A)-binding protein (PABP). GST pull-down using glutathione-sepharose beads was conducted by incubating crude HeLa cell lysates with recombinant GST, GST-Paip2 106–127 and GST-HELZ 1023–1199 ( A ), GST and the indicated GST-HELZ fragments ( D ), or crude HEK293 cell lysates with GST and GST-PABP 554–636 ( C ). Hypoxic HeLa cell lysates were incubated with GST or GST-HELZ 1023–1199 ( B ). Eluates were subjected to SDS-PAGE and immunoblotting using the indicated antibodies.

    Journal: PLoS ONE

    Article Title: The Putative RNA Helicase HELZ Promotes Cell Proliferation, Translation Initiation and Ribosomal Protein S6 Phosphorylation

    doi: 10.1371/journal.pone.0022107

    Figure Lengend Snippet: HELZ interacts with the poly(A)-binding protein (PABP). GST pull-down using glutathione-sepharose beads was conducted by incubating crude HeLa cell lysates with recombinant GST, GST-Paip2 106–127 and GST-HELZ 1023–1199 ( A ), GST and the indicated GST-HELZ fragments ( D ), or crude HEK293 cell lysates with GST and GST-PABP 554–636 ( C ). Hypoxic HeLa cell lysates were incubated with GST or GST-HELZ 1023–1199 ( B ). Eluates were subjected to SDS-PAGE and immunoblotting using the indicated antibodies.

    Article Snippet: Crude HeLa cell lysates were incubated with recombinant GST, the indicated GST-HELZ fragments, GST-HIF-1α530–826 as well as GST-Paip2106–127 and GST pull–down was conducted using glutathione–sepharose beads.

    Techniques: Binding Assay, Recombinant, Incubation, SDS Page

    Interaction of DDX3 with PAdV-3 and HAdV-5 pVIII. (A) Coomassie blue staining of purified protein. Purified GST.DDX3 protein was separated by 10% SDS-PAGE and stained with 0.25 Coomassie blue stain. (B) GST-pull down assay. Purified GSTor GST.DDX3 fusion protein immobilized on Glutathione-Sepharose 4B beads, incubated individually with in vitro translated [ 35 S] methionine labeled PAdV-3 pVIII or HAdV-5 pVIII, separated by 10% SDS-PAGE and detected by autoradiography. (C) Co-immunoprecipitation. Radio labeled in vitro transcribed and translated HAdV5 pVIII or PAdV-3 pVIII was incubated with in vitro transcribed and translated unlabeled DDX3 protein. Proteins were immunoprecipitated with either anti-DDX3 serum or rabbit pre immune sera, separated by 10% SDS-PAGE and auto radio-graphed. Immunoprecipitation (IP).

    Journal: Frontiers in Microbiology

    Article Title: Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap

    doi: 10.3389/fmicb.2016.02119

    Figure Lengend Snippet: Interaction of DDX3 with PAdV-3 and HAdV-5 pVIII. (A) Coomassie blue staining of purified protein. Purified GST.DDX3 protein was separated by 10% SDS-PAGE and stained with 0.25 Coomassie blue stain. (B) GST-pull down assay. Purified GSTor GST.DDX3 fusion protein immobilized on Glutathione-Sepharose 4B beads, incubated individually with in vitro translated [ 35 S] methionine labeled PAdV-3 pVIII or HAdV-5 pVIII, separated by 10% SDS-PAGE and detected by autoradiography. (C) Co-immunoprecipitation. Radio labeled in vitro transcribed and translated HAdV5 pVIII or PAdV-3 pVIII was incubated with in vitro transcribed and translated unlabeled DDX3 protein. Proteins were immunoprecipitated with either anti-DDX3 serum or rabbit pre immune sera, separated by 10% SDS-PAGE and auto radio-graphed. Immunoprecipitation (IP).

    Article Snippet: pVIII Protein Interacts with eIFs Indirectly via Its Interaction with DDX3 To determine if the interaction of pVIII and DDX3 also affects the level of eIFs, the pellet and supernatant fractions of the rabbit-reticulo Lysates incubated with glutathione sepharose beads preloaded with purified GST.pVIII or GST were analyzed by Western blot using protein specific antibodies.

    Techniques: Staining, Purification, SDS Page, Pull Down Assay, Incubation, In Vitro, Labeling, Autoradiography, Immunoprecipitation

    Effect of pVIII on capped mRNA translation. (A). In vitro . The TNT ® T7 luciferase DNA (Promega) (i) was transcribed in vitro in the absence (uncapped) or presence (capped) of 40 mM Ribo m7GpppG cap analog (Promega) using RiboMAX RNA production system-T7 (Promega). The in vitro synthesized capped and uncapped luciferase mRNAs (ii) were translated in the supernatant collected after centrifugation of mixture of Flexi Rabbit Reticulo Lysate (Promega) incubated with Glutathione sepharose beads preloaded with GST.VIII or GST protein alone. The level of luciferase activity was measured using a luciferase kit (Promega) on a Luminometer (Turner Designs, Inc.). The results are shown as relative luciferase activity (iii). Error bars indicate SE of means for separate experiments. The relative luciferase intensity is determined based on GST compared to GST.pVIII. (B) In vivo . 293T cells were transfected with plasmid DNAs (2 μg of pcDNA3-RLuc-POLIRES-FLuc (i) and either 4 μg of pEY.pVIII or 4 μg of pEYFPN1). At 36 h post transfection, Firefly luciferase (FLuc) and Renilla reniformis luciferase (RLuc) activities were measured in a luminometer by using a dual luciferase assay kit (Promega) as per the company’s procedure. Expression of EYFP was used to normalize the transfection efficiency. The results are shown as relative luciferase activity (iii). The level of cytoplasmic RLuc-POLIRES-FLuc mRNA both in EY.pVIII and EYFP expressing plasmid transfected cells was quantified by RT-PCR (ii). Error bars indicate SE of means for three separate experiments. ∗ statistically significant.

    Journal: Frontiers in Microbiology

    Article Title: Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap

    doi: 10.3389/fmicb.2016.02119

    Figure Lengend Snippet: Effect of pVIII on capped mRNA translation. (A). In vitro . The TNT ® T7 luciferase DNA (Promega) (i) was transcribed in vitro in the absence (uncapped) or presence (capped) of 40 mM Ribo m7GpppG cap analog (Promega) using RiboMAX RNA production system-T7 (Promega). The in vitro synthesized capped and uncapped luciferase mRNAs (ii) were translated in the supernatant collected after centrifugation of mixture of Flexi Rabbit Reticulo Lysate (Promega) incubated with Glutathione sepharose beads preloaded with GST.VIII or GST protein alone. The level of luciferase activity was measured using a luciferase kit (Promega) on a Luminometer (Turner Designs, Inc.). The results are shown as relative luciferase activity (iii). Error bars indicate SE of means for separate experiments. The relative luciferase intensity is determined based on GST compared to GST.pVIII. (B) In vivo . 293T cells were transfected with plasmid DNAs (2 μg of pcDNA3-RLuc-POLIRES-FLuc (i) and either 4 μg of pEY.pVIII or 4 μg of pEYFPN1). At 36 h post transfection, Firefly luciferase (FLuc) and Renilla reniformis luciferase (RLuc) activities were measured in a luminometer by using a dual luciferase assay kit (Promega) as per the company’s procedure. Expression of EYFP was used to normalize the transfection efficiency. The results are shown as relative luciferase activity (iii). The level of cytoplasmic RLuc-POLIRES-FLuc mRNA both in EY.pVIII and EYFP expressing plasmid transfected cells was quantified by RT-PCR (ii). Error bars indicate SE of means for three separate experiments. ∗ statistically significant.

    Article Snippet: pVIII Protein Interacts with eIFs Indirectly via Its Interaction with DDX3 To determine if the interaction of pVIII and DDX3 also affects the level of eIFs, the pellet and supernatant fractions of the rabbit-reticulo Lysates incubated with glutathione sepharose beads preloaded with purified GST.pVIII or GST were analyzed by Western blot using protein specific antibodies.

    Techniques: In Vitro, Luciferase, Synthesized, Centrifugation, Incubation, Activity Assay, In Vivo, Transfection, Plasmid Preparation, Expressing, Reverse Transcription Polymerase Chain Reaction

    m7GTP-sepharose binding assay. (A) The supernatant of the lysates of the cells collected at 36 h post BAdV-3 infection of MDBK cells (mock or BAdV-3) or transfection of 293T cells with plasmid DNAs (pEY.pVIII or pEYFPN1) were incubated with m7GTP sepharose cap affinity beads. After washing, the bound proteins were analyzed by Western blot using indicated protein specific antibodies and IRDye 800 conjugated goat anti-mouse IgG or Alexa Flour 680 goat anti-rabbit IgG as secondary antibody. The intensity of the bands of the Western blot in all cases was analyzed by Odyssey Software v2.1. The relative amount of proteins in BAdV-3 infected or pEY.VIII transfected cell lysates that are retained in the 7-methyl GTP resins as compared to mock infected or pEYFPN1 transfected cells, respectively (i.e., considering the amount of protein in mock infected or pEYFPN1 transfected cell lysates that are retained in the m7GTP resins as 100%) is plotted. Error bars indicate SE of means for three separate experiments. Proteins from the lysates of BAdV-3 infected or transfected cells were separated by 10% SDS-PAGE and probed in Western blot using anti-pVIII serum. (B) Proteins from the lysates of mock infected or BAdV-3 infected MDBK cells collected at 36 h post infection were separated by 10% SDS-PAGE and analyzed by Western blot using protein specific antibody and anti-rabbit IRDye 800 conjugated goat anti-mouse IgG (Li-COR biosciences) or Alexa Flour 680 goat anti-rabbit IgG as secondary antibody. β-actin was used as a loading control.

    Journal: Frontiers in Microbiology

    Article Title: Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap

    doi: 10.3389/fmicb.2016.02119

    Figure Lengend Snippet: m7GTP-sepharose binding assay. (A) The supernatant of the lysates of the cells collected at 36 h post BAdV-3 infection of MDBK cells (mock or BAdV-3) or transfection of 293T cells with plasmid DNAs (pEY.pVIII or pEYFPN1) were incubated with m7GTP sepharose cap affinity beads. After washing, the bound proteins were analyzed by Western blot using indicated protein specific antibodies and IRDye 800 conjugated goat anti-mouse IgG or Alexa Flour 680 goat anti-rabbit IgG as secondary antibody. The intensity of the bands of the Western blot in all cases was analyzed by Odyssey Software v2.1. The relative amount of proteins in BAdV-3 infected or pEY.VIII transfected cell lysates that are retained in the 7-methyl GTP resins as compared to mock infected or pEYFPN1 transfected cells, respectively (i.e., considering the amount of protein in mock infected or pEYFPN1 transfected cell lysates that are retained in the m7GTP resins as 100%) is plotted. Error bars indicate SE of means for three separate experiments. Proteins from the lysates of BAdV-3 infected or transfected cells were separated by 10% SDS-PAGE and probed in Western blot using anti-pVIII serum. (B) Proteins from the lysates of mock infected or BAdV-3 infected MDBK cells collected at 36 h post infection were separated by 10% SDS-PAGE and analyzed by Western blot using protein specific antibody and anti-rabbit IRDye 800 conjugated goat anti-mouse IgG (Li-COR biosciences) or Alexa Flour 680 goat anti-rabbit IgG as secondary antibody. β-actin was used as a loading control.

    Article Snippet: pVIII Protein Interacts with eIFs Indirectly via Its Interaction with DDX3 To determine if the interaction of pVIII and DDX3 also affects the level of eIFs, the pellet and supernatant fractions of the rabbit-reticulo Lysates incubated with glutathione sepharose beads preloaded with purified GST.pVIII or GST were analyzed by Western blot using protein specific antibodies.

    Techniques: Binding Assay, Infection, Transfection, Plasmid Preparation, Incubation, Western Blot, Software, SDS Page

    Interaction of DDX3 with BAdV-3 pVIII. (A) Glutathione S-transferase (GST) pull down assay. Purified GST or GST.pVIII fusion protein immobilized on Glutathione-Sepharose 4B beads, incubated with in vitro translated [ 35 S] methionine labeled HA tagged DDX3 were separated by 10% SDS-PAGE and detected by autoradiography. (B,C) Co-immunoprecipitation in transfected cells. Proteins from the lysates of cells co-transfected with either pHA.DX3 and pEY.pVIII or pHA.DX3 and pEYFPN1 were immunoprecipitated with anti-pVIII serum (B) or anti-HA MAb (C) , separated by 10% SDS-PAGE and transferred to nitrocellulose membrane. The separated proteins were probed in Western blot using anti-HA MAb (B) or anti-pVIII serum (C) . (D) Co-immunoprecipitation in BAdV-3 infected cells. Proteins from the lysates of mock or BAdV-3 infected Madin-Darby Bovine Kidney (MDBK) cells were immunoprecipitated with anti-pVIII serum, separated by 10% SDS-PAGE, transferred to nitrocellulose membrane and probed in Western blot using anti-DDX3 MAb. Immunoprecipitation (IP). WB (Western blot). Ctl (Control) . (E–G) Confocal microscopy. MDBK cells mock infected (panels a and f) or infected with BAdV-3 (panels d and g1–g4) VERO cells untransfected (panel b) or transfected with indicated plasmid (panels c, e, and h1–h4) DNA, were fixed 36 h post-infection/transfection. The subcellular localization of DDX3 (panels a–c, g2, and h2) protein was visualized by indirect immunofluorescence (panels a–c, g2, h2) using anti-DDX3 MAb and fluorescein conjugated goat anti-mouse IgG-FITC (panels a and g2), anti-DDX3 MAb and Cy3 conjugated goat anti-mouse (pane b) secondary antibody, anti-HA MAb and Cy3 conjugated goat anti-mouse secondary antibody (panel c and h2). The subcellular localization of pVIII (panels d, e, f, g1, and h1) was visualized by direct fluorescence (panels e and h1) or indirect immunofluorescence using anti-pVIII serum and Cy3 conjugated goat anti-rabbit secondary antibody (panels d, f, and g1). Nuclei were stained with DAPI in each panel. A merge of the images is shown. Enlargement of panel g4 and h4 is shown, arrows in white shows few of the colocalization of pVIII and DDX3.

    Journal: Frontiers in Microbiology

    Article Title: Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap

    doi: 10.3389/fmicb.2016.02119

    Figure Lengend Snippet: Interaction of DDX3 with BAdV-3 pVIII. (A) Glutathione S-transferase (GST) pull down assay. Purified GST or GST.pVIII fusion protein immobilized on Glutathione-Sepharose 4B beads, incubated with in vitro translated [ 35 S] methionine labeled HA tagged DDX3 were separated by 10% SDS-PAGE and detected by autoradiography. (B,C) Co-immunoprecipitation in transfected cells. Proteins from the lysates of cells co-transfected with either pHA.DX3 and pEY.pVIII or pHA.DX3 and pEYFPN1 were immunoprecipitated with anti-pVIII serum (B) or anti-HA MAb (C) , separated by 10% SDS-PAGE and transferred to nitrocellulose membrane. The separated proteins were probed in Western blot using anti-HA MAb (B) or anti-pVIII serum (C) . (D) Co-immunoprecipitation in BAdV-3 infected cells. Proteins from the lysates of mock or BAdV-3 infected Madin-Darby Bovine Kidney (MDBK) cells were immunoprecipitated with anti-pVIII serum, separated by 10% SDS-PAGE, transferred to nitrocellulose membrane and probed in Western blot using anti-DDX3 MAb. Immunoprecipitation (IP). WB (Western blot). Ctl (Control) . (E–G) Confocal microscopy. MDBK cells mock infected (panels a and f) or infected with BAdV-3 (panels d and g1–g4) VERO cells untransfected (panel b) or transfected with indicated plasmid (panels c, e, and h1–h4) DNA, were fixed 36 h post-infection/transfection. The subcellular localization of DDX3 (panels a–c, g2, and h2) protein was visualized by indirect immunofluorescence (panels a–c, g2, h2) using anti-DDX3 MAb and fluorescein conjugated goat anti-mouse IgG-FITC (panels a and g2), anti-DDX3 MAb and Cy3 conjugated goat anti-mouse (pane b) secondary antibody, anti-HA MAb and Cy3 conjugated goat anti-mouse secondary antibody (panel c and h2). The subcellular localization of pVIII (panels d, e, f, g1, and h1) was visualized by direct fluorescence (panels e and h1) or indirect immunofluorescence using anti-pVIII serum and Cy3 conjugated goat anti-rabbit secondary antibody (panels d, f, and g1). Nuclei were stained with DAPI in each panel. A merge of the images is shown. Enlargement of panel g4 and h4 is shown, arrows in white shows few of the colocalization of pVIII and DDX3.

    Article Snippet: pVIII Protein Interacts with eIFs Indirectly via Its Interaction with DDX3 To determine if the interaction of pVIII and DDX3 also affects the level of eIFs, the pellet and supernatant fractions of the rabbit-reticulo Lysates incubated with glutathione sepharose beads preloaded with purified GST.pVIII or GST were analyzed by Western blot using protein specific antibodies.

    Techniques: Pull Down Assay, Purification, Incubation, In Vitro, Labeling, SDS Page, Autoradiography, Immunoprecipitation, Transfection, Western Blot, Infection, CTL Assay, Confocal Microscopy, Plasmid Preparation, Immunofluorescence, Fluorescence, Staining