glutathione sepharose  (Millipore)


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    Name:
    Glutathione
    Description:
    Pharmaceutical secondary standards for application in quality control provide pharma laboratories and manufacturers with a convenient and cost effective alternative to the preparation of in house working standards
    Catalog Number:
    phr1359
    Price:
    None
    Applications:
    May be used at 5-10 mM to elute glutathione S-transferase (GST) from glutathione agarose.
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    Structured Review

    Millipore glutathione sepharose
    Glutathione
    Pharmaceutical secondary standards for application in quality control provide pharma laboratories and manufacturers with a convenient and cost effective alternative to the preparation of in house working standards
    https://www.bioz.com/result/glutathione sepharose/product/Millipore
    Average 99 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    glutathione sepharose - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Drug discovery with an RBM20 dependent titin splice reporter identifies cardenolides as lead structures to improve cardiac filling"

    Article Title: Drug discovery with an RBM20 dependent titin splice reporter identifies cardenolides as lead structures to improve cardiac filling

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0198492

    Inhibitors of titin splicing identified by HTS. For each compound concentration dependent activity in the dual luciferase reporter assay and cell viability are plotted. Dashed lines indicate the concentrations used for validation on RNA level. The tables provide kinetic information. Validation by RT-PCR (agarose gel) is quantified by calculating the percent spliced in values (PSI). (a-c) Inhibition of alternative splicing was validated manually in the 96-well format using the DLR assay and conventional as well as quantitative RT-qPCR using an independent genomic minigene derived from TTN exons 241-43 (N = 4). Cardenolides efficiently reduce titin splicing by RBM20 with different potency (splicing IC50). The effect on splicing translates to reduced viability of HEK293 cells (IC50 values splicing vs. viability). (d) The steroid hydrocortisone does not interfere with splicing activity (N = 4). Compared to the cardenolides it lacks the lactone ring at C17 (chemical structures provided on the right). * P
    Figure Legend Snippet: Inhibitors of titin splicing identified by HTS. For each compound concentration dependent activity in the dual luciferase reporter assay and cell viability are plotted. Dashed lines indicate the concentrations used for validation on RNA level. The tables provide kinetic information. Validation by RT-PCR (agarose gel) is quantified by calculating the percent spliced in values (PSI). (a-c) Inhibition of alternative splicing was validated manually in the 96-well format using the DLR assay and conventional as well as quantitative RT-qPCR using an independent genomic minigene derived from TTN exons 241-43 (N = 4). Cardenolides efficiently reduce titin splicing by RBM20 with different potency (splicing IC50). The effect on splicing translates to reduced viability of HEK293 cells (IC50 values splicing vs. viability). (d) The steroid hydrocortisone does not interfere with splicing activity (N = 4). Compared to the cardenolides it lacks the lactone ring at C17 (chemical structures provided on the right). * P

    Techniques Used: Concentration Assay, Activity Assay, Luciferase, Reporter Assay, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Inhibition, Quantitative RT-PCR, Derivative Assay

    2) Product Images from "Filamin A Phosphorylation at Serine 2152 by the Serine/Threonine Kinase Ndr2 Controls TCR-Induced LFA-1 Activation in T Cells"

    Article Title: Filamin A Phosphorylation at Serine 2152 by the Serine/Threonine Kinase Ndr2 Controls TCR-Induced LFA-1 Activation in T Cells

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02852

    Mutation of S2152A within FLNa abolishes TCR-mediated T-cell adhesion, interaction with APCs and LFA-1 activation. (A) Jurkat T cells were transiently transfected with dsRed C1 vector (dsRed) or plasmids encoding wild type (WT) dsRed-tagged FLNa (FLNa WT) or dsRed-tagged S2152A or dsRed-tagged S2152E FLNa mutants (FLNa S215A and FLNa S2152E). After 24 h the expression of WT and its mutants were analyzed by anti-dsRed and FLNa immunoblotting. Detection of β-actin served as loading control. (B) Jurkat T cells transfected as described in (A) were left untreated (non) or stimulated for 30 min with CD3 antibodies. Cells were analyzed for adhesion to ICAM-1-coated 96 well plates. Bound cells were counted and calculated as percentage of input ( n = 4) (mean ± SEM; * p ≤ 0.05, *** p ≤ 0.001). (C) Cells were transfected as described in (A) and analyzed for their ability to form conjugates with DDAO-SE (red)-stained Raji B cells that were pulsed without (non) or with superantigen (SA) for 30 min at 37°C. The percentage of conjugates was defined as the number of double-positive events in the upper right quadrant ( n = 4) (mean ± SEM; *** p ≤ 0.001). (D) Jurkat T cells transfected as described in (A) were left untreated (non) or stimulated with anti-CD3 antibodies (CD3), followed by staining with the anti-LFA-1 antibody mAb24 to detect the high affinity conformation of LFA-1. mAb24 epitope expression was assessed by flow cytometry and data are normalized against LFA-1 expression detected by MEM48 ( n = 4) (mean ± SEM; *** p ≤ 0.001). (E) HEK 293T cells were transfected with either dsRed, dsRed-tagged FLNa wild type (FLAa WT) or its mutants (FLNa S2152A and FLNa S2152E). 24 h after transfection, whole cell extracts were prepared and analyzed for the expression of dsRed and dsRed-tagged FLNa forms by Western blotting using the indicated antibodies (left panel). Lysates were incubated with GST-fusion proteins bound to glutathione-sepharose beads. Precipitates were analyzed by Western blotting using the indicated antibodies (right panel). One representative experiment of 3 is shown. (mean ± SEM; * p ≤ 0.05, *** p ≤ 0.001).
    Figure Legend Snippet: Mutation of S2152A within FLNa abolishes TCR-mediated T-cell adhesion, interaction with APCs and LFA-1 activation. (A) Jurkat T cells were transiently transfected with dsRed C1 vector (dsRed) or plasmids encoding wild type (WT) dsRed-tagged FLNa (FLNa WT) or dsRed-tagged S2152A or dsRed-tagged S2152E FLNa mutants (FLNa S215A and FLNa S2152E). After 24 h the expression of WT and its mutants were analyzed by anti-dsRed and FLNa immunoblotting. Detection of β-actin served as loading control. (B) Jurkat T cells transfected as described in (A) were left untreated (non) or stimulated for 30 min with CD3 antibodies. Cells were analyzed for adhesion to ICAM-1-coated 96 well plates. Bound cells were counted and calculated as percentage of input ( n = 4) (mean ± SEM; * p ≤ 0.05, *** p ≤ 0.001). (C) Cells were transfected as described in (A) and analyzed for their ability to form conjugates with DDAO-SE (red)-stained Raji B cells that were pulsed without (non) or with superantigen (SA) for 30 min at 37°C. The percentage of conjugates was defined as the number of double-positive events in the upper right quadrant ( n = 4) (mean ± SEM; *** p ≤ 0.001). (D) Jurkat T cells transfected as described in (A) were left untreated (non) or stimulated with anti-CD3 antibodies (CD3), followed by staining with the anti-LFA-1 antibody mAb24 to detect the high affinity conformation of LFA-1. mAb24 epitope expression was assessed by flow cytometry and data are normalized against LFA-1 expression detected by MEM48 ( n = 4) (mean ± SEM; *** p ≤ 0.001). (E) HEK 293T cells were transfected with either dsRed, dsRed-tagged FLNa wild type (FLAa WT) or its mutants (FLNa S2152A and FLNa S2152E). 24 h after transfection, whole cell extracts were prepared and analyzed for the expression of dsRed and dsRed-tagged FLNa forms by Western blotting using the indicated antibodies (left panel). Lysates were incubated with GST-fusion proteins bound to glutathione-sepharose beads. Precipitates were analyzed by Western blotting using the indicated antibodies (right panel). One representative experiment of 3 is shown. (mean ± SEM; * p ≤ 0.05, *** p ≤ 0.001).

    Techniques Used: Mutagenesis, Activation Assay, Transfection, Plasmid Preparation, Expressing, Staining, Flow Cytometry, Cytometry, Western Blot, Incubation

    3) Product Images from "Cellular Redistribution of Protein Tyrosine Phosphatases LAR and PTP? by Inducible Proteolytic Processing "

    Article Title: Cellular Redistribution of Protein Tyrosine Phosphatases LAR and PTP? by Inducible Proteolytic Processing

    Journal: The Journal of Cell Biology

    doi:

    Shedding of LAR and PTPσ E subunits in A431 cells. A431 cells were starved for 2 d, washed once with starvation medium, and treated with or without A23187 (10 −5 M, 1 h) or TPA (1 μM, 40 min). Cell lysates ( TRITON ) were either immunoprecipitated with antiserum specific for LAR and PTPσ (IP:320, 0.9 mg protein) or were bound to WGA-sepharose beads (WGA, 0.6 mg protein). Proteins of the tissue culture supernatant of these cells ( MEDIUM , corresponding to 1.5 mg protein of cell lysate) were bound to WGA-sepharose beads ( WGA ). Immunoprecipitates and WGA-bound proteins were separated by 8% SDS-PAGE, transferred to nitrocellulose, and analyzed by immunoblotting of the membrane with antiserum specific for the COOH terminus of LAR and PTPσ ( 320 ), the NH 2 terminus of PTPσ ( 322 ), or the COOH terminus of the LAR E subunit ( αLAR EC ). Arrows at the right indicate the position of the LAR or PTPσ subunits.
    Figure Legend Snippet: Shedding of LAR and PTPσ E subunits in A431 cells. A431 cells were starved for 2 d, washed once with starvation medium, and treated with or without A23187 (10 −5 M, 1 h) or TPA (1 μM, 40 min). Cell lysates ( TRITON ) were either immunoprecipitated with antiserum specific for LAR and PTPσ (IP:320, 0.9 mg protein) or were bound to WGA-sepharose beads (WGA, 0.6 mg protein). Proteins of the tissue culture supernatant of these cells ( MEDIUM , corresponding to 1.5 mg protein of cell lysate) were bound to WGA-sepharose beads ( WGA ). Immunoprecipitates and WGA-bound proteins were separated by 8% SDS-PAGE, transferred to nitrocellulose, and analyzed by immunoblotting of the membrane with antiserum specific for the COOH terminus of LAR and PTPσ ( 320 ), the NH 2 terminus of PTPσ ( 322 ), or the COOH terminus of the LAR E subunit ( αLAR EC ). Arrows at the right indicate the position of the LAR or PTPσ subunits.

    Techniques Used: Immunoprecipitation, Whole Genome Amplification, SDS Page

    In vitro association of LAR with plakoglobin and β-catenin. β-catenin and plakoglobin were transiently expressed in 293 cells, and cells were stimulated for 10 min with pervanadate before lysis. Equal amounts of lysates were incubated with the LAR–GST-fusion protein, GST–hPTP LAR i , or a threefold molar excess of GST, complexes were immobilized on glutathione–sepharose, and precipitates were separated by SDS-PAGE. Lysates of control plasmid-transfected 293 cells were bound in the same way to GST–hPTP LAR i -glutathione–sepharose. Bound proteins were analyzed by immunoblotting with antibodies specific for β-catenin ( A ) or plakoglobin ( B ). Arrows indicate the proteins of interest; molecular size standards in kD are shown on the left.
    Figure Legend Snippet: In vitro association of LAR with plakoglobin and β-catenin. β-catenin and plakoglobin were transiently expressed in 293 cells, and cells were stimulated for 10 min with pervanadate before lysis. Equal amounts of lysates were incubated with the LAR–GST-fusion protein, GST–hPTP LAR i , or a threefold molar excess of GST, complexes were immobilized on glutathione–sepharose, and precipitates were separated by SDS-PAGE. Lysates of control plasmid-transfected 293 cells were bound in the same way to GST–hPTP LAR i -glutathione–sepharose. Bound proteins were analyzed by immunoblotting with antibodies specific for β-catenin ( A ) or plakoglobin ( B ). Arrows indicate the proteins of interest; molecular size standards in kD are shown on the left.

    Techniques Used: In Vitro, Lysis, Incubation, SDS Page, Plasmid Preparation, Transfection

    Physiological- and calcium ionophore-induced processing of LAR and PTPσ after transfection into 293 cells. ( A ) Schematic representation of the biosynthesis of LAR and PTPσ. Dashed lines indicate a gap introduced for alignment purposes. Antibody 320 specifically recognizes the P subunit, while α LAR EC and 322 recognize the E subunits of LAR and PTPσ, respectively. ( B ) LAR or PTPσ were transiently expressed in 293 cells and before lysis cells were treated for 1 h with or without 10 −5 M A23187, as indicated. Control cells were transfected with the expression plasmid pRK5. Lysates were either separated by 8% SDS-PAGE ( TRITON ) or after binding to WGA–sepharose beads. Proteins were transferred to nitrocellulose and analyzed by immunoblotting of the membrane with antisera specific for the COOH terminus of LAR and PTPσ ( 320 ), the NH 2 terminus of PTPσ ( 322 ), or the COOH terminus of the LAR E subunit ( αLAR EC ). Arrows on the left indicate molecular weight standards, and arrows on the right indicate the position of the LAR and PTPσ subunits. ( C ) 293 cells transfected with LAR, PTPσ, or control plasmid were labeled with [ 35 S]methionine (16 h) and incubated with or without A23187, as described above. Lysates were immunoprecipitated with antiserum specific for LAR and PTPσ ( 320 ) or with nonimmune serum ( NI ). Immunoprecipitates were separated by 8% SDS-PAGE, and the dried gel was exposed to X-ray film for 24 h. Arrows on the left indicate molecular weight and on the right the position of the P subunits of LAR and PTPσ, respectively.
    Figure Legend Snippet: Physiological- and calcium ionophore-induced processing of LAR and PTPσ after transfection into 293 cells. ( A ) Schematic representation of the biosynthesis of LAR and PTPσ. Dashed lines indicate a gap introduced for alignment purposes. Antibody 320 specifically recognizes the P subunit, while α LAR EC and 322 recognize the E subunits of LAR and PTPσ, respectively. ( B ) LAR or PTPσ were transiently expressed in 293 cells and before lysis cells were treated for 1 h with or without 10 −5 M A23187, as indicated. Control cells were transfected with the expression plasmid pRK5. Lysates were either separated by 8% SDS-PAGE ( TRITON ) or after binding to WGA–sepharose beads. Proteins were transferred to nitrocellulose and analyzed by immunoblotting of the membrane with antisera specific for the COOH terminus of LAR and PTPσ ( 320 ), the NH 2 terminus of PTPσ ( 322 ), or the COOH terminus of the LAR E subunit ( αLAR EC ). Arrows on the left indicate molecular weight standards, and arrows on the right indicate the position of the LAR and PTPσ subunits. ( C ) 293 cells transfected with LAR, PTPσ, or control plasmid were labeled with [ 35 S]methionine (16 h) and incubated with or without A23187, as described above. Lysates were immunoprecipitated with antiserum specific for LAR and PTPσ ( 320 ) or with nonimmune serum ( NI ). Immunoprecipitates were separated by 8% SDS-PAGE, and the dried gel was exposed to X-ray film for 24 h. Arrows on the left indicate molecular weight and on the right the position of the P subunits of LAR and PTPσ, respectively.

    Techniques Used: Transfection, Lysis, Expressing, Plasmid Preparation, SDS Page, Binding Assay, Whole Genome Amplification, Molecular Weight, Labeling, Incubation, Immunoprecipitation

    4) Product Images from "Yeast Nop15p is an RNA-binding protein required for pre-rRNA processing and cytokinesis"

    Article Title: Yeast Nop15p is an RNA-binding protein required for pre-rRNA processing and cytokinesis

    Journal: The EMBO Journal

    doi: 10.1093/emboj/cdg616

    Fig. 2. Pulse–chase analysis of rRNA synthesis. ( A ) Structure and processing sites of the 35S pre-rRNA. This precursor contains the sequences for the mature 18S, 5.8S and 25S, which are separated by the two internal transcribed spacers ITS1 and ITS2 and flanked by the two external transcribed spacers 5′ETS and 3′ETS. The positions of oligonucleotide probes utilized in northern hybridization and primer extension analyses are indicated. ( B . ( C and D ) Pre-rRNA was pulse-labelled with [ 3 H]uracil for 2 min at 30°C and chased with a large excess of unlabelled uracil for the times indicated. Labelling was performed for the GAL::nop15 strain and a wild-type strain 16 h after transfer to glucose medium. (C) High molecular weight RNA separated on a 1.2% agarose/formaldehyde gel. (D) Low molecular weight RNA separated on an 8% polyacrylamide/urea gel.
    Figure Legend Snippet: Fig. 2. Pulse–chase analysis of rRNA synthesis. ( A ) Structure and processing sites of the 35S pre-rRNA. This precursor contains the sequences for the mature 18S, 5.8S and 25S, which are separated by the two internal transcribed spacers ITS1 and ITS2 and flanked by the two external transcribed spacers 5′ETS and 3′ETS. The positions of oligonucleotide probes utilized in northern hybridization and primer extension analyses are indicated. ( B . ( C and D ) Pre-rRNA was pulse-labelled with [ 3 H]uracil for 2 min at 30°C and chased with a large excess of unlabelled uracil for the times indicated. Labelling was performed for the GAL::nop15 strain and a wild-type strain 16 h after transfer to glucose medium. (C) High molecular weight RNA separated on a 1.2% agarose/formaldehyde gel. (D) Low molecular weight RNA separated on an 8% polyacrylamide/urea gel.

    Techniques Used: Pulse Chase, Northern Blot, Hybridization, Molecular Weight

    Fig. 4. Nop15p associates with pre-rRNAs in vivo and in vitro . ( A – C ) Nop15p-TAP co-precipitates with pre-rRNAs. Lane 1, total RNA control (5 µg); lane 2, mock precipitate from a wild-type control strain; lane 3, precipitate from a strain expressing Nop15p-TAP. (A) Northern hybridization of high and low molecular weight RNAs separated on a 1.2% agarose/formaldehyde gel or 8% polyacrylamide/urea gel, respectively. (B and C) Primer extension analyses. Nop15p-TAP was immunoprecipitated from cell lysates using IgG–agarose, with release of bound RNA–protein complexes by cleavage of the protein A linker by TEV protease. RNA was recovered from the released material, and from a mock-treated, isogenic wild-type control strain. Oligonucleotides used are indicated in parentheses. ( D ) Nop15p binds to pre-rRNA in vitro . Gel mobility shift assay performed with an in vitro transcribed pre-rRNA fragment, extending from the 5′ region of ITS1 to the 3′ region of ITS2. Nop15p was expressed in E.coli as a GST fusion with a thrombin-sensitive linker and eluted from a glutathione–Sepharose column by cleavage with thrombin. Lanes 1–4, 10 fmol pre-rRNA incubated with 0–30 fmol Nop15p as indicated; lanes 5–8, competition experiment. The gel shift assay was performed in the presence of 200-fold molar excess of cold pre-rRNA (lane 7) and tRNA (lane 8). Reactions in lanes 6–8 contained 10 fmol RNA and 15 fmol Nop15p. Complexes were resolved by electrophoresis in native 6% acrylamide/bisacrylamide (80:1) gels containing 0.5× TBE.
    Figure Legend Snippet: Fig. 4. Nop15p associates with pre-rRNAs in vivo and in vitro . ( A – C ) Nop15p-TAP co-precipitates with pre-rRNAs. Lane 1, total RNA control (5 µg); lane 2, mock precipitate from a wild-type control strain; lane 3, precipitate from a strain expressing Nop15p-TAP. (A) Northern hybridization of high and low molecular weight RNAs separated on a 1.2% agarose/formaldehyde gel or 8% polyacrylamide/urea gel, respectively. (B and C) Primer extension analyses. Nop15p-TAP was immunoprecipitated from cell lysates using IgG–agarose, with release of bound RNA–protein complexes by cleavage of the protein A linker by TEV protease. RNA was recovered from the released material, and from a mock-treated, isogenic wild-type control strain. Oligonucleotides used are indicated in parentheses. ( D ) Nop15p binds to pre-rRNA in vitro . Gel mobility shift assay performed with an in vitro transcribed pre-rRNA fragment, extending from the 5′ region of ITS1 to the 3′ region of ITS2. Nop15p was expressed in E.coli as a GST fusion with a thrombin-sensitive linker and eluted from a glutathione–Sepharose column by cleavage with thrombin. Lanes 1–4, 10 fmol pre-rRNA incubated with 0–30 fmol Nop15p as indicated; lanes 5–8, competition experiment. The gel shift assay was performed in the presence of 200-fold molar excess of cold pre-rRNA (lane 7) and tRNA (lane 8). Reactions in lanes 6–8 contained 10 fmol RNA and 15 fmol Nop15p. Complexes were resolved by electrophoresis in native 6% acrylamide/bisacrylamide (80:1) gels containing 0.5× TBE.

    Techniques Used: In Vivo, In Vitro, Expressing, Northern Blot, Hybridization, Molecular Weight, Immunoprecipitation, Mobility Shift, Incubation, Electrophoretic Mobility Shift Assay, Electrophoresis

    Fig. 3. Analysis of pre-rRNA processing. ( A ) Northern analysis. Lanes 1 and 2, wild-type strain in RGS medium and 24 h after transfer to glucose; lanes 3–7, GAL::nop15 strain in RGS medium and after transfer to glucose medium for the times indicated. ( B ) Northern analysis. Lane 1, wild-type strain 24 h after transfer to glucose; lanes 2–7, GAL::nop15 strain in RGS medium and after transfer to glucose medium for the times indicated. RNA was separated on a 1.2% agarose/formaldehyde gel (A) or 8% polyacrylamide/urea gel (B). Probe names are indicated in parentheses on the left. ( C ) Primer extension using oligo 006, which hybridizes within ITS2, 3′ to site C 2 (the 3′ end of the 7S pre-rRNA). Primer extension stops at sites A 2 , A 3 , B 1S and B 1L show levels of the 27SA 2 , 27SA 3 , 27SB S and 27SB L pre-rRNAs, respectively. Lanes 1 and 2, wild-type strain in RGS medium and 20 h after transfer to glucose medium; lanes 3 and 4, GAL::nop15 strain in RGS medium and 20 h after transfer to glucose medium.
    Figure Legend Snippet: Fig. 3. Analysis of pre-rRNA processing. ( A ) Northern analysis. Lanes 1 and 2, wild-type strain in RGS medium and 24 h after transfer to glucose; lanes 3–7, GAL::nop15 strain in RGS medium and after transfer to glucose medium for the times indicated. ( B ) Northern analysis. Lane 1, wild-type strain 24 h after transfer to glucose; lanes 2–7, GAL::nop15 strain in RGS medium and after transfer to glucose medium for the times indicated. RNA was separated on a 1.2% agarose/formaldehyde gel (A) or 8% polyacrylamide/urea gel (B). Probe names are indicated in parentheses on the left. ( C ) Primer extension using oligo 006, which hybridizes within ITS2, 3′ to site C 2 (the 3′ end of the 7S pre-rRNA). Primer extension stops at sites A 2 , A 3 , B 1S and B 1L show levels of the 27SA 2 , 27SA 3 , 27SB S and 27SB L pre-rRNAs, respectively. Lanes 1 and 2, wild-type strain in RGS medium and 20 h after transfer to glucose medium; lanes 3 and 4, GAL::nop15 strain in RGS medium and 20 h after transfer to glucose medium.

    Techniques Used: Northern Blot

    5) Product Images from "Multiple roles for the C-terminal domain of eIF5 in translation initiation complex assembly and GTPase activation"

    Article Title: Multiple roles for the C-terminal domain of eIF5 in translation initiation complex assembly and GTPase activation

    Journal: The EMBO Journal

    doi: 10.1093/emboj/20.9.2326

    Fig. 3. eIF5 interacts with eIF4G in vitro . ( A ) Binding of GST–eIF4G to native eIF2 or recombinant eIF5-FL. Lanes 1–3, Coomassie Blue staining following SDS–PAGE of GST, GST–eIF4G1 and GST–eIF4G2 proteins used in the assays. Aliquots containing ∼5 µg of GST or ∼1 µg of the full-length GST–eIF4G fusion proteins were incubated with 100 ng of either recombinant eIF5-FL (lanes 4–7) or eIF5-FL-7A (lanes 8–11), or 1 µg of eIF2 (lanes 12–15). Proteins bound to the GST fusions were isolated with glutathione–Sepharose beads (GST pull-down) and analyzed by immunoblotting with the appropriate polyclonal antibodies, except that anti-FLAG antibodies were used for detecting eIF5-FL and eIF5-FL-7A. Lanes 4, 8, 12 and 16, 20% of input (In) amount of the indicated proteins. ( B ) Binding of GST–eIF5 to segments of eIF4G2 in GST pull-down assays. Aliquots containing ∼5 µg of GST (C) or GST–eIF5 (5), shown in a Coomassie Blue-stained gel following SDS–PAGE in lanes 1 and 2, were incubated with [ 35 ).
    Figure Legend Snippet: Fig. 3. eIF5 interacts with eIF4G in vitro . ( A ) Binding of GST–eIF4G to native eIF2 or recombinant eIF5-FL. Lanes 1–3, Coomassie Blue staining following SDS–PAGE of GST, GST–eIF4G1 and GST–eIF4G2 proteins used in the assays. Aliquots containing ∼5 µg of GST or ∼1 µg of the full-length GST–eIF4G fusion proteins were incubated with 100 ng of either recombinant eIF5-FL (lanes 4–7) or eIF5-FL-7A (lanes 8–11), or 1 µg of eIF2 (lanes 12–15). Proteins bound to the GST fusions were isolated with glutathione–Sepharose beads (GST pull-down) and analyzed by immunoblotting with the appropriate polyclonal antibodies, except that anti-FLAG antibodies were used for detecting eIF5-FL and eIF5-FL-7A. Lanes 4, 8, 12 and 16, 20% of input (In) amount of the indicated proteins. ( B ) Binding of GST–eIF5 to segments of eIF4G2 in GST pull-down assays. Aliquots containing ∼5 µg of GST (C) or GST–eIF5 (5), shown in a Coomassie Blue-stained gel following SDS–PAGE in lanes 1 and 2, were incubated with [ 35 ).

    Techniques Used: In Vitro, Binding Assay, Recombinant, Staining, SDS Page, Incubation, Isolation

    6) Product Images from "LKB1 tumor suppressor protein regulates actin filament assembly through Rho and its exchange factor Dbl independently of kinase activity"

    Article Title: LKB1 tumor suppressor protein regulates actin filament assembly through Rho and its exchange factor Dbl independently of kinase activity

    Journal: BMC Cell Biology

    doi: 10.1186/1471-2121-11-77

    LKB1 interacts with endogenous Dbl . A . Schematic representation of proto-Dbl and the constructs used in this work. B . HeLa-S3 cells transfected with vector (pRK5myc), or pRK5myc-wild type LKB1. LKB1 was immunoprecipitated from cell lysates with anti-myc (lanes 1 and 3), or control IgG (lane 2) antibody and analyzed by western blot analysis. Dbl was detected using a Dbl antibody. Approximately 20% of endogenous Dbl was precipitated with myc antibody. C . HeLa-S3 cells transfected with pRK5myc-LKB1. Cell lysates were subjected to immunoprecipitation using either control rabbit IgG (lane 1), or anti-Dbl antibody (lane 2). Upper two panels are total cell lysates blotted with anti-Dbl and anti-myc antibodies. Lower two panels are Dbl and IgG immunoprecipitates blotted with anti-Dbl and anti-myc antibodies. Around 1% of the overexpressed myc-LKB1 was precipitated with endogenous Dbl using anti-Dbl. D . HeLa-S3 cells transfected with pRK5myc, or pRK5myc with LKB1 cDNAs. Bottom two panels: Endogenous Dbl immunoprecipitated from cells lysates with anti-Dbl antibody, followed by western blot using either an anti-myc, or anti-Dbl antibody. Top panel: Expression of LKB1 in total cell lysates using anti-myc. E . Dbl constructs expressed in HEK293T cells as GST fusion proteins and purified using glutathione agarose. Equal amounts of cell lysate isolated from cells expressing pRK5myc-wild type LKB1 were added to the beads, and bead-associated LKB1 detected on western blot analysis using anti-myc antibody (bottom panel). GST-Dbl and LKB1 were detected with anti-GST and anti-myc antibody, respectively.
    Figure Legend Snippet: LKB1 interacts with endogenous Dbl . A . Schematic representation of proto-Dbl and the constructs used in this work. B . HeLa-S3 cells transfected with vector (pRK5myc), or pRK5myc-wild type LKB1. LKB1 was immunoprecipitated from cell lysates with anti-myc (lanes 1 and 3), or control IgG (lane 2) antibody and analyzed by western blot analysis. Dbl was detected using a Dbl antibody. Approximately 20% of endogenous Dbl was precipitated with myc antibody. C . HeLa-S3 cells transfected with pRK5myc-LKB1. Cell lysates were subjected to immunoprecipitation using either control rabbit IgG (lane 1), or anti-Dbl antibody (lane 2). Upper two panels are total cell lysates blotted with anti-Dbl and anti-myc antibodies. Lower two panels are Dbl and IgG immunoprecipitates blotted with anti-Dbl and anti-myc antibodies. Around 1% of the overexpressed myc-LKB1 was precipitated with endogenous Dbl using anti-Dbl. D . HeLa-S3 cells transfected with pRK5myc, or pRK5myc with LKB1 cDNAs. Bottom two panels: Endogenous Dbl immunoprecipitated from cells lysates with anti-Dbl antibody, followed by western blot using either an anti-myc, or anti-Dbl antibody. Top panel: Expression of LKB1 in total cell lysates using anti-myc. E . Dbl constructs expressed in HEK293T cells as GST fusion proteins and purified using glutathione agarose. Equal amounts of cell lysate isolated from cells expressing pRK5myc-wild type LKB1 were added to the beads, and bead-associated LKB1 detected on western blot analysis using anti-myc antibody (bottom panel). GST-Dbl and LKB1 were detected with anti-GST and anti-myc antibody, respectively.

    Techniques Used: Construct, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Expressing, Purification, Isolation

    7) Product Images from "Filamin A Phosphorylation at Serine 2152 by the Serine/Threonine Kinase Ndr2 Controls TCR-Induced LFA-1 Activation in T Cells"

    Article Title: Filamin A Phosphorylation at Serine 2152 by the Serine/Threonine Kinase Ndr2 Controls TCR-Induced LFA-1 Activation in T Cells

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02852

    Mutation of S2152A within FLNa abolishes TCR-mediated T-cell adhesion, interaction with APCs and LFA-1 activation. (A) Jurkat T cells were transiently transfected with dsRed C1 vector (dsRed) or plasmids encoding wild type (WT) dsRed-tagged FLNa (FLNa WT) or dsRed-tagged S2152A or dsRed-tagged S2152E FLNa mutants (FLNa S215A and FLNa S2152E). After 24 h the expression of WT and its mutants were analyzed by anti-dsRed and FLNa immunoblotting. Detection of β-actin served as loading control. (B) Jurkat T cells transfected as described in (A) were left untreated (non) or stimulated for 30 min with CD3 antibodies. Cells were analyzed for adhesion to ICAM-1-coated 96 well plates. Bound cells were counted and calculated as percentage of input ( n = 4) (mean ± SEM; * p ≤ 0.05, *** p ≤ 0.001). (C) Cells were transfected as described in (A) and analyzed for their ability to form conjugates with DDAO-SE (red)-stained Raji B cells that were pulsed without (non) or with superantigen (SA) for 30 min at 37°C. The percentage of conjugates was defined as the number of double-positive events in the upper right quadrant ( n = 4) (mean ± SEM; *** p ≤ 0.001). (D) Jurkat T cells transfected as described in (A) were left untreated (non) or stimulated with anti-CD3 antibodies (CD3), followed by staining with the anti-LFA-1 antibody mAb24 to detect the high affinity conformation of LFA-1. mAb24 epitope expression was assessed by flow cytometry and data are normalized against LFA-1 expression detected by MEM48 ( n = 4) (mean ± SEM; *** p ≤ 0.001). (E) HEK 293T cells were transfected with either dsRed, dsRed-tagged FLNa wild type (FLAa WT) or its mutants (FLNa S2152A and FLNa S2152E). 24 h after transfection, whole cell extracts were prepared and analyzed for the expression of dsRed and dsRed-tagged FLNa forms by Western blotting using the indicated antibodies (left panel). Lysates were incubated with GST-fusion proteins bound to glutathione-sepharose beads. Precipitates were analyzed by Western blotting using the indicated antibodies (right panel). One representative experiment of 3 is shown. (mean ± SEM; * p ≤ 0.05, *** p ≤ 0.001).
    Figure Legend Snippet: Mutation of S2152A within FLNa abolishes TCR-mediated T-cell adhesion, interaction with APCs and LFA-1 activation. (A) Jurkat T cells were transiently transfected with dsRed C1 vector (dsRed) or plasmids encoding wild type (WT) dsRed-tagged FLNa (FLNa WT) or dsRed-tagged S2152A or dsRed-tagged S2152E FLNa mutants (FLNa S215A and FLNa S2152E). After 24 h the expression of WT and its mutants were analyzed by anti-dsRed and FLNa immunoblotting. Detection of β-actin served as loading control. (B) Jurkat T cells transfected as described in (A) were left untreated (non) or stimulated for 30 min with CD3 antibodies. Cells were analyzed for adhesion to ICAM-1-coated 96 well plates. Bound cells were counted and calculated as percentage of input ( n = 4) (mean ± SEM; * p ≤ 0.05, *** p ≤ 0.001). (C) Cells were transfected as described in (A) and analyzed for their ability to form conjugates with DDAO-SE (red)-stained Raji B cells that were pulsed without (non) or with superantigen (SA) for 30 min at 37°C. The percentage of conjugates was defined as the number of double-positive events in the upper right quadrant ( n = 4) (mean ± SEM; *** p ≤ 0.001). (D) Jurkat T cells transfected as described in (A) were left untreated (non) or stimulated with anti-CD3 antibodies (CD3), followed by staining with the anti-LFA-1 antibody mAb24 to detect the high affinity conformation of LFA-1. mAb24 epitope expression was assessed by flow cytometry and data are normalized against LFA-1 expression detected by MEM48 ( n = 4) (mean ± SEM; *** p ≤ 0.001). (E) HEK 293T cells were transfected with either dsRed, dsRed-tagged FLNa wild type (FLAa WT) or its mutants (FLNa S2152A and FLNa S2152E). 24 h after transfection, whole cell extracts were prepared and analyzed for the expression of dsRed and dsRed-tagged FLNa forms by Western blotting using the indicated antibodies (left panel). Lysates were incubated with GST-fusion proteins bound to glutathione-sepharose beads. Precipitates were analyzed by Western blotting using the indicated antibodies (right panel). One representative experiment of 3 is shown. (mean ± SEM; * p ≤ 0.05, *** p ≤ 0.001).

    Techniques Used: Mutagenesis, Activation Assay, Transfection, Plasmid Preparation, Expressing, Staining, Flow Cytometry, Cytometry, Western Blot, Incubation

    8) Product Images from "Two Novel Src Homology 2 Domain Proteins Interact to Regulate Dictyostelium Gene Expression during Growth and Early Development *"

    Article Title: Two Novel Src Homology 2 Domain Proteins Interact to Regulate Dictyostelium Gene Expression during Growth and Early Development *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.139733

    Binding of 14-3-3 to LrrB-CTAP. A , Ax2 cells transformed with LrrB-CTAP were harvested and shaken in suspension in KK2 at 1 × 10 7 cells/ml, and then aliquots were harvested at the time points indicated. LrrB-CTAP in the cell extracts was precipitated using IgG-agarose beads, and the 14-3-3 pulled down was detected by Western analysis using 14-3-3 antibody ( lower panel ). LrrB-CTAP in cell extracts was also assessed by Western analysis, using TAP antibody, to monitor LrrB-CTAP levels during the time course of the experiment. Similar results, with cells developed on agar, show the same drop in 14-3-3 binding, and low level was maintained up to 18 h. B , Ax2 cells transformed with either LrrB-CTAP or ΔCLrrB-CTAP were harvested during vegetative growth, and LrrB-CTAP or ΔCLrrB-CTAP in the cell extracts was precipitated (as above). Precipitated extracts were analyzed for 14-3-3 (by Western analysis) and quantified using an Odyssey infrared imaging system (LI-COR Biosciences). The amount of 14-3-3 pulled down was quantified then normalized to the LrrB-CTAP (or ΔCLrrB-CTAP) in corresponding cell extracts. Two independent experiments are shown. The top two panels show the LrrB-CTAP/ΔCLrrB-CTAP in cell extracts and 14-3-3 pulled down, and the bar graph represents the normalized values ( filled bars , LrrB-CTAP; empty bars , ΔCLrrB-CTAP).
    Figure Legend Snippet: Binding of 14-3-3 to LrrB-CTAP. A , Ax2 cells transformed with LrrB-CTAP were harvested and shaken in suspension in KK2 at 1 × 10 7 cells/ml, and then aliquots were harvested at the time points indicated. LrrB-CTAP in the cell extracts was precipitated using IgG-agarose beads, and the 14-3-3 pulled down was detected by Western analysis using 14-3-3 antibody ( lower panel ). LrrB-CTAP in cell extracts was also assessed by Western analysis, using TAP antibody, to monitor LrrB-CTAP levels during the time course of the experiment. Similar results, with cells developed on agar, show the same drop in 14-3-3 binding, and low level was maintained up to 18 h. B , Ax2 cells transformed with either LrrB-CTAP or ΔCLrrB-CTAP were harvested during vegetative growth, and LrrB-CTAP or ΔCLrrB-CTAP in the cell extracts was precipitated (as above). Precipitated extracts were analyzed for 14-3-3 (by Western analysis) and quantified using an Odyssey infrared imaging system (LI-COR Biosciences). The amount of 14-3-3 pulled down was quantified then normalized to the LrrB-CTAP (or ΔCLrrB-CTAP) in corresponding cell extracts. Two independent experiments are shown. The top two panels show the LrrB-CTAP/ΔCLrrB-CTAP in cell extracts and 14-3-3 pulled down, and the bar graph represents the normalized values ( filled bars , LrrB-CTAP; empty bars , ΔCLrrB-CTAP).

    Techniques Used: Binding Assay, Transformation Assay, Western Blot, Imaging

    9) Product Images from "Phospho-epitope binding by the BRCT domains of hPTIP controls multiple aspects of the cellular response to DNA damage"

    Article Title: Phospho-epitope binding by the BRCT domains of hPTIP controls multiple aspects of the cellular response to DNA damage

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm493

    Effect of mutation of conserved residues in the hPTIP BRCT domains on binding to phospho-53BP1 ( A ) The binding of bacterially expressed hPTIP-BRCT pair C1 + C2 proteins: wild type, W676A, R910Q and W929A to the 53BP1 phospho-Ser25 peptide (biotin-DTPCLIIEDpSQPESQVLEDD) was analysed using BiaCore as described in the Materials and Methods section and in the legend to Figure 3 A. The inset shows a Coomassie-stained gel of the GST fusion proteins used in this analysis. ( B ) HEK293 cells were co-transfected with full-length HA-53BP1 and FLAG-hPTIP bearing the indicated mutations. Cells were exposed to IR (0 Gy or 20 Gy) and after cell lysis anti-FLAG immunoprecipitates were subjected to SDS–PAGE and western blotting with the indicated antibodies. The lowest panel in each case shows HA-53BP1 levels in cell extracts. ( C ) Bacterially expressed versions of GST-hPTIP-BRCT pair C1 + C2: wild-type (WT) and Trp676Ala (W676A), GST-hPTIP-BRCT pair C1 and GST-hPTIP-BRCT pair C2, or GST alone were immobilized on glutathione-sepharose and incubated with extracts of HEK 293 cells that had been exposed, or not, to 10 Gy IR. After extensive washing, beads were subjected to SDS–PAGE and western blot analysis with antibodies against 53BP1. Low and high exposures of the blot are shown in the top and middle panels, respectively and the bottom panel shows a Coomassie-stained gel of the GST-fusions used in this experiment. NS, non-specific.
    Figure Legend Snippet: Effect of mutation of conserved residues in the hPTIP BRCT domains on binding to phospho-53BP1 ( A ) The binding of bacterially expressed hPTIP-BRCT pair C1 + C2 proteins: wild type, W676A, R910Q and W929A to the 53BP1 phospho-Ser25 peptide (biotin-DTPCLIIEDpSQPESQVLEDD) was analysed using BiaCore as described in the Materials and Methods section and in the legend to Figure 3 A. The inset shows a Coomassie-stained gel of the GST fusion proteins used in this analysis. ( B ) HEK293 cells were co-transfected with full-length HA-53BP1 and FLAG-hPTIP bearing the indicated mutations. Cells were exposed to IR (0 Gy or 20 Gy) and after cell lysis anti-FLAG immunoprecipitates were subjected to SDS–PAGE and western blotting with the indicated antibodies. The lowest panel in each case shows HA-53BP1 levels in cell extracts. ( C ) Bacterially expressed versions of GST-hPTIP-BRCT pair C1 + C2: wild-type (WT) and Trp676Ala (W676A), GST-hPTIP-BRCT pair C1 and GST-hPTIP-BRCT pair C2, or GST alone were immobilized on glutathione-sepharose and incubated with extracts of HEK 293 cells that had been exposed, or not, to 10 Gy IR. After extensive washing, beads were subjected to SDS–PAGE and western blot analysis with antibodies against 53BP1. Low and high exposures of the blot are shown in the top and middle panels, respectively and the bottom panel shows a Coomassie-stained gel of the GST-fusions used in this experiment. NS, non-specific.

    Techniques Used: Mutagenesis, Binding Assay, Staining, Transfection, Lysis, SDS Page, Western Blot, Incubation

    10) Product Images from "Expression of plant protein phosphatase 2C interferes with nuclear import of the Agrobacterium T-complex protein VirD2"

    Article Title: Expression of plant protein phosphatase 2C interferes with nuclear import of the Agrobacterium T-complex protein VirD2

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

    doi: 10.1073/pnas.0300084101

    Strength and specificity of DIG3-3 interaction with VirD2. ( A ) Yeast strains harboring the DIG3-3 prey were transformed with cVirD2, mcVirD2, flVirD2, or the A. nidulans GTPase baits and assayed for β-galactosidase activity as described in Materials and Methods . ( B ) In vitro interaction of DIG3-3 with VirD2. Purified T7-epitope tagged DIG3-3 was passed over glutathione-Sepharose columns that had bound GST, GST-cVirD2, or GST-flVirD2. Bound fractions were eluted and analyzed on Western blots with anti-T7 monoclonal antibodies. Lane 1, GST-cVirD2; lane 2, GST-flVirD2; lane 3, GST; lane 4, purified T7-tagged DIG3-3. Molecular mass markers are indicated in kDa on the left.
    Figure Legend Snippet: Strength and specificity of DIG3-3 interaction with VirD2. ( A ) Yeast strains harboring the DIG3-3 prey were transformed with cVirD2, mcVirD2, flVirD2, or the A. nidulans GTPase baits and assayed for β-galactosidase activity as described in Materials and Methods . ( B ) In vitro interaction of DIG3-3 with VirD2. Purified T7-epitope tagged DIG3-3 was passed over glutathione-Sepharose columns that had bound GST, GST-cVirD2, or GST-flVirD2. Bound fractions were eluted and analyzed on Western blots with anti-T7 monoclonal antibodies. Lane 1, GST-cVirD2; lane 2, GST-flVirD2; lane 3, GST; lane 4, purified T7-tagged DIG3-3. Molecular mass markers are indicated in kDa on the left.

    Techniques Used: Transformation Assay, Activity Assay, In Vitro, Purification, Western Blot

    11) Product Images from "Identification of Nucleolin as New ErbB Receptors- Interacting Protein"

    Article Title: Identification of Nucleolin as New ErbB Receptors- Interacting Protein

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0002310

    Nucleolin binds the cytoplasmic tail of ErbB4. (A) SDS-PAGE of Coomassie blue–stained proteins isolated from PC12 cell extracts, loaded on GST-ErbB4 agarose affinity matrix or control GST agarose matrix. The arrow indicates a specific 110-kD band, which was identified as nucleolin by mass spectroscopy. (B) COS7 cells were transiently transfected with expression vector of Myc-Nucleolin. Cell lysates were incubated with immobilized GST-ErbB4 or GST. Proteins retained on the beads were resolved by SDS-PAGE and then processed for Western blot using anti-Myc antibodies. (C) Cell lysates prepared from Du145 cells were incubated with immobilized GST-ErbB4 or GST. Eluates from GST-ErbB4 and GST control affinity matrices were resolved by SDS-PAGE and then processed for Western blot using a monoclonal mouse anti-nucleolin antibody.
    Figure Legend Snippet: Nucleolin binds the cytoplasmic tail of ErbB4. (A) SDS-PAGE of Coomassie blue–stained proteins isolated from PC12 cell extracts, loaded on GST-ErbB4 agarose affinity matrix or control GST agarose matrix. The arrow indicates a specific 110-kD band, which was identified as nucleolin by mass spectroscopy. (B) COS7 cells were transiently transfected with expression vector of Myc-Nucleolin. Cell lysates were incubated with immobilized GST-ErbB4 or GST. Proteins retained on the beads were resolved by SDS-PAGE and then processed for Western blot using anti-Myc antibodies. (C) Cell lysates prepared from Du145 cells were incubated with immobilized GST-ErbB4 or GST. Eluates from GST-ErbB4 and GST control affinity matrices were resolved by SDS-PAGE and then processed for Western blot using a monoclonal mouse anti-nucleolin antibody.

    Techniques Used: SDS Page, Staining, Isolation, Mass Spectrometry, Transfection, Expressing, Plasmid Preparation, Incubation, Western Blot

    12) Product Images from "Termination of non‐coding transcription in yeast relies on both an RNA Pol II CTD interaction domain and a CTD‐mimicking region in Sen1"

    Article Title: Termination of non‐coding transcription in yeast relies on both an RNA Pol II CTD interaction domain and a CTD‐mimicking region in Sen1

    Journal: The EMBO Journal

    doi: 10.15252/embj.2019101548

    The N‐terminal domain of Sen1 can recognize the S5‐phosphorylated form of RNAPII CTD and Sen1 C‐terminal domain Deletion of Sen1 N‐terminal domain does not prevent the interaction of Sen1 with RNAPII. CoIP experiments using Rbp3‐FLAG as the bait. Assays were performed in a Sen1‐AID strain harbouring a plasmid expressing either SEN1 or sen1∆Nter upon depletion of Sen1‐AID in the presence of IAA for 2 h. An asterisk denotes a major proteolytic Sen1 fragment detected in the extracts of roughly the size of sen1∆Nter . Nrd1 is detected as a positive control. Representative gel of one out of two independent experiments. Protein extracts were treated with RNaseA before immunoprecipitation. Deletion of the Sen1 N‐terminal domain reduces the interaction of Sen1 with the S5P‐CTD. CoIP experiments using TAP‐Sen1 as the bait. Sen1 proteins were expressed from pGAL in the presence of galactose. Nab3 is detected as a positive control. Representative gel of one out of three independent experiments. Protein extracts were treated with RNaseA before immunoprecipitation. Replacing the Nter of Sen1 by the CID of Nrd1 restores viability. Growth test performed in the same conditions as in Fig 3 A but in the presence of a TRP1 ‐plasmid carrying the SEN1 versions indicated in the scheme on the left. The growth of the strain expressing the Nrd1 CID‐sen1 ∆ Nter chimera in 5‐FOA implies that this gene can support viability. Substituting the Nter of Sen1 by Nrd1 CID but not Pcf11 CID partially suppresses the termination defects detected in the sen1∆Nter mutant. Northern blot assays performed in a Sen1‐AID strain carrying an empty vector or a plasmid expressing the indicated versions of SEN1 upon depletion of the endogenous Sen1 protein as in Fig 3 B. Experiments performed in a ∆rrp6 background. Representative gel of one out of two independent experiments. The U4 RNA is used as a loading control. Probes used for RNA detection are described in Appendix Table S6 . Sen1 Nter interacts with the C‐terminal domain (Cter) of Sen1 both in the presence and in the absence of the NIM in vitro . Pull‐down experiments using either a wt or a ∆ NIM version of recombinant Sen1 Cter immobilized on glutathione sepharose beads and a TAP‐tagged version of Sen1 Nter expressed in yeast. Representative gel of one out of three independent experiments. Data information: Antibodies used for protein detection are listed in Appendix Table S3 .
    Figure Legend Snippet: The N‐terminal domain of Sen1 can recognize the S5‐phosphorylated form of RNAPII CTD and Sen1 C‐terminal domain Deletion of Sen1 N‐terminal domain does not prevent the interaction of Sen1 with RNAPII. CoIP experiments using Rbp3‐FLAG as the bait. Assays were performed in a Sen1‐AID strain harbouring a plasmid expressing either SEN1 or sen1∆Nter upon depletion of Sen1‐AID in the presence of IAA for 2 h. An asterisk denotes a major proteolytic Sen1 fragment detected in the extracts of roughly the size of sen1∆Nter . Nrd1 is detected as a positive control. Representative gel of one out of two independent experiments. Protein extracts were treated with RNaseA before immunoprecipitation. Deletion of the Sen1 N‐terminal domain reduces the interaction of Sen1 with the S5P‐CTD. CoIP experiments using TAP‐Sen1 as the bait. Sen1 proteins were expressed from pGAL in the presence of galactose. Nab3 is detected as a positive control. Representative gel of one out of three independent experiments. Protein extracts were treated with RNaseA before immunoprecipitation. Replacing the Nter of Sen1 by the CID of Nrd1 restores viability. Growth test performed in the same conditions as in Fig 3 A but in the presence of a TRP1 ‐plasmid carrying the SEN1 versions indicated in the scheme on the left. The growth of the strain expressing the Nrd1 CID‐sen1 ∆ Nter chimera in 5‐FOA implies that this gene can support viability. Substituting the Nter of Sen1 by Nrd1 CID but not Pcf11 CID partially suppresses the termination defects detected in the sen1∆Nter mutant. Northern blot assays performed in a Sen1‐AID strain carrying an empty vector or a plasmid expressing the indicated versions of SEN1 upon depletion of the endogenous Sen1 protein as in Fig 3 B. Experiments performed in a ∆rrp6 background. Representative gel of one out of two independent experiments. The U4 RNA is used as a loading control. Probes used for RNA detection are described in Appendix Table S6 . Sen1 Nter interacts with the C‐terminal domain (Cter) of Sen1 both in the presence and in the absence of the NIM in vitro . Pull‐down experiments using either a wt or a ∆ NIM version of recombinant Sen1 Cter immobilized on glutathione sepharose beads and a TAP‐tagged version of Sen1 Nter expressed in yeast. Representative gel of one out of three independent experiments. Data information: Antibodies used for protein detection are listed in Appendix Table S3 .

    Techniques Used: Co-Immunoprecipitation Assay, Plasmid Preparation, Expressing, Positive Control, Immunoprecipitation, Mutagenesis, Northern Blot, RNA Detection, In Vitro, Recombinant

    13) Product Images from "Involvement of Phospholipase C?1 in Mouse Egg Activation Induced by a Truncated Form of the C-kit Tyrosine Kinase Present in Spermatozoa "

    Article Title: Involvement of Phospholipase C?1 in Mouse Egg Activation Induced by a Truncated Form of the C-kit Tyrosine Kinase Present in Spermatozoa

    Journal: The Journal of Cell Biology

    doi:

    Tr-kit does not stably associate with PLCγ1. ( A ) COS cells were transfected with no DNA ( mock ), or 20 μg/dish pCMV5-c-kit ( c-kit ), or 20 μg/dish pCMV5-tr-kit ( tr-kit ). C-kit–transfected cells were incubated for the final 10 min with or without 100 ng/ml SCF. Cell extracts were either analyzed immediately in Western blot (50 μg in each lane) with an anti-kit antibody ( right side of the panel ), or incubated for 2 h with a GST-PLCγ1-SH2SH2SH3 fusion protein linked to glutathione–agarose beads. Proteins bound to the beads were eluted as described under Materials and Methods and analyzed in Western blot using an anti-kit antibody ( left side of the panel ). ( B ) Cells were transfected as described in A with tr-kit or c-kit expression vectors. Cell extracts were immunoprecipitated using an anti-kit antibody preadsorbed to protein A–Sepharose beads. Immunoprecipitated proteins were analyzed in Western blot using an anti-phosphotyrosine antibody. The band recognized by the anti-phosphotyrosine antibody with a molecular size similar to the one expected for tr-kit is present in all the samples, regardless of tr-kit presence, indicating that this band is due to a different tyrosine-phosphorylated protein present in the anti-kit immunoprecipitates from COS cells. ( C ) Cell extracts (50 μg) from the same samples shown in B were analyzed in Western blot using an anti-kit antibody. All panels are representative of at least three separate experiments.
    Figure Legend Snippet: Tr-kit does not stably associate with PLCγ1. ( A ) COS cells were transfected with no DNA ( mock ), or 20 μg/dish pCMV5-c-kit ( c-kit ), or 20 μg/dish pCMV5-tr-kit ( tr-kit ). C-kit–transfected cells were incubated for the final 10 min with or without 100 ng/ml SCF. Cell extracts were either analyzed immediately in Western blot (50 μg in each lane) with an anti-kit antibody ( right side of the panel ), or incubated for 2 h with a GST-PLCγ1-SH2SH2SH3 fusion protein linked to glutathione–agarose beads. Proteins bound to the beads were eluted as described under Materials and Methods and analyzed in Western blot using an anti-kit antibody ( left side of the panel ). ( B ) Cells were transfected as described in A with tr-kit or c-kit expression vectors. Cell extracts were immunoprecipitated using an anti-kit antibody preadsorbed to protein A–Sepharose beads. Immunoprecipitated proteins were analyzed in Western blot using an anti-phosphotyrosine antibody. The band recognized by the anti-phosphotyrosine antibody with a molecular size similar to the one expected for tr-kit is present in all the samples, regardless of tr-kit presence, indicating that this band is due to a different tyrosine-phosphorylated protein present in the anti-kit immunoprecipitates from COS cells. ( C ) Cell extracts (50 μg) from the same samples shown in B were analyzed in Western blot using an anti-kit antibody. All panels are representative of at least three separate experiments.

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

    14) Product Images from "Covalent Modification of the Transcriptional Repressor Tramtrack by the Ubiquitin-Related Protein Smt3 in Drosophila Flies"

    Article Title: Covalent Modification of the Transcriptional Repressor Tramtrack by the Ubiquitin-Related Protein Smt3 in Drosophila Flies

    Journal: Molecular and Cellular Biology

    doi:

    The Ttk69 transcriptional repressor is covalently modified by dSmt3. (A) Extracts from SL2 cells cotransfected with a vector expressing the Ttk69 protein with a vector expressing dSmt3 HA (lanes 1 and 5), Cactus His (lanes 2 and 6), or dSmt3 His (lanes 3, 4, and 7) were subjected to precipitation with Ni-agarose beads, and the precipitates were analyzed by Western blotting with the rat anti-Ttk69 polyclonal antibody. Aliquots of the corresponding unprecipitated extracts (1/10) were loaded in lanes 1 to 4. In lane 4, the cells had been incubated with 1.25 μM calyculin A prior to protein extraction. The 120-kDa doublet observed in crude extracts (lanes 1 and 3) corresponds to the Ttk69 protein conjugated either to the endogenous dSmt3 protein (lower band of the doublet) or to the transfected HA- or His-tagged dSmt3 product (upper band of the doublet). (B) SDS lysates from untransfected SL2 cells (lane 1) or from SL2 cells cotransfected with dSmt3 and Ttk69 were immunoprecipitated with the rabbit polyclonal anti-PML antibody (lane 3), the rabbit anti-Ttk69 antibody (lane 4), or the rabbit anti-dSmt3 antibody (lane 5). An aliquot of the transfected cell extract (1/200 of the material used for immunoprecipitation) was loaded in lane 2. Immunoprecipitates and cell extracts were fractionated by electrophoresis and analyzed by Western blotting with a rat anti-Ttk69 antibody. Immunoglobulins are marked by an asterisk. The 100-kDa unmodified and 120-kDa dSmt3-modified forms of Ttk69 are indicated.
    Figure Legend Snippet: The Ttk69 transcriptional repressor is covalently modified by dSmt3. (A) Extracts from SL2 cells cotransfected with a vector expressing the Ttk69 protein with a vector expressing dSmt3 HA (lanes 1 and 5), Cactus His (lanes 2 and 6), or dSmt3 His (lanes 3, 4, and 7) were subjected to precipitation with Ni-agarose beads, and the precipitates were analyzed by Western blotting with the rat anti-Ttk69 polyclonal antibody. Aliquots of the corresponding unprecipitated extracts (1/10) were loaded in lanes 1 to 4. In lane 4, the cells had been incubated with 1.25 μM calyculin A prior to protein extraction. The 120-kDa doublet observed in crude extracts (lanes 1 and 3) corresponds to the Ttk69 protein conjugated either to the endogenous dSmt3 protein (lower band of the doublet) or to the transfected HA- or His-tagged dSmt3 product (upper band of the doublet). (B) SDS lysates from untransfected SL2 cells (lane 1) or from SL2 cells cotransfected with dSmt3 and Ttk69 were immunoprecipitated with the rabbit polyclonal anti-PML antibody (lane 3), the rabbit anti-Ttk69 antibody (lane 4), or the rabbit anti-dSmt3 antibody (lane 5). An aliquot of the transfected cell extract (1/200 of the material used for immunoprecipitation) was loaded in lane 2. Immunoprecipitates and cell extracts were fractionated by electrophoresis and analyzed by Western blotting with a rat anti-Ttk69 antibody. Immunoglobulins are marked by an asterisk. The 100-kDa unmodified and 120-kDa dSmt3-modified forms of Ttk69 are indicated.

    Techniques Used: Modification, Plasmid Preparation, Expressing, Western Blot, Incubation, Protein Extraction, Transfection, Immunoprecipitation, Electrophoresis

    dSmt3-protein conjugates in Drosophila and human cells. (A) dSmt3 is conjugated to multiple proteins in SL2 and HeLa cells. dSmt3 HA was transfected in SL2 or HeLa cells. Protein extracts from untransfected (−) and transfected (+) cells were subjected to Western blot analysis using anti-HA (α-HA) MAb, anti-dSmt3 (α-dSmt3) antiserum, or the corresponding preimmune serum (PI). Positions of the free dSmt3 HA and its conjugates are indicated. (B) Modification of PML by dSmt3 in SL2 cells. dSmt3 His or untagged dSmt3 was coexpressed with PML in SL2 cells. Extracts were precipitated by nickel-agarose beads (Ppn Ni). Crude extracts (1/10; lanes 1 to 3) and Ni-agarose precipitates (lanes 4 to 6) were analyzed by Western blotting using anti-PML antibodies. Positions of PML and its dSmt3-conjugated forms are indicated.
    Figure Legend Snippet: dSmt3-protein conjugates in Drosophila and human cells. (A) dSmt3 is conjugated to multiple proteins in SL2 and HeLa cells. dSmt3 HA was transfected in SL2 or HeLa cells. Protein extracts from untransfected (−) and transfected (+) cells were subjected to Western blot analysis using anti-HA (α-HA) MAb, anti-dSmt3 (α-dSmt3) antiserum, or the corresponding preimmune serum (PI). Positions of the free dSmt3 HA and its conjugates are indicated. (B) Modification of PML by dSmt3 in SL2 cells. dSmt3 His or untagged dSmt3 was coexpressed with PML in SL2 cells. Extracts were precipitated by nickel-agarose beads (Ppn Ni). Crude extracts (1/10; lanes 1 to 3) and Ni-agarose precipitates (lanes 4 to 6) were analyzed by Western blotting using anti-PML antibodies. Positions of PML and its dSmt3-conjugated forms are indicated.

    Techniques Used: Transfection, Western Blot, Modification

    15) Product Images from "A mitochondria-anchored isoform of the actin-nucleating spire protein regulates mitochondrial division"

    Article Title: A mitochondria-anchored isoform of the actin-nucleating spire protein regulates mitochondrial division

    Journal: eLife

    doi: 10.7554/eLife.08828

    Spire1C and inverted formin 2 (INF2) directly interact and work together to regulate mitochondrial fission. ( A ) INF2-CT directly binds to Spire1-KIND in vitro. GST pull-down assays in actin polymerization buffer, containing combinations of the following: 20 μM GST or GST-Spire1 KIND bound to glutathione-sepharose beads; 1 μM INF2-CT; and 10 μM INF2-NT. Co-incubation of the GST-Spire1 KIND domain pulls down INF2-CT (third to last lane), but not INF2-NT (second to last lane). INF2-CT pulldown is inhibited by the addition of the INF2-NT (last lane). STDS lane represents 0.2 μM INF2-CT. ( B ) Fluorescence anisotropy binding curve of purified INF2-CT (20 nM) labeled with tetramethylrhodamine succinimide mixed with varying concentrations of Spire1 KIND or bovine serum albumin reveals a direct interaction between Spire1 KIND and INF2-CT. ( C ) Cells overexpressing a constitutively active INF2 mutant (INF2 A149 alone, n cells = 16, n mitochondria = 232) display very short, fragmented mitochondria compared to control cells (p
    Figure Legend Snippet: Spire1C and inverted formin 2 (INF2) directly interact and work together to regulate mitochondrial fission. ( A ) INF2-CT directly binds to Spire1-KIND in vitro. GST pull-down assays in actin polymerization buffer, containing combinations of the following: 20 μM GST or GST-Spire1 KIND bound to glutathione-sepharose beads; 1 μM INF2-CT; and 10 μM INF2-NT. Co-incubation of the GST-Spire1 KIND domain pulls down INF2-CT (third to last lane), but not INF2-NT (second to last lane). INF2-CT pulldown is inhibited by the addition of the INF2-NT (last lane). STDS lane represents 0.2 μM INF2-CT. ( B ) Fluorescence anisotropy binding curve of purified INF2-CT (20 nM) labeled with tetramethylrhodamine succinimide mixed with varying concentrations of Spire1 KIND or bovine serum albumin reveals a direct interaction between Spire1 KIND and INF2-CT. ( C ) Cells overexpressing a constitutively active INF2 mutant (INF2 A149 alone, n cells = 16, n mitochondria = 232) display very short, fragmented mitochondria compared to control cells (p

    Techniques Used: In Vitro, Incubation, Fluorescence, Binding Assay, Purification, Labeling, Mutagenesis

    16) Product Images from "Drug discovery with an RBM20 dependent titin splice reporter identifies cardenolides as lead structures to improve cardiac filling"

    Article Title: Drug discovery with an RBM20 dependent titin splice reporter identifies cardenolides as lead structures to improve cardiac filling

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0198492

    Inhibitors of titin splicing identified by HTS. For each compound concentration dependent activity in the dual luciferase reporter assay and cell viability are plotted. Dashed lines indicate the concentrations used for validation on RNA level. The tables provide kinetic information. Validation by RT-PCR (agarose gel) is quantified by calculating the percent spliced in values (PSI). (a-c) Inhibition of alternative splicing was validated manually in the 96-well format using the DLR assay and conventional as well as quantitative RT-qPCR using an independent genomic minigene derived from TTN exons 241-43 (N = 4). Cardenolides efficiently reduce titin splicing by RBM20 with different potency (splicing IC50). The effect on splicing translates to reduced viability of HEK293 cells (IC50 values splicing vs. viability). (d) The steroid hydrocortisone does not interfere with splicing activity (N = 4). Compared to the cardenolides it lacks the lactone ring at C17 (chemical structures provided on the right). * P
    Figure Legend Snippet: Inhibitors of titin splicing identified by HTS. For each compound concentration dependent activity in the dual luciferase reporter assay and cell viability are plotted. Dashed lines indicate the concentrations used for validation on RNA level. The tables provide kinetic information. Validation by RT-PCR (agarose gel) is quantified by calculating the percent spliced in values (PSI). (a-c) Inhibition of alternative splicing was validated manually in the 96-well format using the DLR assay and conventional as well as quantitative RT-qPCR using an independent genomic minigene derived from TTN exons 241-43 (N = 4). Cardenolides efficiently reduce titin splicing by RBM20 with different potency (splicing IC50). The effect on splicing translates to reduced viability of HEK293 cells (IC50 values splicing vs. viability). (d) The steroid hydrocortisone does not interfere with splicing activity (N = 4). Compared to the cardenolides it lacks the lactone ring at C17 (chemical structures provided on the right). * P

    Techniques Used: Concentration Assay, Activity Assay, Luciferase, Reporter Assay, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Inhibition, Quantitative RT-PCR, Derivative Assay

    17) Product Images from "Vps51p Mediates the Association of the GARP (Vps52/53/54) Complex with the Late Golgi t-SNARE Tlg1p"

    Article Title: Vps51p Mediates the Association of the GARP (Vps52/53/54) Complex with the Late Golgi t-SNARE Tlg1p

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E02-10-0654

    VPS51 is required for the binding of Vps52p to the Tlg1p N-terminal domain in vitro. Binding to the N-terminal (N), C-terminal (C), or full-length cytosolic portion (C+N) of Tlg1p was assessed by incubating lysates from cells expressing Vps52p-HA (1 and 2) or Vps51p-myc (3) with GST-Tlg1 fusion proteins purified from E. coli and bound to sepharose beads. The binding of Vps52p-HA to the full-length and N-terminal domains of Tlg1p was abolished by loss of VPS51 . The following strains were used: 1, LCY325; 2, LCY321; and 3, LCY327.
    Figure Legend Snippet: VPS51 is required for the binding of Vps52p to the Tlg1p N-terminal domain in vitro. Binding to the N-terminal (N), C-terminal (C), or full-length cytosolic portion (C+N) of Tlg1p was assessed by incubating lysates from cells expressing Vps52p-HA (1 and 2) or Vps51p-myc (3) with GST-Tlg1 fusion proteins purified from E. coli and bound to sepharose beads. The binding of Vps52p-HA to the full-length and N-terminal domains of Tlg1p was abolished by loss of VPS51 . The following strains were used: 1, LCY325; 2, LCY321; and 3, LCY327.

    Techniques Used: Binding Assay, In Vitro, Expressing, Purification

    18) Product Images from "Importin ? Protein Acts as a Negative Regulator for Snail Protein Nuclear Import *"

    Article Title: Importin ? Protein Acts as a Negative Regulator for Snail Protein Nuclear Import *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.213579

    Importin α inhibits nuclear import of Snail in vitro and in vivo . A , three importin αs showed direct binding activity on the Snail protein. GST-Snail-GFP was incubated with FLAG-importin α in the presence of anti-FLAG-agarose.
    Figure Legend Snippet: Importin α inhibits nuclear import of Snail in vitro and in vivo . A , three importin αs showed direct binding activity on the Snail protein. GST-Snail-GFP was incubated with FLAG-importin α in the presence of anti-FLAG-agarose.

    Techniques Used: In Vitro, In Vivo, Binding Assay, Activity Assay, Incubation

    19) Product Images from "β-Catenin Associates With Human Parainfluenza Virus Type 3 Ribonucleoprotein Complex and Activates Transcription of Viral Genome RNA In Vitro"

    Article Title: β-Catenin Associates With Human Parainfluenza Virus Type 3 Ribonucleoprotein Complex and Activates Transcription of Viral Genome RNA In Vitro

    Journal: Gene Expression

    doi:

    In vitro transcriptional activation of HPIV-3 genome by immunoprecipitated β-catenin (β-cat). In vitro transcription was carried out as described in Materials and Methods in a transcription reaction mixture containing purified HPIV-3 RNP. Washed immunoprotein A-sepharose bead pellets (P) obtained following immunoprecipitation of uninfected A549 cell lysate with either actin (lane 2) or β-catenin (lane 5) antibodies were added to the transcription reaction either separately (lane 2 and lane 5) or together (lane 3). Similarly, immunopellets obtained following immunoprecipitation with control rabbit serum (lane 4) and the corresponding immunodepleted supernatant (S) obtained from control rabbit serum (lane 6) or β-catenin (devoid of β-catenin) (lane 7) immunoprecipitates were added to the transcription reaction mixture and analyzed as described in the Materials and Methods.
    Figure Legend Snippet: In vitro transcriptional activation of HPIV-3 genome by immunoprecipitated β-catenin (β-cat). In vitro transcription was carried out as described in Materials and Methods in a transcription reaction mixture containing purified HPIV-3 RNP. Washed immunoprotein A-sepharose bead pellets (P) obtained following immunoprecipitation of uninfected A549 cell lysate with either actin (lane 2) or β-catenin (lane 5) antibodies were added to the transcription reaction either separately (lane 2 and lane 5) or together (lane 3). Similarly, immunopellets obtained following immunoprecipitation with control rabbit serum (lane 4) and the corresponding immunodepleted supernatant (S) obtained from control rabbit serum (lane 6) or β-catenin (devoid of β-catenin) (lane 7) immunoprecipitates were added to the transcription reaction mixture and analyzed as described in the Materials and Methods.

    Techniques Used: In Vitro, Activation Assay, Immunoprecipitation, Purification

    Iin vitro binding of his-tagged β-catenin with GST-N and GST-P. (A) Purified his-tagged β-catenin (2 μg) was subjected to 7.5% SDS-PAGE and Coomassie blue staining. (B) Purified his-tagged β-catenin (0.2 μg) (lane 1) and A549 cell lysate (12 μg) (lane 2) were subjected to 7.5% SDS-PAGE and Western blot analysis with β-catenin antibody. (C) GST alone (lane 1), GST-P (lane 2), and GST-N (lane 3) (8 μg each) bound to the glutathione beads were incubated with purified his-tagged β-catenin (0.5 μg). The in vitro binding of β-catenin with GST and GST fusion proteins were visualized following Western blot analysis of glutathione-sepharose-bound proteins with β-catenin antibody. Cell lysate from A549 cells (lane 4) was used as a control.
    Figure Legend Snippet: Iin vitro binding of his-tagged β-catenin with GST-N and GST-P. (A) Purified his-tagged β-catenin (2 μg) was subjected to 7.5% SDS-PAGE and Coomassie blue staining. (B) Purified his-tagged β-catenin (0.2 μg) (lane 1) and A549 cell lysate (12 μg) (lane 2) were subjected to 7.5% SDS-PAGE and Western blot analysis with β-catenin antibody. (C) GST alone (lane 1), GST-P (lane 2), and GST-N (lane 3) (8 μg each) bound to the glutathione beads were incubated with purified his-tagged β-catenin (0.5 μg). The in vitro binding of β-catenin with GST and GST fusion proteins were visualized following Western blot analysis of glutathione-sepharose-bound proteins with β-catenin antibody. Cell lysate from A549 cells (lane 4) was used as a control.

    Techniques Used: Binding Assay, Purification, SDS Page, Staining, Western Blot, Incubation, In Vitro

    20) Product Images from "CD44 Promotes Intoxication by the Clostridial Iota-Family Toxins"

    Article Title: CD44 Promotes Intoxication by the Clostridial Iota-Family Toxins

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0051356

    Binding of iota-family B components to purified CD44 in solution. ( A and B ) The B component (10 µg) of each toxin was added to CD44-IgG (10 µg in 50 µl) at room temperature for 60 min. Protein A - agarose beads were then added for 5 min at room temperature, gently centrifuged, and washed with buffer. SDS-PAGE sample buffer containing reducing agent was added to the beads, the mixture heated, and protein separated from the beads by centrifugation. Supernatant proteins were then resolved on a 10% gel, transferred onto nitrocellulose, and clostridial B component detected with either 1∶1000 dilutions of rabbit anti-Ib (Panel A) or anti-C2II sera (Panel B). Protein A - peroxidase conjugate was used to detect bound antibody, and following washes, specific bands were visualized with chemiluminescent substrate. (C) Like the CD44-IgG construct, Ib also binds specifically to a CD44-GST construct. A GST version of C. botulinum C3 exoenzyme, used as a negative control, does not bind to Ib in pull-down experiments done similarly for panels A and B, with an exception being the use of glutathione-sepharose (instead of Protein A-agarose) beads.
    Figure Legend Snippet: Binding of iota-family B components to purified CD44 in solution. ( A and B ) The B component (10 µg) of each toxin was added to CD44-IgG (10 µg in 50 µl) at room temperature for 60 min. Protein A - agarose beads were then added for 5 min at room temperature, gently centrifuged, and washed with buffer. SDS-PAGE sample buffer containing reducing agent was added to the beads, the mixture heated, and protein separated from the beads by centrifugation. Supernatant proteins were then resolved on a 10% gel, transferred onto nitrocellulose, and clostridial B component detected with either 1∶1000 dilutions of rabbit anti-Ib (Panel A) or anti-C2II sera (Panel B). Protein A - peroxidase conjugate was used to detect bound antibody, and following washes, specific bands were visualized with chemiluminescent substrate. (C) Like the CD44-IgG construct, Ib also binds specifically to a CD44-GST construct. A GST version of C. botulinum C3 exoenzyme, used as a negative control, does not bind to Ib in pull-down experiments done similarly for panels A and B, with an exception being the use of glutathione-sepharose (instead of Protein A-agarose) beads.

    Techniques Used: Binding Assay, Purification, SDS Page, Centrifugation, Construct, Negative Control

    21) Product Images from "Interaction of the synprint site of N-type Ca2+ channels with the C2B domain of synaptotagmin I"

    Article Title: Interaction of the synprint site of N-type Ca2+ channels with the C2B domain of synaptotagmin I

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

    doi:

    Displacement of synaptotagmin binding to syntaxin by the synprint peptide. ( A ) Synprint peptide competes with the binding of recombinant syt I to syntaxin 1A. GST-syntaxin 1A bound to glutathione-Sepharose beads was incubated with a constant amount of the purified recombinant cytoplasmic domain (residues 80–421) of syt I and increasing amounts of His-L II–III (718–963) as indicated, in a binding buffer containing 20 μM of free Ca 2+ . Beads were washed, and bound proteins were eluted with 15 mM reduced glutathione in 50 mM of Tris⋅HCl (pH 8), and analyzed by immunoblotting with 1D12, a mAb against the carboxyl terminals of both syt I and syt II, and T7.Tag antibody as described. ( B ) The synprint peptide competes for the interaction of native synaptotagmin with syntaxin. GST-VAMP/synaptobrevin 2 bound to glutathione-Sepharose beads was incubated with a constant amount of solubilized synaptosomes and an increasing amount of His-L II–III (718–963) as indicated, in a binding buffer with 20 μM free Ca 2+ ) to form an SDS-resistant complex, which serves as an acceptor for binding either recombinant L II–III (718–963) or native synaptotagmin from solubilized synaptosomes. After extensive washing with incubation buffer, GST-VAMP/synaptobrevin 2-bound protein complexes were eluted with reduced glutathione and analyzed by immunoblotting with T7.Tag, 1D12, and 10H5, a syntaxin 1 antibody. Each lane contains an equivalent amount of GST-syntaxin 1A ( A ) and GST-VAMP/synaptobrevin 2 ( B ), bound to beads, as confirmed by anti-GST immunoblotting (data not shown).
    Figure Legend Snippet: Displacement of synaptotagmin binding to syntaxin by the synprint peptide. ( A ) Synprint peptide competes with the binding of recombinant syt I to syntaxin 1A. GST-syntaxin 1A bound to glutathione-Sepharose beads was incubated with a constant amount of the purified recombinant cytoplasmic domain (residues 80–421) of syt I and increasing amounts of His-L II–III (718–963) as indicated, in a binding buffer containing 20 μM of free Ca 2+ . Beads were washed, and bound proteins were eluted with 15 mM reduced glutathione in 50 mM of Tris⋅HCl (pH 8), and analyzed by immunoblotting with 1D12, a mAb against the carboxyl terminals of both syt I and syt II, and T7.Tag antibody as described. ( B ) The synprint peptide competes for the interaction of native synaptotagmin with syntaxin. GST-VAMP/synaptobrevin 2 bound to glutathione-Sepharose beads was incubated with a constant amount of solubilized synaptosomes and an increasing amount of His-L II–III (718–963) as indicated, in a binding buffer with 20 μM free Ca 2+ ) to form an SDS-resistant complex, which serves as an acceptor for binding either recombinant L II–III (718–963) or native synaptotagmin from solubilized synaptosomes. After extensive washing with incubation buffer, GST-VAMP/synaptobrevin 2-bound protein complexes were eluted with reduced glutathione and analyzed by immunoblotting with T7.Tag, 1D12, and 10H5, a syntaxin 1 antibody. Each lane contains an equivalent amount of GST-syntaxin 1A ( A ) and GST-VAMP/synaptobrevin 2 ( B ), bound to beads, as confirmed by anti-GST immunoblotting (data not shown).

    Techniques Used: Binding Assay, Recombinant, Incubation, Purification

    22) Product Images from "Elongation factor 2 kinase promotes cell survival by inhibiting protein synthesis without inducing autophagy"

    Article Title: Elongation factor 2 kinase promotes cell survival by inhibiting protein synthesis without inducing autophagy

    Journal: Cellular Signalling

    doi: 10.1016/j.cellsig.2016.01.005

    eEF2K does not regulate autophagy in lung carcinoma cells (A) A549 cells were cultured in the presence or absence of 1 mM IPTG for 5 days to induce the knockdown of eEF2K. Cells were then treated with 10 mM 2-deoxyglucose (2DG), rapamycin (100 nM), AZD8055 (1 μM) or MG132 (10 μM) for 16 h. Cells were then lysed and samples containing equal amounts of protein were analysed by western blot using the indicated antibodies. Values given below each lane show quantitation of the signal for LC3II, corrected for the actin signal from multiple experiments as in (A) expressed as LC3II normalized to actin, mean ± SEM (control cells without treatment = 1; n = 3). (B) A549 cells were cultured as in (A) to induce eEF2K knockdown. A549 cells were transiently transfected with the GST-BHMT construct and maintained in full media in the presence or absence of AZD8055 (1 μM) for 16 h, in the presence of leupeptin and E64d. GST-BHMT was precipitated with glutathione agarose from the whole cell extracts (bottom panel). Values given below show the quantitation of data from multiple experiments as in (B) expressed as the level of BHMT fragment normalized to the control without IPTG, mean ± SEM (control cells without treatment = 1; n = 3). Values given below each lane show quantitation of the signal for P-eEF2, corrected for the eEF2 signal from multiple experiments as in (B) expressed as P-eEF2 normalized to eEF2, mean ± SEM (control cells without treatment = 1; n = 3). (C) Cells were treated as described in Supplemental Fig. S3, visualized by confocal microscopy and the rate of autophagosome–lysosome fusion was measured according to green/red fluorescence ratio which reflect the percentage of unfused LC3. Scale bar = 20 μm. (D) Data quantification of C, data are presented as means ± SEM. ( n = 5) ***P
    Figure Legend Snippet: eEF2K does not regulate autophagy in lung carcinoma cells (A) A549 cells were cultured in the presence or absence of 1 mM IPTG for 5 days to induce the knockdown of eEF2K. Cells were then treated with 10 mM 2-deoxyglucose (2DG), rapamycin (100 nM), AZD8055 (1 μM) or MG132 (10 μM) for 16 h. Cells were then lysed and samples containing equal amounts of protein were analysed by western blot using the indicated antibodies. Values given below each lane show quantitation of the signal for LC3II, corrected for the actin signal from multiple experiments as in (A) expressed as LC3II normalized to actin, mean ± SEM (control cells without treatment = 1; n = 3). (B) A549 cells were cultured as in (A) to induce eEF2K knockdown. A549 cells were transiently transfected with the GST-BHMT construct and maintained in full media in the presence or absence of AZD8055 (1 μM) for 16 h, in the presence of leupeptin and E64d. GST-BHMT was precipitated with glutathione agarose from the whole cell extracts (bottom panel). Values given below show the quantitation of data from multiple experiments as in (B) expressed as the level of BHMT fragment normalized to the control without IPTG, mean ± SEM (control cells without treatment = 1; n = 3). Values given below each lane show quantitation of the signal for P-eEF2, corrected for the eEF2 signal from multiple experiments as in (B) expressed as P-eEF2 normalized to eEF2, mean ± SEM (control cells without treatment = 1; n = 3). (C) Cells were treated as described in Supplemental Fig. S3, visualized by confocal microscopy and the rate of autophagosome–lysosome fusion was measured according to green/red fluorescence ratio which reflect the percentage of unfused LC3. Scale bar = 20 μm. (D) Data quantification of C, data are presented as means ± SEM. ( n = 5) ***P

    Techniques Used: Cell Culture, Western Blot, Quantitation Assay, Transfection, Construct, Confocal Microscopy, Fluorescence

    23) Product Images from "The N-Terminal Domain of Aliivibrio fischeri LuxR Is a Target of the GroEL Chaperonin ▿"

    Article Title: The N-Terminal Domain of Aliivibrio fischeri LuxR Is a Target of the GroEL Chaperonin ▿

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00754-10

    SDS electrophoresis, in 12% gel, of the protein fractions obtained by affinity chromatography on a column with glutathione Sepharose. Proteins were isolated from E. coli TG-1 containing pGEX-LuxRΔN (lane 1), pGEX-LuxR (lane 2), or pGEX-LuxRΔC (lane 3). The bands of glycerol kinase (56.2 kDa; lanes 1 and 3) and of elongation factor EF-Tu (43.3 kDa; lanes 1 and 2) are present also (according to mass-spectrometry analysis, these bands correspond to the glycerol kinase and EF-Tu). These proteins accompanied the GST protein purified using gentle washing conditions.
    Figure Legend Snippet: SDS electrophoresis, in 12% gel, of the protein fractions obtained by affinity chromatography on a column with glutathione Sepharose. Proteins were isolated from E. coli TG-1 containing pGEX-LuxRΔN (lane 1), pGEX-LuxR (lane 2), or pGEX-LuxRΔC (lane 3). The bands of glycerol kinase (56.2 kDa; lanes 1 and 3) and of elongation factor EF-Tu (43.3 kDa; lanes 1 and 2) are present also (according to mass-spectrometry analysis, these bands correspond to the glycerol kinase and EF-Tu). These proteins accompanied the GST protein purified using gentle washing conditions.

    Techniques Used: Electrophoresis, Affinity Chromatography, Isolation, Mass Spectrometry, Purification

    24) Product Images from "Protein Kinase C-Mediated Phosphorylation of a Single Serine Residue on the Rat Glial Glutamine Transporter SN1 Governs Its Membrane Trafficking"

    Article Title: Protein Kinase C-Mediated Phosphorylation of a Single Serine Residue on the Rat Glial Glutamine Transporter SN1 Governs Its Membrane Trafficking

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.3694-10.2011

    PKCα and PKCγ phosphorylate the N-terminal part of SN1 in vitro . Recombinant N-terminal SN1–GST fusion protein bound to glutathione Sepharose underwent phosphorylation in vitro with [γ- 32 P]ATP and PKC-M (catalytic subunit from rat brain) or three recombinant PKC isoforms. After washing of the beads, elution, and SDS-PAGE to purify the fusion protein, the corresponding radioactive bands (localized by phospho-imaging) were cut out of the gel and subjected to thermolysin proteolysis, and phosphopeptides were applied to thin-layer silica sheets and analyzed by horizontal electrophoresis and vertical chromatography (phosphopeptide mapping). A–C , A single phosphopeptide (indicated by arrows) is detected at the same spot during stimulation with PKC-M ( A ), PKCα ( B ), and PKCγ ( C ). D , PKCβ1 does not phosphorylate the N terminal of SN1 (arrow).
    Figure Legend Snippet: PKCα and PKCγ phosphorylate the N-terminal part of SN1 in vitro . Recombinant N-terminal SN1–GST fusion protein bound to glutathione Sepharose underwent phosphorylation in vitro with [γ- 32 P]ATP and PKC-M (catalytic subunit from rat brain) or three recombinant PKC isoforms. After washing of the beads, elution, and SDS-PAGE to purify the fusion protein, the corresponding radioactive bands (localized by phospho-imaging) were cut out of the gel and subjected to thermolysin proteolysis, and phosphopeptides were applied to thin-layer silica sheets and analyzed by horizontal electrophoresis and vertical chromatography (phosphopeptide mapping). A–C , A single phosphopeptide (indicated by arrows) is detected at the same spot during stimulation with PKC-M ( A ), PKCα ( B ), and PKCγ ( C ). D , PKCβ1 does not phosphorylate the N terminal of SN1 (arrow).

    Techniques Used: In Vitro, Recombinant, SDS Page, Imaging, Electrophoresis, Chromatography

    25) Product Images from "Interplay between components of a novel LIM kinase-slingshot phosphatase complex regulates cofilin"

    Article Title: Interplay between components of a novel LIM kinase-slingshot phosphatase complex regulates cofilin

    Journal:

    doi: 10.1038/sj.emboj.7600543

    Slingshot is a LIMK1 phosphatase. Active and inactive myc-SSH-1L proteins were immunopurified with anti-myc mAb and GST-LIMK1 was affinity-purified with glutathione Sepharose. The purified proteins were incubated in the presence of 5 μCi [γ
    Figure Legend Snippet: Slingshot is a LIMK1 phosphatase. Active and inactive myc-SSH-1L proteins were immunopurified with anti-myc mAb and GST-LIMK1 was affinity-purified with glutathione Sepharose. The purified proteins were incubated in the presence of 5 μCi [γ

    Techniques Used: Affinity Purification, Purification, Incubation

    26) Product Images from "The infected cell protein 0 of herpes simplex virus 1 dynamically interacts with proteasomes, binds and activates the cdc34 E2 ubiquitin-conjugating enzyme, and possesses in vitro E3 ubiquitin ligase activity"

    Article Title: The infected cell protein 0 of herpes simplex virus 1 dynamically interacts with proteasomes, binds and activates the cdc34 E2 ubiquitin-conjugating enzyme, and possesses in vitro E3 ubiquitin ligase activity

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

    doi: 10.1073/pnas.161283098

    The domain encoded by the carboxyl terminus of exon 3 of ICP0 acts as an E3 ligase, whereas the sequences encoded by exon 2 bind cdc34 ubiquitin-conjugating enzyme. ( A ) Immunoblots of electrophoretically separated products of substrate-independent in vitro ubiquitination reactions. GST (lanes 1 and 2), GST-exon 2 (lanes 3 and 4), GST-exon 3 (lanes 5 and 6), and no additional protein (lane 7) were added to the substrate-independent in vitro ubiquitination reaction master mix (MM) containing recombinant Uba1 (E1), recombinant cdc34, biotinylated ubiquitin, and ubiquitination buffer in the presence and absence of ATP and an ATP regenerating system, as described in Materials and Methods . The reaction was stopped after 90 min, and the reaction mixture was electrophoretically separated in a denaturing polyacrylamide gel and probed with streptavidin. ( B ) Electrophoretically separated reaction mixtures containing the indicated GST fusion protein in addition to the master mix (lanes 8–13) or the master mix alone (lane 14) in the presence and absence of ATP and an ATP regenerating system were probed with a rabbit polyclonal antibody directed against GST. ( C ) cdc34 was precipitated from reactions containing the indicated GST fusion protein in addition to the master mix (lanes 15–17) or the master mix alone (lane 18) in the presence of ATP and an ATP regenerating system. The precipitate was electrophoretically separated in a denaturing polyacrylamide gel and probed with streptavidin. ( D ) GST or GST fusion proteins were precipitated from reactions containing the indicated GST fusion protein in addition to the master mix (lanes 19–21) or the master mix alone (lane 22) in the presence of ATP and an ATP regenerating system by using glutathione Sepharose beads. The precipitate was electrophoretically separated in a denaturing polyacrylamide gel and probed with a rabbit polyclonal antibody directed against cdc34. The dots to the right of high molecular weight bands in lanes 6 and 17 identify ubiquitinated proteins; G, GST; 2, GST-exon 2 chimeric protein; 3, GST-carboxyl-terminal domain of exon 3 fusion protein.
    Figure Legend Snippet: The domain encoded by the carboxyl terminus of exon 3 of ICP0 acts as an E3 ligase, whereas the sequences encoded by exon 2 bind cdc34 ubiquitin-conjugating enzyme. ( A ) Immunoblots of electrophoretically separated products of substrate-independent in vitro ubiquitination reactions. GST (lanes 1 and 2), GST-exon 2 (lanes 3 and 4), GST-exon 3 (lanes 5 and 6), and no additional protein (lane 7) were added to the substrate-independent in vitro ubiquitination reaction master mix (MM) containing recombinant Uba1 (E1), recombinant cdc34, biotinylated ubiquitin, and ubiquitination buffer in the presence and absence of ATP and an ATP regenerating system, as described in Materials and Methods . The reaction was stopped after 90 min, and the reaction mixture was electrophoretically separated in a denaturing polyacrylamide gel and probed with streptavidin. ( B ) Electrophoretically separated reaction mixtures containing the indicated GST fusion protein in addition to the master mix (lanes 8–13) or the master mix alone (lane 14) in the presence and absence of ATP and an ATP regenerating system were probed with a rabbit polyclonal antibody directed against GST. ( C ) cdc34 was precipitated from reactions containing the indicated GST fusion protein in addition to the master mix (lanes 15–17) or the master mix alone (lane 18) in the presence of ATP and an ATP regenerating system. The precipitate was electrophoretically separated in a denaturing polyacrylamide gel and probed with streptavidin. ( D ) GST or GST fusion proteins were precipitated from reactions containing the indicated GST fusion protein in addition to the master mix (lanes 19–21) or the master mix alone (lane 22) in the presence of ATP and an ATP regenerating system by using glutathione Sepharose beads. The precipitate was electrophoretically separated in a denaturing polyacrylamide gel and probed with a rabbit polyclonal antibody directed against cdc34. The dots to the right of high molecular weight bands in lanes 6 and 17 identify ubiquitinated proteins; G, GST; 2, GST-exon 2 chimeric protein; 3, GST-carboxyl-terminal domain of exon 3 fusion protein.

    Techniques Used: Western Blot, In Vitro, Recombinant, Molecular Weight

    27) Product Images from "Covalent Modification of the Transcriptional Repressor Tramtrack by the Ubiquitin-Related Protein Smt3 in Drosophila Flies"

    Article Title: Covalent Modification of the Transcriptional Repressor Tramtrack by the Ubiquitin-Related Protein Smt3 in Drosophila Flies

    Journal: Molecular and Cellular Biology

    doi:

    The Ttk69 transcriptional repressor is covalently modified by dSmt3. (A) Extracts from SL2 cells cotransfected with a vector expressing the Ttk69 protein with a vector expressing dSmt3 HA (lanes 1 and 5), Cactus His (lanes 2 and 6), or dSmt3 His (lanes 3, 4, and 7) were subjected to precipitation with Ni-agarose beads, and the precipitates were analyzed by Western blotting with the rat anti-Ttk69 polyclonal antibody. Aliquots of the corresponding unprecipitated extracts (1/10) were loaded in lanes 1 to 4. In lane 4, the cells had been incubated with 1.25 μM calyculin A prior to protein extraction. The 120-kDa doublet observed in crude extracts (lanes 1 and 3) corresponds to the Ttk69 protein conjugated either to the endogenous dSmt3 protein (lower band of the doublet) or to the transfected HA- or His-tagged dSmt3 product (upper band of the doublet). (B) SDS lysates from untransfected SL2 cells (lane 1) or from SL2 cells cotransfected with dSmt3 and Ttk69 were immunoprecipitated with the rabbit polyclonal anti-PML antibody (lane 3), the rabbit anti-Ttk69 antibody (lane 4), or the rabbit anti-dSmt3 antibody (lane 5). An aliquot of the transfected cell extract (1/200 of the material used for immunoprecipitation) was loaded in lane 2. Immunoprecipitates and cell extracts were fractionated by electrophoresis and analyzed by Western blotting with a rat anti-Ttk69 antibody. Immunoglobulins are marked by an asterisk. The 100-kDa unmodified and 120-kDa dSmt3-modified forms of Ttk69 are indicated.
    Figure Legend Snippet: The Ttk69 transcriptional repressor is covalently modified by dSmt3. (A) Extracts from SL2 cells cotransfected with a vector expressing the Ttk69 protein with a vector expressing dSmt3 HA (lanes 1 and 5), Cactus His (lanes 2 and 6), or dSmt3 His (lanes 3, 4, and 7) were subjected to precipitation with Ni-agarose beads, and the precipitates were analyzed by Western blotting with the rat anti-Ttk69 polyclonal antibody. Aliquots of the corresponding unprecipitated extracts (1/10) were loaded in lanes 1 to 4. In lane 4, the cells had been incubated with 1.25 μM calyculin A prior to protein extraction. The 120-kDa doublet observed in crude extracts (lanes 1 and 3) corresponds to the Ttk69 protein conjugated either to the endogenous dSmt3 protein (lower band of the doublet) or to the transfected HA- or His-tagged dSmt3 product (upper band of the doublet). (B) SDS lysates from untransfected SL2 cells (lane 1) or from SL2 cells cotransfected with dSmt3 and Ttk69 were immunoprecipitated with the rabbit polyclonal anti-PML antibody (lane 3), the rabbit anti-Ttk69 antibody (lane 4), or the rabbit anti-dSmt3 antibody (lane 5). An aliquot of the transfected cell extract (1/200 of the material used for immunoprecipitation) was loaded in lane 2. Immunoprecipitates and cell extracts were fractionated by electrophoresis and analyzed by Western blotting with a rat anti-Ttk69 antibody. Immunoglobulins are marked by an asterisk. The 100-kDa unmodified and 120-kDa dSmt3-modified forms of Ttk69 are indicated.

    Techniques Used: Modification, Plasmid Preparation, Expressing, Western Blot, Incubation, Protein Extraction, Transfection, Immunoprecipitation, Electrophoresis

    dSmt3-protein conjugates in Drosophila and human cells. (A) dSmt3 is conjugated to multiple proteins in SL2 and HeLa cells. dSmt3 HA was transfected in SL2 or HeLa cells. Protein extracts from untransfected (−) and transfected (+) cells were subjected to Western blot analysis using anti-HA (α-HA) MAb, anti-dSmt3 (α-dSmt3) antiserum, or the corresponding preimmune serum (PI). Positions of the free dSmt3 HA and its conjugates are indicated. (B) Modification of PML by dSmt3 in SL2 cells. dSmt3 His or untagged dSmt3 was coexpressed with PML in SL2 cells. Extracts were precipitated by nickel-agarose beads (Ppn Ni). Crude extracts (1/10; lanes 1 to 3) and Ni-agarose precipitates (lanes 4 to 6) were analyzed by Western blotting using anti-PML antibodies. Positions of PML and its dSmt3-conjugated forms are indicated.
    Figure Legend Snippet: dSmt3-protein conjugates in Drosophila and human cells. (A) dSmt3 is conjugated to multiple proteins in SL2 and HeLa cells. dSmt3 HA was transfected in SL2 or HeLa cells. Protein extracts from untransfected (−) and transfected (+) cells were subjected to Western blot analysis using anti-HA (α-HA) MAb, anti-dSmt3 (α-dSmt3) antiserum, or the corresponding preimmune serum (PI). Positions of the free dSmt3 HA and its conjugates are indicated. (B) Modification of PML by dSmt3 in SL2 cells. dSmt3 His or untagged dSmt3 was coexpressed with PML in SL2 cells. Extracts were precipitated by nickel-agarose beads (Ppn Ni). Crude extracts (1/10; lanes 1 to 3) and Ni-agarose precipitates (lanes 4 to 6) were analyzed by Western blotting using anti-PML antibodies. Positions of PML and its dSmt3-conjugated forms are indicated.

    Techniques Used: Transfection, Western Blot, Modification

    28) Product Images from "Association of the von Hippel-Lindau protein with AUF1 and post-transcriptional regulation of Vascular Endothelial Growth Factor A mRNA"

    Article Title: Association of the von Hippel-Lindau protein with AUF1 and post-transcriptional regulation of Vascular Endothelial Growth Factor A mRNA

    Journal: Molecular Cancer Research

    doi: 10.1158/1541-7786.MCR-11-0435

    In vitro interaction domain mapping of pVHL and AUF1. A, Each AUF1 isoform (as indicated) was translated in vitro in the presence of 35 S-methionine and tested for binding to GST or GST-VHL proteins that had been pre-bound to glutathione-Sepharose. GST,
    Figure Legend Snippet: In vitro interaction domain mapping of pVHL and AUF1. A, Each AUF1 isoform (as indicated) was translated in vitro in the presence of 35 S-methionine and tested for binding to GST or GST-VHL proteins that had been pre-bound to glutathione-Sepharose. GST,

    Techniques Used: In Vitro, Binding Assay

    29) Product Images from "CUGBP2 directly interacts with U2 17S snRNP components and promotes U2 snRNA binding to cardiac troponin T pre-mRNA"

    Article Title: CUGBP2 directly interacts with U2 17S snRNP components and promotes U2 snRNA binding to cardiac troponin T pre-mRNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp346

    CUGBP2 enhances complex A formation of a chicken cTNT pre-mRNA. ( A ) Diagram of chicken cTNT pre-mRNA substrate E46NB. ( B ) Spliceosome assembly during a time course for E46NB. 32 P-UTP labeled E46NB was incubated under splicing conditions for the times indicated. Ten percent of each splicing reaction was loaded on 0.4% agarose–4% acrylamide non-denaturing gel (left). RNA was purified from what remained and was separated on an 8 M urea, 5% acrylamide gel (right). For both assays, RNA was visualized by autoradiography. ( C ) CUGBP2 increases complex A formation (dose response). Purified recombinant His-CUGBP2 (145 ng and 725 ng) is shown to be > 95% pure by Coomassie staining (left). The indicated amounts of CUGBP2 were added to the splicing reactions, which were incubated for 30 min (right). ( D ) CUGBP2 (1 µg) increases complex A formation from the earliest time point tested. The complex enhanced by CUGBP2 is complex A based on ( E ) ATP-dependence and ( F ) U1 dependence. In F, complex assembly reactions were performed using HeLa nuclear extracts in which the first 12 nt of U1 snRNA were digested using a complementary oligo and RNase H. A non-specific DNA nucleotide (R5S) was used as a control ( 39 ).
    Figure Legend Snippet: CUGBP2 enhances complex A formation of a chicken cTNT pre-mRNA. ( A ) Diagram of chicken cTNT pre-mRNA substrate E46NB. ( B ) Spliceosome assembly during a time course for E46NB. 32 P-UTP labeled E46NB was incubated under splicing conditions for the times indicated. Ten percent of each splicing reaction was loaded on 0.4% agarose–4% acrylamide non-denaturing gel (left). RNA was purified from what remained and was separated on an 8 M urea, 5% acrylamide gel (right). For both assays, RNA was visualized by autoradiography. ( C ) CUGBP2 increases complex A formation (dose response). Purified recombinant His-CUGBP2 (145 ng and 725 ng) is shown to be > 95% pure by Coomassie staining (left). The indicated amounts of CUGBP2 were added to the splicing reactions, which were incubated for 30 min (right). ( D ) CUGBP2 (1 µg) increases complex A formation from the earliest time point tested. The complex enhanced by CUGBP2 is complex A based on ( E ) ATP-dependence and ( F ) U1 dependence. In F, complex assembly reactions were performed using HeLa nuclear extracts in which the first 12 nt of U1 snRNA were digested using a complementary oligo and RNase H. A non-specific DNA nucleotide (R5S) was used as a control ( 39 ).

    Techniques Used: Labeling, Incubation, Purification, Acrylamide Gel Assay, Autoradiography, Recombinant, Staining

    Enhanced complex A formation of cTNT exon 5 by CUGBP2 requires both introns 4 and 5. (A ) Diagram of E46NB, E45NA and E56NB RNAs. ( B ) 32 P-UTP labeled E46NB, E45NA and E56BD RNAs were incubated for the indicated times under splicing conditions with or without CUGBP2. Spliceosome complexes were resolved on 1.5% native agarose gels and visualized by autoradiography.
    Figure Legend Snippet: Enhanced complex A formation of cTNT exon 5 by CUGBP2 requires both introns 4 and 5. (A ) Diagram of E46NB, E45NA and E56NB RNAs. ( B ) 32 P-UTP labeled E46NB, E45NA and E56BD RNAs were incubated for the indicated times under splicing conditions with or without CUGBP2. Spliceosome complexes were resolved on 1.5% native agarose gels and visualized by autoradiography.

    Techniques Used: Labeling, Incubation, Autoradiography

    CUGBP2 enhances formation of an A-like complex on an RNA containing only cTNT exon 5 and portions of its flanking introns. (A ) Diagram of the MSE1-4 RNA. ( B ) Time course with and without 1 µg His-CUGBP2. ( C ) Dose-response to His-CUGBP2 in 30 min reactions. 32 P-UTP labeled MSE1-4 RNA was incubated under splicing conditions and 10% of each reaction was displayed on a 0.4% agarose–4% acrylamide non-denaturing gel and detected by autoradiography. A-like complex indicated as A*.
    Figure Legend Snippet: CUGBP2 enhances formation of an A-like complex on an RNA containing only cTNT exon 5 and portions of its flanking introns. (A ) Diagram of the MSE1-4 RNA. ( B ) Time course with and without 1 µg His-CUGBP2. ( C ) Dose-response to His-CUGBP2 in 30 min reactions. 32 P-UTP labeled MSE1-4 RNA was incubated under splicing conditions and 10% of each reaction was displayed on a 0.4% agarose–4% acrylamide non-denaturing gel and detected by autoradiography. A-like complex indicated as A*.

    Techniques Used: Labeling, Incubation, Autoradiography

    CUGBP2 binds directly to SF3b145 and SF3b49. ( A ) GST-CUGBP2 and GST-CUGBP1 (Coomassie staining gels left side or each panel) coupled on glutathione-sepharose were incubated with [ 35 S] Methionine/Cysteine-labeled SF3b145 (A) and SF3b49 ( B ). Bound proteins were separated on 10% SDS–PAGE gel and visualized with Cyclone Phosphorimager. ( C ) RNase A (50 μg/ml) reduced interactions between CELF and SF3b proteins in vitro . ( D ) Bound proteins in GST pull down were treated with the reversible protein cross-linker (DSP) prior to RNase A treatment. M: protein marker.
    Figure Legend Snippet: CUGBP2 binds directly to SF3b145 and SF3b49. ( A ) GST-CUGBP2 and GST-CUGBP1 (Coomassie staining gels left side or each panel) coupled on glutathione-sepharose were incubated with [ 35 S] Methionine/Cysteine-labeled SF3b145 (A) and SF3b49 ( B ). Bound proteins were separated on 10% SDS–PAGE gel and visualized with Cyclone Phosphorimager. ( C ) RNase A (50 μg/ml) reduced interactions between CELF and SF3b proteins in vitro . ( D ) Bound proteins in GST pull down were treated with the reversible protein cross-linker (DSP) prior to RNase A treatment. M: protein marker.

    Techniques Used: Staining, Incubation, Labeling, SDS Page, In Vitro, Marker

    CUGBP2 binds to SF3b145 (SF3B2) and SF3b49 (SF3B4) but not SF3b125. Nuclear extracts were prepared from HeLa cells stably expressing CUGBP2-TAP (DE5) or TAP alone (CA1) as a control. Affinity purification using IgG sepharose with or without RNase A treatment was performed. Proteins bound to IgG Sepharose were released by TEV cleavage and precipitated by TCA and labeled as bound fractions. Western blots were performed using anti-SF3b145 ( A ), SF3b49 ( B ) and SF3b125 ( C ). For inputs, 10% of the total was loaded.
    Figure Legend Snippet: CUGBP2 binds to SF3b145 (SF3B2) and SF3b49 (SF3B4) but not SF3b125. Nuclear extracts were prepared from HeLa cells stably expressing CUGBP2-TAP (DE5) or TAP alone (CA1) as a control. Affinity purification using IgG sepharose with or without RNase A treatment was performed. Proteins bound to IgG Sepharose were released by TEV cleavage and precipitated by TCA and labeled as bound fractions. Western blots were performed using anti-SF3b145 ( A ), SF3b49 ( B ) and SF3b125 ( C ). For inputs, 10% of the total was loaded.

    Techniques Used: Stable Transfection, Expressing, Affinity Purification, Labeling, Western Blot

    30) Product Images from "Convergent Use of RhoGAP Toxins by Eukaryotic Parasites and Bacterial Pathogens"

    Article Title: Convergent Use of RhoGAP Toxins by Eukaryotic Parasites and Bacterial Pathogens

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.0030203

    LbGAP Interacts with Rac1 and Rac2 (A) Results based on growth on selective medium lacking histidine and qualitative ß-galactosidase overlay assays. x: non-tested; -: no interaction; (+): very weak interaction; ++: mean interaction; ++++: strong interaction. (B) Interactions with Rac1G12V, Rac2G12V, Cdc42G12V, and RhoAG14V were assayed by measuring ß-galactosidase activity in total protein extracts. Grey bars: ß-galactosidase activity in the presence of LbGAP. Black bars: ß-galactosidase activity in the presence of T antigen. Open bar: Positive control interaction between Ras and Raf. For each value, bars represent the standard error of three measurements. (C) GST pull-down assay. LbGAP protein was synthesized using rabbit reticulocyte lysates and mixed with equal amounts of GST-Rac1G12V or GST proteins bound to Glutathione Sepharose beads. After incubation, the mixtures were subjected to SDS PAGE analysis. 1: LbGAP + GST-Rac1G12V. 2: LbGAP + GST-Rac2G12V. 3: LbGAP + GST. 4: LbGAP alone. 5: GST-Rac1G12V alone. 6: GST-Rac1G12V alone.
    Figure Legend Snippet: LbGAP Interacts with Rac1 and Rac2 (A) Results based on growth on selective medium lacking histidine and qualitative ß-galactosidase overlay assays. x: non-tested; -: no interaction; (+): very weak interaction; ++: mean interaction; ++++: strong interaction. (B) Interactions with Rac1G12V, Rac2G12V, Cdc42G12V, and RhoAG14V were assayed by measuring ß-galactosidase activity in total protein extracts. Grey bars: ß-galactosidase activity in the presence of LbGAP. Black bars: ß-galactosidase activity in the presence of T antigen. Open bar: Positive control interaction between Ras and Raf. For each value, bars represent the standard error of three measurements. (C) GST pull-down assay. LbGAP protein was synthesized using rabbit reticulocyte lysates and mixed with equal amounts of GST-Rac1G12V or GST proteins bound to Glutathione Sepharose beads. After incubation, the mixtures were subjected to SDS PAGE analysis. 1: LbGAP + GST-Rac1G12V. 2: LbGAP + GST-Rac2G12V. 3: LbGAP + GST. 4: LbGAP alone. 5: GST-Rac1G12V alone. 6: GST-Rac1G12V alone.

    Techniques Used: Activity Assay, Positive Control, Pull Down Assay, Synthesized, Incubation, SDS Page

    31) Product Images from "TSGΔ154-1054 splice variant increases TSG101 oncogenicity by inhibiting its E3-ligase-mediated proteasomal degradation"

    Article Title: TSGΔ154-1054 splice variant increases TSG101 oncogenicity by inhibiting its E3-ligase-mediated proteasomal degradation

    Journal: Oncotarget

    doi: 10.18632/oncotarget.6973

    TSGΔ154-1054 competes with TSG101 for binding Tal thereby diminishing Tal-mediated polyubiquitination of TSG101 A. Binding of TSGΔ154-1054 to Tal but not TSG101. Glutathione-Sepharose-bound GST-TSGΔ154-1054 and the in vitro translated HA-Tal or TSG101 were applied to in vitro binding assay followed by western blotting. B. Binding of Tal and its deletion mutants to TSGΔ154-1054 and TSG101. A schematic illustration of the domain structures of Tal and its deletion mutants is shown (top). Tal comprises a leucine-rich repeat (LRR), ezrin-radixin-moesin (ERM) domain, coiled-coil (CC) region, sterile alpha motif (SAM), RING finger (RF), and a double PTAP motif. These HA-Tal mutants were subjected to in vitro transcription and translation, and applied to in vitro binding assay together with bead-bound GST-TSGΔ154-1054 or GST-TSG101. The result was visualized by western blotting (bottom). C. Competitive binding between TSGΔ154-1054 and TSG101 to Tal. Glutathione-Sepharose-bound GST-TSG101 and the in vitro translated HA-Tal were subjected to an in vitro competitive binding assay in the presence of increasing amounts of in vitro translated TSGΔ154-1054, and the result was revealed by western blotting (upper panel). An in vivo competitive binding between TSGΔ154-1054 and TSG101 to Tal was accomplished on TW01 cells co-transfected with HA-Tal, TSG101 and increasing level of GFP-TSGΔ154-1054 using co-immunoprecipitation coupled with western blot (lower panel). Co-immunoprecipitation was performed using anti-TSG101 antibody, and anti-GST antibody as an irrelevant antibody control. D. Effect of TSGΔ154-1054 on Tal-mediated ubiquitination of TSG101. The bead-bound GST-TSG101 was incubated with an i n vitro ubiquitination reaction containing E1, E2 (UbcH5a), Ub, immunopurified HA-Tal, and in vitro translated TSGΔ154-1054. Immunoblot was performed using anti-Ub antibody.
    Figure Legend Snippet: TSGΔ154-1054 competes with TSG101 for binding Tal thereby diminishing Tal-mediated polyubiquitination of TSG101 A. Binding of TSGΔ154-1054 to Tal but not TSG101. Glutathione-Sepharose-bound GST-TSGΔ154-1054 and the in vitro translated HA-Tal or TSG101 were applied to in vitro binding assay followed by western blotting. B. Binding of Tal and its deletion mutants to TSGΔ154-1054 and TSG101. A schematic illustration of the domain structures of Tal and its deletion mutants is shown (top). Tal comprises a leucine-rich repeat (LRR), ezrin-radixin-moesin (ERM) domain, coiled-coil (CC) region, sterile alpha motif (SAM), RING finger (RF), and a double PTAP motif. These HA-Tal mutants were subjected to in vitro transcription and translation, and applied to in vitro binding assay together with bead-bound GST-TSGΔ154-1054 or GST-TSG101. The result was visualized by western blotting (bottom). C. Competitive binding between TSGΔ154-1054 and TSG101 to Tal. Glutathione-Sepharose-bound GST-TSG101 and the in vitro translated HA-Tal were subjected to an in vitro competitive binding assay in the presence of increasing amounts of in vitro translated TSGΔ154-1054, and the result was revealed by western blotting (upper panel). An in vivo competitive binding between TSGΔ154-1054 and TSG101 to Tal was accomplished on TW01 cells co-transfected with HA-Tal, TSG101 and increasing level of GFP-TSGΔ154-1054 using co-immunoprecipitation coupled with western blot (lower panel). Co-immunoprecipitation was performed using anti-TSG101 antibody, and anti-GST antibody as an irrelevant antibody control. D. Effect of TSGΔ154-1054 on Tal-mediated ubiquitination of TSG101. The bead-bound GST-TSG101 was incubated with an i n vitro ubiquitination reaction containing E1, E2 (UbcH5a), Ub, immunopurified HA-Tal, and in vitro translated TSGΔ154-1054. Immunoblot was performed using anti-Ub antibody.

    Techniques Used: Binding Assay, In Vitro, Western Blot, Competitive Binding Assay, In Vivo, Transfection, Immunoprecipitation, Incubation

    32) Product Images from "Deficiency of Sorting Nexin 27 (SNX27) Leads to Growth Retardation and Elevated Levels of N-Methyl-d-Aspartate Receptor 2C (NR2C) ▿-Aspartate Receptor 2C (NR2C) ▿ ‡"

    Article Title: Deficiency of Sorting Nexin 27 (SNX27) Leads to Growth Retardation and Elevated Levels of N-Methyl-d-Aspartate Receptor 2C (NR2C) ▿-Aspartate Receptor 2C (NR2C) ▿ ‡

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.01044-10

    The PDZ domain of SNX27 mediates interaction with the C-terminal PDZ-binding motif of NR2C. (A) Alignment of the C-terminal amino acid sequences of human, rat, and mouse NR2C showing the conserved class I PDZ-binding motif (SEV). (B) Diagrammatic representation of the NR2C and SNX27a deletion mutants. (C) HEK-293 cells were cotransfected with HA-SNX27 (or its indicated mutants) and Myc-NR2C-CT or Myc-NR2C-CTΔSEV. Total cell lysates were prepared and incubated with monoclonal antibody against Myc cross-linked to protein G-Sepharose (BD). The immunoprecipitates were resolved by SDS-PAGE and then processed for immunoblotting using rabbit polyclonal antibody against the HA tag (top) to assess the efficiency of coimmunoprecipitation as a measure of interaction. Following stripping, the immunoblots were reprobed with rabbit polyclonal antibody against the Myc tag (middle) to ensure the efficiency of immunoprecipitation of the Myc-tagged proteins. Five percent of the lysates used in the immunoprecipitations were also analyzed for the expression of HA-tagged SNX27 or its mutants (bottom). IP, immunoprecipitation; WB, Western blotting.
    Figure Legend Snippet: The PDZ domain of SNX27 mediates interaction with the C-terminal PDZ-binding motif of NR2C. (A) Alignment of the C-terminal amino acid sequences of human, rat, and mouse NR2C showing the conserved class I PDZ-binding motif (SEV). (B) Diagrammatic representation of the NR2C and SNX27a deletion mutants. (C) HEK-293 cells were cotransfected with HA-SNX27 (or its indicated mutants) and Myc-NR2C-CT or Myc-NR2C-CTΔSEV. Total cell lysates were prepared and incubated with monoclonal antibody against Myc cross-linked to protein G-Sepharose (BD). The immunoprecipitates were resolved by SDS-PAGE and then processed for immunoblotting using rabbit polyclonal antibody against the HA tag (top) to assess the efficiency of coimmunoprecipitation as a measure of interaction. Following stripping, the immunoblots were reprobed with rabbit polyclonal antibody against the Myc tag (middle) to ensure the efficiency of immunoprecipitation of the Myc-tagged proteins. Five percent of the lysates used in the immunoprecipitations were also analyzed for the expression of HA-tagged SNX27 or its mutants (bottom). IP, immunoprecipitation; WB, Western blotting.

    Techniques Used: Binding Assay, Incubation, SDS Page, Stripping Membranes, Western Blot, Immunoprecipitation, Expressing

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    Cell Culture:

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    Purification:

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    Produced:

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    Concentration Assay:

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    Expressing:

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    Millipore raf1 ras binding domain glutathione agarose
    ERM proteins are necessary for PDGFR signaling. A , Down-regulation of ERM proteins reduces PDGF-dependent Erk phosphorylation. NIH 3T3 cells plated at low density were treated with a combination of siRNA SMARTpools against mouse ERM proteins for 24 hours. For exogenous reconstitution of human ERMs, cells were transfected with plasmid DNA coding for human ezrin-VSVg, radixin-Flag and untagged moesin or ezrin-VSVg alone. Cells were serum starved overnight prior to treatment with PDGF for 5 min. Lysates were immunoblotted as indicated. B , Schematic representation of the architecture of ezrin mutants. C , Ezrin mutants inhibit PDGF-dependent Erk phosphorylation. RT4 cells at low density were transfected with either empty vector (control) or ezrin mutants (ezrinNterm-GFP or ezrin deleted in the Actin-Binding-Domain ezrinΔABD-GFP) (left panel) ezrin wild type or ezrin mutant R579A (right panel). Cells with high GFP expression were sorted by FACS, replated at low cell density and serum starved overnight prior to induction with PDGF for 5 min. Lysates were immunoblotted as indicated. D , NIH 3T3 cells were plated at a low density, co-transfected in a 5∶1 ratio with constructs encoding either ezrin wild type-GFP or ezrin mutant-GFP and with a hygromycin resistance construct, selected by hygromycin for 1 day, and serum starved overnight prior to treatment with PDGF or EGF for 3 min. For Ras-GTP levels, lysates were treated with <t>GST-Raf1-RBD</t> (Ras-binding domain, RBD). Co-precipitated proteins were immunoblotted with antibodies against Ras. Lysates were immunoblotted as indicated. E , Ezrin R579A also inhibits PDGF-dependent Ras activation in RT4 cells. Experiment as in D, except ezrin constructs encoding either ezrin wild type-VSVg or ezrin mutant-VSVg were used. F , Down-regulation of individual ERM proteins using a cocktail of specific siRNAs reduces PDGF-dependent Ras activation in NIH 3T3 cells. Ras activity was determined as in D . G , Ezrin mutant, but not wild type ezrin, inhibits agar colony formation in RT4 cells. Dox-inducible ezrin wild type- or mutant-expressing cells were generated and placed in soft agar (− and + dox). Results represent mean absolute colony number ± s.d. of at least three independent experiments, ** P
    Raf1 Ras Binding Domain Glutathione Agarose, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore his tagged hrv 3c protease
    . (B) SDS-PAGE separation of buffer-exchanged Nup84 complex obtained from affinity isolation and elution by <t>HRV</t> 3C protease.
    His Tagged Hrv 3c Protease, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore glutathione gsh
    (A–B) Levels of serum <t>SOD</t> ( A ) and <t>GSH</t> ( B ) in control, TAA, basil leaves extract plus TAA and basil leaves extract treated rats after six weeks. * Indicates a significant difference between control and treated groups. ** Indicates a significant difference between rats treated with TAA and basil leaves extract plus TAA and basil leaves extract. ***indicates a significant difference between rats treated with basil leaves extract plus TAA and basil leaves extract.
    Glutathione Gsh, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ERM proteins are necessary for PDGFR signaling. A , Down-regulation of ERM proteins reduces PDGF-dependent Erk phosphorylation. NIH 3T3 cells plated at low density were treated with a combination of siRNA SMARTpools against mouse ERM proteins for 24 hours. For exogenous reconstitution of human ERMs, cells were transfected with plasmid DNA coding for human ezrin-VSVg, radixin-Flag and untagged moesin or ezrin-VSVg alone. Cells were serum starved overnight prior to treatment with PDGF for 5 min. Lysates were immunoblotted as indicated. B , Schematic representation of the architecture of ezrin mutants. C , Ezrin mutants inhibit PDGF-dependent Erk phosphorylation. RT4 cells at low density were transfected with either empty vector (control) or ezrin mutants (ezrinNterm-GFP or ezrin deleted in the Actin-Binding-Domain ezrinΔABD-GFP) (left panel) ezrin wild type or ezrin mutant R579A (right panel). Cells with high GFP expression were sorted by FACS, replated at low cell density and serum starved overnight prior to induction with PDGF for 5 min. Lysates were immunoblotted as indicated. D , NIH 3T3 cells were plated at a low density, co-transfected in a 5∶1 ratio with constructs encoding either ezrin wild type-GFP or ezrin mutant-GFP and with a hygromycin resistance construct, selected by hygromycin for 1 day, and serum starved overnight prior to treatment with PDGF or EGF for 3 min. For Ras-GTP levels, lysates were treated with GST-Raf1-RBD (Ras-binding domain, RBD). Co-precipitated proteins were immunoblotted with antibodies against Ras. Lysates were immunoblotted as indicated. E , Ezrin R579A also inhibits PDGF-dependent Ras activation in RT4 cells. Experiment as in D, except ezrin constructs encoding either ezrin wild type-VSVg or ezrin mutant-VSVg were used. F , Down-regulation of individual ERM proteins using a cocktail of specific siRNAs reduces PDGF-dependent Ras activation in NIH 3T3 cells. Ras activity was determined as in D . G , Ezrin mutant, but not wild type ezrin, inhibits agar colony formation in RT4 cells. Dox-inducible ezrin wild type- or mutant-expressing cells were generated and placed in soft agar (− and + dox). Results represent mean absolute colony number ± s.d. of at least three independent experiments, ** P

    Journal: PLoS ONE

    Article Title: Activation of Ras Requires the ERM-Dependent Link of Actin to the Plasma Membrane

    doi: 10.1371/journal.pone.0027511

    Figure Lengend Snippet: ERM proteins are necessary for PDGFR signaling. A , Down-regulation of ERM proteins reduces PDGF-dependent Erk phosphorylation. NIH 3T3 cells plated at low density were treated with a combination of siRNA SMARTpools against mouse ERM proteins for 24 hours. For exogenous reconstitution of human ERMs, cells were transfected with plasmid DNA coding for human ezrin-VSVg, radixin-Flag and untagged moesin or ezrin-VSVg alone. Cells were serum starved overnight prior to treatment with PDGF for 5 min. Lysates were immunoblotted as indicated. B , Schematic representation of the architecture of ezrin mutants. C , Ezrin mutants inhibit PDGF-dependent Erk phosphorylation. RT4 cells at low density were transfected with either empty vector (control) or ezrin mutants (ezrinNterm-GFP or ezrin deleted in the Actin-Binding-Domain ezrinΔABD-GFP) (left panel) ezrin wild type or ezrin mutant R579A (right panel). Cells with high GFP expression were sorted by FACS, replated at low cell density and serum starved overnight prior to induction with PDGF for 5 min. Lysates were immunoblotted as indicated. D , NIH 3T3 cells were plated at a low density, co-transfected in a 5∶1 ratio with constructs encoding either ezrin wild type-GFP or ezrin mutant-GFP and with a hygromycin resistance construct, selected by hygromycin for 1 day, and serum starved overnight prior to treatment with PDGF or EGF for 3 min. For Ras-GTP levels, lysates were treated with GST-Raf1-RBD (Ras-binding domain, RBD). Co-precipitated proteins were immunoblotted with antibodies against Ras. Lysates were immunoblotted as indicated. E , Ezrin R579A also inhibits PDGF-dependent Ras activation in RT4 cells. Experiment as in D, except ezrin constructs encoding either ezrin wild type-VSVg or ezrin mutant-VSVg were used. F , Down-regulation of individual ERM proteins using a cocktail of specific siRNAs reduces PDGF-dependent Ras activation in NIH 3T3 cells. Ras activity was determined as in D . G , Ezrin mutant, but not wild type ezrin, inhibits agar colony formation in RT4 cells. Dox-inducible ezrin wild type- or mutant-expressing cells were generated and placed in soft agar (− and + dox). Results represent mean absolute colony number ± s.d. of at least three independent experiments, ** P

    Article Snippet: Growth factors, antibodies and reagents Recombinant human platelet-derived growth factor BB (PDGF) (Biomol); recombinant human interleukin-6 (IL-6), epidermal growth factor (EGF), lysophosphatidic acid (LPA), 12-o-tetradecanoyl-phorbol-13-acetate (TPA), Igepal CA-630, Triton-X-100, GDP, GTPγS and doxycycline (dox; Sigma); mantGDP, mantGTP and GST protein (Jena Bioscience); Lubrol 17A17 (Uniqema); ATP (Roche); GST-Grb2 glutathione agarose (GST-Grb2), Raf1-Ras-binding domain glutathione agarose (GST-Raf1-RBD), GST-Ras- and GST-agarose (Upstate); latrunculin B (Calbiochem); glutathione agarose (Santa Cruz).

    Techniques: Transfection, Plasmid Preparation, Binding Assay, Mutagenesis, Expressing, FACS, Construct, Activation Assay, Activity Assay, Generated

    Latrunculin B mimics the ezrin mutants. A Reduction in actin filaments by treatment with latrunculin B. The parental schwannoma cells RT4 were plated at low density and treated with latrunculin B (1.25 µM, 10 min). Cells were processed as described in material and methods (scale bar 10 µm). B Latrunculin B inhibits signaling. RT4 cells at low density were serum starved overnight, then treated with latrunculin B (1.25 µM, 5 min) prior to treatment with PDGF (10 ng/ml, 5 min). Lysates were treated with GST-Raf1-RBD (to pulldown Ras-GTP). Co-precipitated proteins were immunoblotted with antibodies against Ras. Lysates immunoblotted as indicated. C , D , E Specific interference with signaling by latrunculin B. RT4 cells prepared and treated with latrunculin B as in panel A , then stimulated with LPA (20 µM, 5 min, panel B ), IL-6 (1 ng/ml, 5 min, panel C ) or TPA (100 ng/ml, 5 min, panel D ). Lysates immunoblotted as indicated. The results are representative of at least three independent assays and each panel represents experiments from the same blot and the same exposure.

    Journal: PLoS ONE

    Article Title: Activation of Ras Requires the ERM-Dependent Link of Actin to the Plasma Membrane

    doi: 10.1371/journal.pone.0027511

    Figure Lengend Snippet: Latrunculin B mimics the ezrin mutants. A Reduction in actin filaments by treatment with latrunculin B. The parental schwannoma cells RT4 were plated at low density and treated with latrunculin B (1.25 µM, 10 min). Cells were processed as described in material and methods (scale bar 10 µm). B Latrunculin B inhibits signaling. RT4 cells at low density were serum starved overnight, then treated with latrunculin B (1.25 µM, 5 min) prior to treatment with PDGF (10 ng/ml, 5 min). Lysates were treated with GST-Raf1-RBD (to pulldown Ras-GTP). Co-precipitated proteins were immunoblotted with antibodies against Ras. Lysates immunoblotted as indicated. C , D , E Specific interference with signaling by latrunculin B. RT4 cells prepared and treated with latrunculin B as in panel A , then stimulated with LPA (20 µM, 5 min, panel B ), IL-6 (1 ng/ml, 5 min, panel C ) or TPA (100 ng/ml, 5 min, panel D ). Lysates immunoblotted as indicated. The results are representative of at least three independent assays and each panel represents experiments from the same blot and the same exposure.

    Article Snippet: Growth factors, antibodies and reagents Recombinant human platelet-derived growth factor BB (PDGF) (Biomol); recombinant human interleukin-6 (IL-6), epidermal growth factor (EGF), lysophosphatidic acid (LPA), 12-o-tetradecanoyl-phorbol-13-acetate (TPA), Igepal CA-630, Triton-X-100, GDP, GTPγS and doxycycline (dox; Sigma); mantGDP, mantGTP and GST protein (Jena Bioscience); Lubrol 17A17 (Uniqema); ATP (Roche); GST-Grb2 glutathione agarose (GST-Grb2), Raf1-Ras-binding domain glutathione agarose (GST-Raf1-RBD), GST-Ras- and GST-agarose (Upstate); latrunculin B (Calbiochem); glutathione agarose (Santa Cruz).

    Techniques:

    . (B) SDS-PAGE separation of buffer-exchanged Nup84 complex obtained from affinity isolation and elution by HRV 3C protease.

    Journal: Analytical chemistry

    Article Title: A robust workflow for native mass spectrometric analysis of affinity-isolated endogenous protein assemblies

    doi: 10.1021/acs.analchem.5b04477

    Figure Lengend Snippet: . (B) SDS-PAGE separation of buffer-exchanged Nup84 complex obtained from affinity isolation and elution by HRV 3C protease.

    Article Snippet: Depending on the engineered cleavage site available, either the His-tagged HRV 3C protease (1 μg/μL stock; EMD Biosciences) or the His-tagged AcTEV protease (1 μg/μL stock; Life Technologies) was used.

    Techniques: SDS Page, Isolation

    Elution with HRV 3C protease

    Journal: Analytical chemistry

    Article Title: A robust workflow for native mass spectrometric analysis of affinity-isolated endogenous protein assemblies

    doi: 10.1021/acs.analchem.5b04477

    Figure Lengend Snippet: Elution with HRV 3C protease

    Article Snippet: Depending on the engineered cleavage site available, either the His-tagged HRV 3C protease (1 μg/μL stock; EMD Biosciences) or the His-tagged AcTEV protease (1 μg/μL stock; Life Technologies) was used.

    Techniques:

    Analysis of the MDA5:TRIM65 complex structure. (A) Comparison of the MDA5 Hel2 structures in the filamentous state with dsRNA and TRIM65 (left, this study), in the monomeric state with dsRNA (center, PDB: 4GL2) and in complex with the viral protein V without dsRNA (right, PDB: 4I1S). The Cα RMSD between the left and center Hel2 structures, and between left and right Hel2 structures are 1.54 Å and 1.09Å, respectively. (B) Native gel shift assays to examine the interaction between MDA5 and TRIM65 in the absence of dsRNA. TRIM65 was N-terminally labeled with fluorescein (*) for visualization in the native gel. Mobility shift of TRIM65 (CC-PSpry, 0.8 μM) was monitored upon incubation with MDA5 fused with GST (GST-MDA5, 1.6 μM). Various MDA5 constructs were used to confirm the interaction between MDA5 Hel2 and TRIM65: MDA5ΔN, helicase domain, Hel2i-Hel2 (Hel2i2) and isolated a1 helix. Note that isolated Hel2 could not be tested due to its insolubility. We instead used Hel2i2, which was soluble. The GST tag is cleavable by the HRV 3C protease, allowing comparison of the MDA5:TRIM65 interaction in the monomeric (without GST) vs. dimeric (with GST) states. The results showed that MDA5ΔN, helicase domain and Hel2i2 all bind TRIM65 in a manner dependent on GST fusion. Isolated a1 helix did not bind TRIM65, with or without GST, suggesting that a3 is also required. All proteins were recombinantly expressed in E. coli and purified to homogeneity. Bottom: Input samples analyzed by SDS-PAGE and Coommassie Brilliant Blue (CBB) stain. (C) Electrostatic potential of TRIM65 PSpry in surface representation. The a1/a3 helices of MDA5ΔN bound by TRIM65 PSpry are shown in ribbon representation (green). (D) Structures of other PSpry domains in complex with their respective substrates. Left: TRIM21 PSpry in complex with IgG Fc (PDB:2IWG ( James et al., 2007 )). Right: GUSTAVUS PSpry in complex with a peptide isolated from VASA (PDB:2IHS ( Woo et al., 2006 )). VLs involved in substrate recognition are indicated by VL labels. TRIM21 PSpry utilizes VL1 and 3-6 to recognize a domain-domain interface of the IgG antibody, whereas GUSTAVUS PSpry utilizes VL1-3 and 6 to recognize a linear peptide in VASA. Note that VASA is a helicase, but the PSpry epitope resides outside the helicase domain.

    Journal: bioRxiv

    Article Title: Structural analysis of RIG-I-like receptors reveals ancient rules of engagement between diverse RNA helicases and TRIM ubiquitin ligases

    doi: 10.1101/2020.08.26.268649

    Figure Lengend Snippet: Analysis of the MDA5:TRIM65 complex structure. (A) Comparison of the MDA5 Hel2 structures in the filamentous state with dsRNA and TRIM65 (left, this study), in the monomeric state with dsRNA (center, PDB: 4GL2) and in complex with the viral protein V without dsRNA (right, PDB: 4I1S). The Cα RMSD between the left and center Hel2 structures, and between left and right Hel2 structures are 1.54 Å and 1.09Å, respectively. (B) Native gel shift assays to examine the interaction between MDA5 and TRIM65 in the absence of dsRNA. TRIM65 was N-terminally labeled with fluorescein (*) for visualization in the native gel. Mobility shift of TRIM65 (CC-PSpry, 0.8 μM) was monitored upon incubation with MDA5 fused with GST (GST-MDA5, 1.6 μM). Various MDA5 constructs were used to confirm the interaction between MDA5 Hel2 and TRIM65: MDA5ΔN, helicase domain, Hel2i-Hel2 (Hel2i2) and isolated a1 helix. Note that isolated Hel2 could not be tested due to its insolubility. We instead used Hel2i2, which was soluble. The GST tag is cleavable by the HRV 3C protease, allowing comparison of the MDA5:TRIM65 interaction in the monomeric (without GST) vs. dimeric (with GST) states. The results showed that MDA5ΔN, helicase domain and Hel2i2 all bind TRIM65 in a manner dependent on GST fusion. Isolated a1 helix did not bind TRIM65, with or without GST, suggesting that a3 is also required. All proteins were recombinantly expressed in E. coli and purified to homogeneity. Bottom: Input samples analyzed by SDS-PAGE and Coommassie Brilliant Blue (CBB) stain. (C) Electrostatic potential of TRIM65 PSpry in surface representation. The a1/a3 helices of MDA5ΔN bound by TRIM65 PSpry are shown in ribbon representation (green). (D) Structures of other PSpry domains in complex with their respective substrates. Left: TRIM21 PSpry in complex with IgG Fc (PDB:2IWG ( James et al., 2007 )). Right: GUSTAVUS PSpry in complex with a peptide isolated from VASA (PDB:2IHS ( Woo et al., 2006 )). VLs involved in substrate recognition are indicated by VL labels. TRIM21 PSpry utilizes VL1 and 3-6 to recognize a domain-domain interface of the IgG antibody, whereas GUSTAVUS PSpry utilizes VL1-3 and 6 to recognize a linear peptide in VASA. Note that VASA is a helicase, but the PSpry epitope resides outside the helicase domain.

    Article Snippet: The proteins were purified by Ni-NTA agarose and treated with HRV 3C protease to cleave the His6 -MBP-tag.

    Techniques: Electrophoretic Mobility Shift Assay, Labeling, Mobility Shift, Incubation, Construct, Isolation, Purification, SDS Page, Staining

    (A–B) Levels of serum SOD ( A ) and GSH ( B ) in control, TAA, basil leaves extract plus TAA and basil leaves extract treated rats after six weeks. * Indicates a significant difference between control and treated groups. ** Indicates a significant difference between rats treated with TAA and basil leaves extract plus TAA and basil leaves extract. ***indicates a significant difference between rats treated with basil leaves extract plus TAA and basil leaves extract.

    Journal: Saudi Journal of Biological Sciences

    Article Title: Physiological and histopathological study on the influence of Ocimum basilicum leaves extract on thioacetamide-induced nephrotoxicity in male rats

    doi: 10.1016/j.sjbs.2020.05.034

    Figure Lengend Snippet: (A–B) Levels of serum SOD ( A ) and GSH ( B ) in control, TAA, basil leaves extract plus TAA and basil leaves extract treated rats after six weeks. * Indicates a significant difference between control and treated groups. ** Indicates a significant difference between rats treated with TAA and basil leaves extract plus TAA and basil leaves extract. ***indicates a significant difference between rats treated with basil leaves extract plus TAA and basil leaves extract.

    Article Snippet: The methods of , were used to evaluate the levels of serum superoxide dismutase (SOD) and glutathione (GSH) respectively.

    Techniques: