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  • 99
    GE Healthcare glutathione sepharose beads
    PARP1 ADP-ribosylates, whereas PARG de-ADP-ribosylates Smad1 and Smad5. A , in vitro ADP-ribosylation assay of Smad1, Smad5, Smad4, and Smad3. GST-Smad proteins were incubated with 32 P-β-NAD + and recombinant PARP1. After <t>glutathione-agarose</t> pulldown, ADP-ribosylated GST-Smad1/5/4/3 were imaged by autoradiography. The radioactive protein bands of PARP1 and GST-Smads are marked. The lower panel shows GST-Smad proteins stained with Coomassie Brilliant Blue after SDS-PAGE. M , molecular size marker. A representative autoradiogram of four assays is shown. Molecular size markers in kDa are also marked. B , in vitro de-PARylation of GST-Smad1 and GST-Smad5. PARG or vehicle were incubated with equal amounts of GST-Smad1/5, 32 P-β-NAD + , and recombinant PARP1 for 30 min at 37 °C. ADP-ribosylated proteins were imaged by autoradiography. The radioactive protein bands of PARP1 and GST-Smads are marked. The lower panel shows total GST proteins stained with Coomassie Brilliant Blue. M , molecular size marker. A representative autoradiogram of five assays is shown. Molecular size markers in kDa are also marked. C , immunoblot of endogenous PARP1 from HEK293T cell extracts bound to the indicated GST-Smad1 MH1 domain mutants. TCL shows the levels of endogenous PARP1. Total GST-Smad1 mutant proteins used for immunoblotting of endogenous PARP1 are stained with Coomassie Brilliant Blue in the middle panel . The Smad1 sequence motif that was mutated ( red letters ) and that represents a genuine ADP-ribosylation target sequence is shown in the bottom panel . A representative immunoblot of three repeats is shown. Molecular size markers in kDa are also marked. D , in vitro ADP-ribosylation assay of GST-Smad1-MH1 domain mutants. Control GST, beads, WT-Smad1-MH1 domain, and three mutants (as shown in C ) were incubated with 32 P-β-NAD + and recombinant PARP1. ADP-ribosylated proteins were imaged via autoradiography. The radioactive protein bands of PARP1 and GST-Smad1-MH1 are marked. Total GST proteins were checked by Coomassie Brilliant Blue staining. Lane 1/3 WT indicates a reaction where one-third of the GST-Smad1-MH1 protein was used compared with the WT lanes. A representative autoradiogram of two assays is shown. Molecular size markers in kDa are also marked. E , immunoblot of recombinant PARP1 (20 ng) bound to the indicated GST-Smad1 MH1 domain mutants. The experiment is a repeat of the ribosylation assay of Fig. 8 D , except that only cold β-NAD + was used during incubation, followed by pulldown and immunoblotting. On the side, increasing amounts of recombinant PARP1 along with TCL from HEK293T cells show the levels of recombinant PARP1 used in the assay relative to endogenous PARP1. Total GST-Smad1 mutant proteins checked by Coomassie Brilliant Blue staining, used for immunoblotting of recombinant PARP1. A representative immunoblot of two repeats is shown. Molecular size markers in kDa are also marked. F , molecular model adapted to a detail from the crystal structure of two Smad3 MH1 domains bound to the Smad-binding DNA element (PDB code 1mhd ). Shown is a ribbon diagram of the whole Smad3 MH1 domain with colored amino acids and the acceptor glutamate ( red ) and lysine ( blue ) residues drawn as stick and ball structures on the bottom side of the surface of the regulatory α-helix of one Smad3 MH1 subunit ( white arrow ). The β-hairpin that contacts DNA is also indicated ( white arrow ). WB , Western blotting.
    Glutathione Sepharose Beads, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 18588 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare glutathione conjugated sepharose beads
    TXNIP molecules form interprotomer disulphide bonds between Cys63 and Cys247. ( a , b ) Co-immunoprecipitation assays were performed using lysates from 293 T cells transfected with combinations of FLAG-tagged (F), HA-tagged, and GST-tagged full-length TXNIP, T–TXNIP, or mutant TXNIP. Immobilized proteins on <t>FLAG-agarose</t> beads or glutathione beads were visualized by immunoblotting using anti-HA, anti-FLAG, or anti-GST antibodies. One percent of the WCL was used as the input. ( c ) Proteomic analysis of the interprotomer-interacting TXNIP molecules fractionated by SDS–PAGE under non-reducing conditions. The MS/MS spectrum shows the interprotomer disulphide bond between Cys63 and Cys247 identified as 54 VLWMQGSQQ C K 64 - 240 GNHISGT C ASWR 251 . Doubly charged [M+2H] + peptide ions at m/z 1353.64 were fragmented via higher-energy collisional dissociation. Matched peaks are shown in red. The ion types of matched peaks are written in red for b- and y-ions and blue for ions from C-S and S-S bond cleavages. Annotations used are: P, strand VLWMQGSQQCK; p, strand GNHISGTCASWR; B and Y, ions from P; b and y, ions from p; p+32, persulfide ion of p formed by C-S bond cleavage reactions. ( d ) The effect of TRX on the interaction between TXNIP molecules. Co-immunoprecipitation assays were performed using lysates from 293 T cells transfected with FLAG-tagged TXNIP, HA-tagged TXNIP and FLAG-tagged TRX. Immobilized proteins on HA-agarose beads were visualized by immunoblotting using anti-HA and anti-FLAG antibodies. ( a , b , d ) The results are representative of at least two independent experiments with similar results.
    Glutathione Conjugated Sepharose Beads, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 1477 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    GE Healthcare glutathione sepharose high performance beads
    High NaCl reduces binding of MKP-1 to p38 and has no significant effect on MKP-1 protein expression. ( A–C ) High NaCl reduces binding of MKP-1 to p38. Osmolality bathing mIMCD3 cells stably expressing GST, GST-MKP-1, or GST-p38 was increased to 550 mosmol/kg (NaCl added) for 30 min, then GST-MKP-1 or GST-p38 was pulled down with glutathione <t>Sepharose</t> beads, and the amount of accompanying native MKP-1 or p38 was measured by Western analysis (*, P
    Glutathione Sepharose High Performance Beads, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 96/100, based on 179 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare glutathione s sepharose 4b beads
    Anti-AQP1 specificity of the identified autoantibodies. (A) Patient’s autoantibodies recognize the AQP1 moiety of AQP1-GST. Five sera that had tested positive for binding to the AQP1-GST fusion protein were preincubated with an excess of GST immobilized on <t>Sepharose-glutathione</t> beads, then were tested by RIPA using 125 I-streptavidin labeled AQP1-GST. (B) Binding of anti-AQP1 autoantibodies is specifically inhibited by an extract from AQP1-expressing HEK293 cells, but not control HEK293 cells. Four anti-AQP1-positive sera were preincubated with extracts prepared from either EGFP-transfected or AQP1-GFP-transfected HEK293 cells, then were tested by RIPA for binding to the commercial AQP1 preparation. (C) Binding of anti-AQP1 autoantibodies is specifically inhibited by yeast-expressed human AQP1. Four exclusively anti-AQP1-positive sera were preincubated with human AQP1 or AQP4 that had been expressed in yeast and purified or with BSA as control, then were tested by RIPA using 125 I-streptavidin-labeled commercial AQP1-GST fusion protein. (D) AQP1 autoantibody binding is independent of the source of AQP1. Both the commercial AQP1-GST fusion protein and the in house AQP1 purified from yeast were biotinylated, indirectly labeled by preincubation with 125 I-streptavidin, and used in the RIPA. Five anti-AQP1-positive sera and one serum sample from a healthy control (HC) were tested.
    Glutathione S Sepharose 4b Beads, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 51 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare glutathione sepharose cl 4b beads
    Anti-AQP1 specificity of the identified autoantibodies. (A) Patient’s autoantibodies recognize the AQP1 moiety of AQP1-GST. Five sera that had tested positive for binding to the AQP1-GST fusion protein were preincubated with an excess of GST immobilized on <t>Sepharose-glutathione</t> beads, then were tested by RIPA using 125 I-streptavidin labeled AQP1-GST. (B) Binding of anti-AQP1 autoantibodies is specifically inhibited by an extract from AQP1-expressing HEK293 cells, but not control HEK293 cells. Four anti-AQP1-positive sera were preincubated with extracts prepared from either EGFP-transfected or AQP1-GFP-transfected HEK293 cells, then were tested by RIPA for binding to the commercial AQP1 preparation. (C) Binding of anti-AQP1 autoantibodies is specifically inhibited by yeast-expressed human AQP1. Four exclusively anti-AQP1-positive sera were preincubated with human AQP1 or AQP4 that had been expressed in yeast and purified or with BSA as control, then were tested by RIPA using 125 I-streptavidin-labeled commercial AQP1-GST fusion protein. (D) AQP1 autoantibody binding is independent of the source of AQP1. Both the commercial AQP1-GST fusion protein and the in house AQP1 purified from yeast were biotinylated, indirectly labeled by preincubation with 125 I-streptavidin, and used in the RIPA. Five anti-AQP1-positive sera and one serum sample from a healthy control (HC) were tested.
    Glutathione Sepharose Cl 4b Beads, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PARP1 ADP-ribosylates, whereas PARG de-ADP-ribosylates Smad1 and Smad5. A , in vitro ADP-ribosylation assay of Smad1, Smad5, Smad4, and Smad3. GST-Smad proteins were incubated with 32 P-β-NAD + and recombinant PARP1. After glutathione-agarose pulldown, ADP-ribosylated GST-Smad1/5/4/3 were imaged by autoradiography. The radioactive protein bands of PARP1 and GST-Smads are marked. The lower panel shows GST-Smad proteins stained with Coomassie Brilliant Blue after SDS-PAGE. M , molecular size marker. A representative autoradiogram of four assays is shown. Molecular size markers in kDa are also marked. B , in vitro de-PARylation of GST-Smad1 and GST-Smad5. PARG or vehicle were incubated with equal amounts of GST-Smad1/5, 32 P-β-NAD + , and recombinant PARP1 for 30 min at 37 °C. ADP-ribosylated proteins were imaged by autoradiography. The radioactive protein bands of PARP1 and GST-Smads are marked. The lower panel shows total GST proteins stained with Coomassie Brilliant Blue. M , molecular size marker. A representative autoradiogram of five assays is shown. Molecular size markers in kDa are also marked. C , immunoblot of endogenous PARP1 from HEK293T cell extracts bound to the indicated GST-Smad1 MH1 domain mutants. TCL shows the levels of endogenous PARP1. Total GST-Smad1 mutant proteins used for immunoblotting of endogenous PARP1 are stained with Coomassie Brilliant Blue in the middle panel . The Smad1 sequence motif that was mutated ( red letters ) and that represents a genuine ADP-ribosylation target sequence is shown in the bottom panel . A representative immunoblot of three repeats is shown. Molecular size markers in kDa are also marked. D , in vitro ADP-ribosylation assay of GST-Smad1-MH1 domain mutants. Control GST, beads, WT-Smad1-MH1 domain, and three mutants (as shown in C ) were incubated with 32 P-β-NAD + and recombinant PARP1. ADP-ribosylated proteins were imaged via autoradiography. The radioactive protein bands of PARP1 and GST-Smad1-MH1 are marked. Total GST proteins were checked by Coomassie Brilliant Blue staining. Lane 1/3 WT indicates a reaction where one-third of the GST-Smad1-MH1 protein was used compared with the WT lanes. A representative autoradiogram of two assays is shown. Molecular size markers in kDa are also marked. E , immunoblot of recombinant PARP1 (20 ng) bound to the indicated GST-Smad1 MH1 domain mutants. The experiment is a repeat of the ribosylation assay of Fig. 8 D , except that only cold β-NAD + was used during incubation, followed by pulldown and immunoblotting. On the side, increasing amounts of recombinant PARP1 along with TCL from HEK293T cells show the levels of recombinant PARP1 used in the assay relative to endogenous PARP1. Total GST-Smad1 mutant proteins checked by Coomassie Brilliant Blue staining, used for immunoblotting of recombinant PARP1. A representative immunoblot of two repeats is shown. Molecular size markers in kDa are also marked. F , molecular model adapted to a detail from the crystal structure of two Smad3 MH1 domains bound to the Smad-binding DNA element (PDB code 1mhd ). Shown is a ribbon diagram of the whole Smad3 MH1 domain with colored amino acids and the acceptor glutamate ( red ) and lysine ( blue ) residues drawn as stick and ball structures on the bottom side of the surface of the regulatory α-helix of one Smad3 MH1 subunit ( white arrow ). The β-hairpin that contacts DNA is also indicated ( white arrow ). WB , Western blotting.

    Journal: The Journal of Biological Chemistry

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

    doi: 10.1074/jbc.M116.729699

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

    Article Snippet: Then proteins were extracted from bacteria using a Triton X-100 containing lysis buffer (50 mm Tris-HCl, pH 7.5, 1 mm EDTA, 100 mm NaCl, 5% glycerol, 0.5% Triton X-100), supplemented with 1 mm DTT and protease inhibitors, and incubated end over end at 4 °C, overnight, with glutathione-Sepharose beads (catalog no. 17-5132-01, lot no. 10172617; GE Healthcare).

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

    TXNIP molecules form interprotomer disulphide bonds between Cys63 and Cys247. ( a , b ) Co-immunoprecipitation assays were performed using lysates from 293 T cells transfected with combinations of FLAG-tagged (F), HA-tagged, and GST-tagged full-length TXNIP, T–TXNIP, or mutant TXNIP. Immobilized proteins on FLAG-agarose beads or glutathione beads were visualized by immunoblotting using anti-HA, anti-FLAG, or anti-GST antibodies. One percent of the WCL was used as the input. ( c ) Proteomic analysis of the interprotomer-interacting TXNIP molecules fractionated by SDS–PAGE under non-reducing conditions. The MS/MS spectrum shows the interprotomer disulphide bond between Cys63 and Cys247 identified as 54 VLWMQGSQQ C K 64 - 240 GNHISGT C ASWR 251 . Doubly charged [M+2H] + peptide ions at m/z 1353.64 were fragmented via higher-energy collisional dissociation. Matched peaks are shown in red. The ion types of matched peaks are written in red for b- and y-ions and blue for ions from C-S and S-S bond cleavages. Annotations used are: P, strand VLWMQGSQQCK; p, strand GNHISGTCASWR; B and Y, ions from P; b and y, ions from p; p+32, persulfide ion of p formed by C-S bond cleavage reactions. ( d ) The effect of TRX on the interaction between TXNIP molecules. Co-immunoprecipitation assays were performed using lysates from 293 T cells transfected with FLAG-tagged TXNIP, HA-tagged TXNIP and FLAG-tagged TRX. Immobilized proteins on HA-agarose beads were visualized by immunoblotting using anti-HA and anti-FLAG antibodies. ( a , b , d ) The results are representative of at least two independent experiments with similar results.

    Journal: Nature Communications

    Article Title: The structural basis for the negative regulation of thioredoxin by thioredoxin-interacting protein

    doi: 10.1038/ncomms3958

    Figure Lengend Snippet: TXNIP molecules form interprotomer disulphide bonds between Cys63 and Cys247. ( a , b ) Co-immunoprecipitation assays were performed using lysates from 293 T cells transfected with combinations of FLAG-tagged (F), HA-tagged, and GST-tagged full-length TXNIP, T–TXNIP, or mutant TXNIP. Immobilized proteins on FLAG-agarose beads or glutathione beads were visualized by immunoblotting using anti-HA, anti-FLAG, or anti-GST antibodies. One percent of the WCL was used as the input. ( c ) Proteomic analysis of the interprotomer-interacting TXNIP molecules fractionated by SDS–PAGE under non-reducing conditions. The MS/MS spectrum shows the interprotomer disulphide bond between Cys63 and Cys247 identified as 54 VLWMQGSQQ C K 64 - 240 GNHISGT C ASWR 251 . Doubly charged [M+2H] + peptide ions at m/z 1353.64 were fragmented via higher-energy collisional dissociation. Matched peaks are shown in red. The ion types of matched peaks are written in red for b- and y-ions and blue for ions from C-S and S-S bond cleavages. Annotations used are: P, strand VLWMQGSQQCK; p, strand GNHISGTCASWR; B and Y, ions from P; b and y, ions from p; p+32, persulfide ion of p formed by C-S bond cleavage reactions. ( d ) The effect of TRX on the interaction between TXNIP molecules. Co-immunoprecipitation assays were performed using lysates from 293 T cells transfected with FLAG-tagged TXNIP, HA-tagged TXNIP and FLAG-tagged TRX. Immobilized proteins on HA-agarose beads were visualized by immunoblotting using anti-HA and anti-FLAG antibodies. ( a , b , d ) The results are representative of at least two independent experiments with similar results.

    Article Snippet: The supernatants were incubated for 12 h at 4 °C with mouse monoclonal anti-FLAG M2 agarose beads (Sigma) for immunoprecipitation, or with glutathione-conjugated sepharose beads (GE Healthcare) for pull-down assays.

    Techniques: Immunoprecipitation, Transfection, Mutagenesis, SDS Page, Mass Spectrometry

    Epo1p binds to two different sites in Scs2p. (A, top) Domain structure of Scs2p. The major sperm domain (MSP) is separated from the C-terminal transmembrane segment (TMD) by a stretch of 93 residues. (bottom) Split-Ub interaction assay as in Fig. 1 B but between Epo1CRU and N ub -Scs2 1–225 and two N ub fusions that should not interact with Epo1CRU. (B) Eluates of Sepharose bead-coupled GST-Scs2 1–225 (lanes 1, 3, and 5) or GST (lanes 2, 4, and 6) incubated with yeast extracts containing GFP-tagged Epo1p (lanes 1 and 2) or bacterial extracts containing MBP-Epo1p (lanes 3 and 4) or MBP-Epo1 1–760 (lanes 5 and 6) were separated by SDS-PAGE. Western blots were probed with anti-GFP (lanes 1 and 2) or anti-MBP antibodies (lanes 3–6). Arrows indicate from top to bottom: MBP-Epo1p, Epo1-GFP, and MBP-Epo1 1–760 . Fragments of Epo1-GFP and MBP-Epo1p running

    Journal: The Journal of Cell Biology

    Article Title: A protein complex containing Epo1p anchors the cortical endoplasmic reticulum to the yeast bud tip

    doi: 10.1083/jcb.201407126

    Figure Lengend Snippet: Epo1p binds to two different sites in Scs2p. (A, top) Domain structure of Scs2p. The major sperm domain (MSP) is separated from the C-terminal transmembrane segment (TMD) by a stretch of 93 residues. (bottom) Split-Ub interaction assay as in Fig. 1 B but between Epo1CRU and N ub -Scs2 1–225 and two N ub fusions that should not interact with Epo1CRU. (B) Eluates of Sepharose bead-coupled GST-Scs2 1–225 (lanes 1, 3, and 5) or GST (lanes 2, 4, and 6) incubated with yeast extracts containing GFP-tagged Epo1p (lanes 1 and 2) or bacterial extracts containing MBP-Epo1p (lanes 3 and 4) or MBP-Epo1 1–760 (lanes 5 and 6) were separated by SDS-PAGE. Western blots were probed with anti-GFP (lanes 1 and 2) or anti-MBP antibodies (lanes 3–6). Arrows indicate from top to bottom: MBP-Epo1p, Epo1-GFP, and MBP-Epo1 1–760 . Fragments of Epo1-GFP and MBP-Epo1p running

    Article Snippet: In vitro binding assay GST-tagged proteins were immobilized on glutathione-coupled Sepharose beads (GE Healthcare).

    Techniques: Incubation, SDS Page, Western Blot

    The N-terminal 100 residues of Bem3p interact with the C-terminal coiled-coil regions of Epo1p. (A) Split-Ub assay as in Fig. 1 B but with cells coexpressing Epo1CRU and N ub fusions to Bem3p and its fragments. The N ub fusion to Guk1p should not interact and served as a control for the specificities of the observed interactions. (B) Cartoon indicating the positions of the N ub fragments and the domains of Bem3p. GAP, GTPase-activating domain. (C) As in Fig. 1 F , but with protein extracts of bacterial cells expressing his 6 -Epo1 852–943 (lanes 1, 3, and 4) or his 6 -Epo1 761–867 (lanes 2, 5, and 6) that were incubated with glutathione-coupled Sepharose beads exposing bacterially expressed GST (lanes 3 and 5) or GST-Bem3 1–100 (lanes 4 and 6). The vertical line indicates the removal of a lane loaded with a molecular mass marker. The inputs of GST and GST-Bem3 1–100 are shown as lanes 7 and 8 of a Coomassie-stained gel. Arrows indicate the running positions of GST-Bem3 1–100 and GST.

    Journal: The Journal of Cell Biology

    Article Title: A protein complex containing Epo1p anchors the cortical endoplasmic reticulum to the yeast bud tip

    doi: 10.1083/jcb.201407126

    Figure Lengend Snippet: The N-terminal 100 residues of Bem3p interact with the C-terminal coiled-coil regions of Epo1p. (A) Split-Ub assay as in Fig. 1 B but with cells coexpressing Epo1CRU and N ub fusions to Bem3p and its fragments. The N ub fusion to Guk1p should not interact and served as a control for the specificities of the observed interactions. (B) Cartoon indicating the positions of the N ub fragments and the domains of Bem3p. GAP, GTPase-activating domain. (C) As in Fig. 1 F , but with protein extracts of bacterial cells expressing his 6 -Epo1 852–943 (lanes 1, 3, and 4) or his 6 -Epo1 761–867 (lanes 2, 5, and 6) that were incubated with glutathione-coupled Sepharose beads exposing bacterially expressed GST (lanes 3 and 5) or GST-Bem3 1–100 (lanes 4 and 6). The vertical line indicates the removal of a lane loaded with a molecular mass marker. The inputs of GST and GST-Bem3 1–100 are shown as lanes 7 and 8 of a Coomassie-stained gel. Arrows indicate the running positions of GST-Bem3 1–100 and GST.

    Article Snippet: In vitro binding assay GST-tagged proteins were immobilized on glutathione-coupled Sepharose beads (GE Healthcare).

    Techniques: Expressing, Incubation, Marker, Staining

    Epo1p interacts with members of the polarisome and Scs2p. (A) Split-Ub interaction assay of 48 yeast strains each coexpressing Epo1CRU with a different N ub fusion. Shown are quadruplets of each strain after 3 d of growth on medium containing 5-FOA. White boxes indicate the fusions that induce the growth of the strain reflecting the interaction between N ub and C ub fusion. The identities of all Nub fusions are revealed in Table S2 . (B) Split-Ub interaction assay between Epo1CRU and selected N ub fusion proteins in WT, Δpea2 , Δspa2 , Δkel1 , and Δbem3 cells. Cells were grown to OD 600 of 1 and 4 µl of this, and 10-fold serial dilutions were spotted on 5-FOA plates. N ub without a C-terminally attached ORF (N ub −) serves as a control for the specificity of the Split-Ub assays. (C) As in B, but selected interactions of Pea2CRU were compared between WT and Δepo1 cells. (D) Domain structure of Epo1p. Shown as blue rectangles are the three predicted coiled-coil (CC) regions. Numbers indicate amino acid positions of the putative start and end points of each domain. (E) As in A, but with fragments of Epo1p as CRU fusions and 16 independently generated diploids for each experiment shown after 4 d of growth. (F) Protein extracts of bacterial cells expressing his 6 -Pea2 (lanes 1–4) or yeast cells expressing MYC-tagged Kel1p (lanes 5–8) were incubated with glutathione-coupled Sepharose beads exposing bacterially expressed GST (lanes 1 and 5), GST-Epo1 761–943 (lanes 2 and 6), GST-Epo1 761–867 (lanes 3 and 7), or GST-Epo1 852–943 (lanes 4 and 8). Glutathione eluates were separated by SDS-PAGE and probed with anti-His (lanes 1–4) or anti-MYC (lanes 5–8) antibodies after Western blotting. (G) As in F, except bacterially expressed MBP-Epo1 (lanes 1 and 2) or MBP-Epo1 1–760 (lanes 3 and 4) were precipitated with bacterially expressed and Sepharose bead immobilized GST (lanes 2 and 4) or GST-Pea2p (lanes 1 and 3). The inputs for the experiments in F and G are shown in Fig. S1 . (H) Pea2p mediates the interaction between Epo1p and Spa2p. As in F, except bacterially expressed his 6 -Spa2 1–535 -SNAP (lanes 1, 2, 7, and 8) or his 6 -Spa2 1–488 -SNAP (lanes 4, 5, 9, and 10) were first incubated with his 6 -Pea2 (lanes 1 and 4) or left untreated (lanes 7 and 9) before being incubated with bacterially expressed and immobilized GST-Epo1 761–943 . The glutathione eluates are shown in lanes 1, 4, 7, and 9. The inputs for the experiment in lane 1 are shown in lanes 2 and 3. The inputs for the experiment in lane 4 are shown in lanes 5 and 6. The inputs for the experiment in lanes 7 and 9 are shown in lanes 8 and 10, respectively. The asterisk indicates a degradation product. Lanes 1–6 show cutouts of the same gel with the vertical line indicating the removal of an empty lane. (I) Architecture of the ER–cell tip tethering complex. Edges connecting nodes indicate direct (black) or potentially indirect (green) interactions. Blue rectangles indicate coiled-coil regions shown in D.

    Journal: The Journal of Cell Biology

    Article Title: A protein complex containing Epo1p anchors the cortical endoplasmic reticulum to the yeast bud tip

    doi: 10.1083/jcb.201407126

    Figure Lengend Snippet: Epo1p interacts with members of the polarisome and Scs2p. (A) Split-Ub interaction assay of 48 yeast strains each coexpressing Epo1CRU with a different N ub fusion. Shown are quadruplets of each strain after 3 d of growth on medium containing 5-FOA. White boxes indicate the fusions that induce the growth of the strain reflecting the interaction between N ub and C ub fusion. The identities of all Nub fusions are revealed in Table S2 . (B) Split-Ub interaction assay between Epo1CRU and selected N ub fusion proteins in WT, Δpea2 , Δspa2 , Δkel1 , and Δbem3 cells. Cells were grown to OD 600 of 1 and 4 µl of this, and 10-fold serial dilutions were spotted on 5-FOA plates. N ub without a C-terminally attached ORF (N ub −) serves as a control for the specificity of the Split-Ub assays. (C) As in B, but selected interactions of Pea2CRU were compared between WT and Δepo1 cells. (D) Domain structure of Epo1p. Shown as blue rectangles are the three predicted coiled-coil (CC) regions. Numbers indicate amino acid positions of the putative start and end points of each domain. (E) As in A, but with fragments of Epo1p as CRU fusions and 16 independently generated diploids for each experiment shown after 4 d of growth. (F) Protein extracts of bacterial cells expressing his 6 -Pea2 (lanes 1–4) or yeast cells expressing MYC-tagged Kel1p (lanes 5–8) were incubated with glutathione-coupled Sepharose beads exposing bacterially expressed GST (lanes 1 and 5), GST-Epo1 761–943 (lanes 2 and 6), GST-Epo1 761–867 (lanes 3 and 7), or GST-Epo1 852–943 (lanes 4 and 8). Glutathione eluates were separated by SDS-PAGE and probed with anti-His (lanes 1–4) or anti-MYC (lanes 5–8) antibodies after Western blotting. (G) As in F, except bacterially expressed MBP-Epo1 (lanes 1 and 2) or MBP-Epo1 1–760 (lanes 3 and 4) were precipitated with bacterially expressed and Sepharose bead immobilized GST (lanes 2 and 4) or GST-Pea2p (lanes 1 and 3). The inputs for the experiments in F and G are shown in Fig. S1 . (H) Pea2p mediates the interaction between Epo1p and Spa2p. As in F, except bacterially expressed his 6 -Spa2 1–535 -SNAP (lanes 1, 2, 7, and 8) or his 6 -Spa2 1–488 -SNAP (lanes 4, 5, 9, and 10) were first incubated with his 6 -Pea2 (lanes 1 and 4) or left untreated (lanes 7 and 9) before being incubated with bacterially expressed and immobilized GST-Epo1 761–943 . The glutathione eluates are shown in lanes 1, 4, 7, and 9. The inputs for the experiment in lane 1 are shown in lanes 2 and 3. The inputs for the experiment in lane 4 are shown in lanes 5 and 6. The inputs for the experiment in lanes 7 and 9 are shown in lanes 8 and 10, respectively. The asterisk indicates a degradation product. Lanes 1–6 show cutouts of the same gel with the vertical line indicating the removal of an empty lane. (I) Architecture of the ER–cell tip tethering complex. Edges connecting nodes indicate direct (black) or potentially indirect (green) interactions. Blue rectangles indicate coiled-coil regions shown in D.

    Article Snippet: In vitro binding assay GST-tagged proteins were immobilized on glutathione-coupled Sepharose beads (GE Healthcare).

    Techniques: Generated, Expressing, Incubation, SDS Page, Western Blot

    Mpr1 functions upstream of the Mcs4 response regulator. (A) Oxidative stress induces physical association between Mpr1 and Mcs4. Strain CA337 has chromosomal mcs4 + tagged with the sequence encoding the myc epitope. This strain was transformed with pREP1-KZ-mpr1 and pREP1-KZ-mpr1HQ plasmids, which express GST fusion proteins of wild-type and His-221→Gln mutant Mpr1, respectively, under the regulation of the thiamine-repressible nmt1 promoter. The transformants were grown in EMM2 medium with 0.03 μM thiamine to induce expression of the GST fusion proteins at a low level and treated with either oxidative stress induced by 0.3 mM H 2 O 2 (left panels) or high-osmolarity stress induced by 0.6 M KCl (right panels) for the indicated times. Cell lysates were absorbed to GSH-Sepharose beads, and after extensive washes, proteins bound to the beads (GSH-Beads) were analyzed by immunoblotting with anti-myc and anti-GST antibodies. The amount of Mcs4 detected in the crude cell lysates (Lysate) did not change significantly after the stress treatments. (B) Wild-type (KS1376), Δmcs4 (CA220), Δmpr1 (CA279), and Δmcs4 Δmpr1 (CA420) strains carrying the spc1:HA6H allele were grown to midlog phase at 30°C in YES medium and treated with oxidative stress induced by 0.3 mM H 2 O 2 . Aliquots of cells were harvested at the indicated times, and Spc1 was purified by Ni-NTA chromatography, followed by immunoblotting with anti-phospho-p38 and anti-HA antibodies. The pattern of Spc1 activation in the Δmcs4 Δmpr1 double mutant is identical to that in the Δmcs4 mutant before and after oxidative stress. (C) Wild-type (KS1376), Δmcs4 (CA220), and Δmpr1 (CA279) strains were treated with high-osmolarity stress induced by 0.6 M KCl, and Spc1 activation was examined as described for B.

    Journal: Molecular Biology of the Cell

    Article Title: Multistep Phosphorelay Proteins Transmit Oxidative Stress Signals to the Fission Yeast Stress-activated Protein Kinase

    doi:

    Figure Lengend Snippet: Mpr1 functions upstream of the Mcs4 response regulator. (A) Oxidative stress induces physical association between Mpr1 and Mcs4. Strain CA337 has chromosomal mcs4 + tagged with the sequence encoding the myc epitope. This strain was transformed with pREP1-KZ-mpr1 and pREP1-KZ-mpr1HQ plasmids, which express GST fusion proteins of wild-type and His-221→Gln mutant Mpr1, respectively, under the regulation of the thiamine-repressible nmt1 promoter. The transformants were grown in EMM2 medium with 0.03 μM thiamine to induce expression of the GST fusion proteins at a low level and treated with either oxidative stress induced by 0.3 mM H 2 O 2 (left panels) or high-osmolarity stress induced by 0.6 M KCl (right panels) for the indicated times. Cell lysates were absorbed to GSH-Sepharose beads, and after extensive washes, proteins bound to the beads (GSH-Beads) were analyzed by immunoblotting with anti-myc and anti-GST antibodies. The amount of Mcs4 detected in the crude cell lysates (Lysate) did not change significantly after the stress treatments. (B) Wild-type (KS1376), Δmcs4 (CA220), Δmpr1 (CA279), and Δmcs4 Δmpr1 (CA420) strains carrying the spc1:HA6H allele were grown to midlog phase at 30°C in YES medium and treated with oxidative stress induced by 0.3 mM H 2 O 2 . Aliquots of cells were harvested at the indicated times, and Spc1 was purified by Ni-NTA chromatography, followed by immunoblotting with anti-phospho-p38 and anti-HA antibodies. The pattern of Spc1 activation in the Δmcs4 Δmpr1 double mutant is identical to that in the Δmcs4 mutant before and after oxidative stress. (C) Wild-type (KS1376), Δmcs4 (CA220), and Δmpr1 (CA279) strains were treated with high-osmolarity stress induced by 0.6 M KCl, and Spc1 activation was examined as described for B.

    Article Snippet: The protein concentration of each lysate was normalized with the use of a Bio-Rad (Richmond, CA) protein assay before immunoblot analysis of Mcs4 (see Figure A, bottom panel) and incubation with 10 μl (bed volume) of glutathione (GSH)-Sepharose beads (Amersham Pharmacia).

    Techniques: Sequencing, Transformation Assay, Mutagenesis, Expressing, Purification, Chromatography, Activation Assay

    ( A ) BiFC assay in 5-d differentiated rat primary myotubes and HeLa cells. BiFC assay was performed on 5-d differentiated myotubes ( a – c ) or HeLa cells ( d – f ) expressing either pBiFC-VN173-JPH1TMD and pBiFC-VC155-JPH1TMD ( a and d ) or pBiFC-VN173-JPH1TMD and pBiFC-VC155-JPH4TMD ( b and e ) or pBiFC-VN173-JPH4TMD and pBiFC-VC155-JPH4TMD ( c and f ). Scale bar, 15 µm. ( B ) FRAP analysis on 12-d differentiated rat primary myotubes expressing GFP-JPH1 and GFP-JPH1 deletion mutants. FRAP analysis was performed on 12-d differentiated myotubes expressing either GFP-JPH1 or GFP-JPH1 deletion mutants. Data are expressed as percentage of mobile fraction ± SEM; n values are as follows: GFP-JPH1 ( n = 20); GFP-JPH1ΔMORN I-VIII ( n = 12), GFP-JPH1ΔTMD ( n = 7), and GFP-TMD-JPH1 ( n = 10). Asterisks indicate statistical significance compared with the mobile fraction of GFP-JPH1, as evaluated by the Kruskal–Wallis multiple comparisons test (** P ≤ 0.05; *** P ≤ 0.01). ( C ) Immunoprecipitation experiments on HEK293T cells coexpressing myc-JPH1 and GFP-JPH1 or myc-JPH1 and GFP-JPH2, or myc-JPH2 and GFP-JPH2. Total lysates from HEK293T cells were immunoprecipitated with anti-myc conjugated agarose beads. Immunocomplexes were separated by SDS/PAGE, transferred to nitrocellulose membranes, and detected by mouse monoclonal anti-myc or anti-GFP antibodies. The vertical black line at Bottom indicates that an unrelated lane was eliminated from the figure. ( D ) GST pull-down experiments on the microsomal fraction of mouse skeletal muscles and of HEK293T cells. A total of 500 µg of the microsomal fraction from mouse skeletal muscles or HEK293T cells expressing either GFP-JPH1 or GFP-JPH2 was used in GST pull-down experiments using GST-joining JPH1 or GST-joining JPH2 fusion proteins. Proteins were separated by SDS/PAGE, transferred to nitrocellulose membranes, and detected by specific antibodies. A total of 30 µg of solubilized microsomes was loaded as control.

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

    Article Title: Molecular determinants of homo- and heteromeric interactions of Junctophilin-1 at triads in adult skeletal muscle fibers

    doi: 10.1073/pnas.1820980116

    Figure Lengend Snippet: ( A ) BiFC assay in 5-d differentiated rat primary myotubes and HeLa cells. BiFC assay was performed on 5-d differentiated myotubes ( a – c ) or HeLa cells ( d – f ) expressing either pBiFC-VN173-JPH1TMD and pBiFC-VC155-JPH1TMD ( a and d ) or pBiFC-VN173-JPH1TMD and pBiFC-VC155-JPH4TMD ( b and e ) or pBiFC-VN173-JPH4TMD and pBiFC-VC155-JPH4TMD ( c and f ). Scale bar, 15 µm. ( B ) FRAP analysis on 12-d differentiated rat primary myotubes expressing GFP-JPH1 and GFP-JPH1 deletion mutants. FRAP analysis was performed on 12-d differentiated myotubes expressing either GFP-JPH1 or GFP-JPH1 deletion mutants. Data are expressed as percentage of mobile fraction ± SEM; n values are as follows: GFP-JPH1 ( n = 20); GFP-JPH1ΔMORN I-VIII ( n = 12), GFP-JPH1ΔTMD ( n = 7), and GFP-TMD-JPH1 ( n = 10). Asterisks indicate statistical significance compared with the mobile fraction of GFP-JPH1, as evaluated by the Kruskal–Wallis multiple comparisons test (** P ≤ 0.05; *** P ≤ 0.01). ( C ) Immunoprecipitation experiments on HEK293T cells coexpressing myc-JPH1 and GFP-JPH1 or myc-JPH1 and GFP-JPH2, or myc-JPH2 and GFP-JPH2. Total lysates from HEK293T cells were immunoprecipitated with anti-myc conjugated agarose beads. Immunocomplexes were separated by SDS/PAGE, transferred to nitrocellulose membranes, and detected by mouse monoclonal anti-myc or anti-GFP antibodies. The vertical black line at Bottom indicates that an unrelated lane was eliminated from the figure. ( D ) GST pull-down experiments on the microsomal fraction of mouse skeletal muscles and of HEK293T cells. A total of 500 µg of the microsomal fraction from mouse skeletal muscles or HEK293T cells expressing either GFP-JPH1 or GFP-JPH2 was used in GST pull-down experiments using GST-joining JPH1 or GST-joining JPH2 fusion proteins. Proteins were separated by SDS/PAGE, transferred to nitrocellulose membranes, and detected by specific antibodies. A total of 30 µg of solubilized microsomes was loaded as control.

    Article Snippet: The fusion proteins were immobilized by incubating 1 mL of the soluble fraction with 100 μL of beads of glutathione-Sepharose 4B (GE Healthcare, Buckinghamshire, United Kingdom) for 10 min and washed 3 times with 1 mL of a buffer containing PBS and 1% Triton X-100.

    Techniques: Bimolecular Fluorescence Complementation Assay, Expressing, Immunoprecipitation, SDS Page

    (A) Arkadia ubiquitinates poly-SUMO2 chains in a SIM- and RING-dependent manner. HEK293 cells were transfected with either Flag empty vector, Flag-Ark-wt, Flag-Ark-RING*, Flag-Ark-SIM123*, or Flag-RNF4 as a positive control. The different Flag constructs were purified on anti-Flag agarose beads, and 10 μl of Flag-purified proteins was incubated in the presence of recombinant poly-SUMO2 chains for 30 min on ice. In vitro ubiquitination was then performed for 45 min at 37°C in the presence of HA-Ub, E1 (UBE1), E2 (UbcH5b and UbcH5c), and ATP. Ubiquitinated poly-SUMO2 was visualized by Western blotting as a shift of higher-molecular-mass species using anti-SUMO2/3 antibody. The relative level of Flag-purified proteins used in the assay was evaluated in parallel by Western blotting using an anti-Flag antibody, as shown in the lower blot. (B) Arkadia and RNF4 independently ubiquitinate poly-SUMO2 chains. HEK293 cells were transfected with siArk or siRNF4. At 48 h after siRNA transfection, cells were transfected with Flag-Ark-wt or Flag-RNF4-wt, as indicated. At 24 h after plasmid transfection, cell lysates were extracted and Arkadia and RNF4 expression were evaluated by Western blotting using anti-Ark and anti-RNF4 as well as antiactin as a loading control. The rest of the lysate was purified with anti-Flag agarose beads. Ten microliters of Flag-purified proteins was used for in vitro ubiquitination assay of poly-SUMO2 chains, as described for panel A. Ubiquitinated poly-SUMO2 was visualized by Western blotting as a shift of higher-molecular-mass species using anti-SUMO2/3 antibody. Numbers to the left of the gels are molecular masses (in kDa).

    Journal: Molecular and Cellular Biology

    Article Title: Arkadia, a Novel SUMO-Targeted Ubiquitin Ligase Involved in PML Degradation

    doi: 10.1128/MCB.01019-12

    Figure Lengend Snippet: (A) Arkadia ubiquitinates poly-SUMO2 chains in a SIM- and RING-dependent manner. HEK293 cells were transfected with either Flag empty vector, Flag-Ark-wt, Flag-Ark-RING*, Flag-Ark-SIM123*, or Flag-RNF4 as a positive control. The different Flag constructs were purified on anti-Flag agarose beads, and 10 μl of Flag-purified proteins was incubated in the presence of recombinant poly-SUMO2 chains for 30 min on ice. In vitro ubiquitination was then performed for 45 min at 37°C in the presence of HA-Ub, E1 (UBE1), E2 (UbcH5b and UbcH5c), and ATP. Ubiquitinated poly-SUMO2 was visualized by Western blotting as a shift of higher-molecular-mass species using anti-SUMO2/3 antibody. The relative level of Flag-purified proteins used in the assay was evaluated in parallel by Western blotting using an anti-Flag antibody, as shown in the lower blot. (B) Arkadia and RNF4 independently ubiquitinate poly-SUMO2 chains. HEK293 cells were transfected with siArk or siRNF4. At 48 h after siRNA transfection, cells were transfected with Flag-Ark-wt or Flag-RNF4-wt, as indicated. At 24 h after plasmid transfection, cell lysates were extracted and Arkadia and RNF4 expression were evaluated by Western blotting using anti-Ark and anti-RNF4 as well as antiactin as a loading control. The rest of the lysate was purified with anti-Flag agarose beads. Ten microliters of Flag-purified proteins was used for in vitro ubiquitination assay of poly-SUMO2 chains, as described for panel A. Ubiquitinated poly-SUMO2 was visualized by Western blotting as a shift of higher-molecular-mass species using anti-SUMO2/3 antibody. Numbers to the left of the gels are molecular masses (in kDa).

    Article Snippet: For GST pulldown, 5 μg of each GST protein was conjugated to 20 μl of a glutathione-Sepharose bead slurry (GE Healthcare), and the conjugated proteins were incubated at 4°C overnight on a wheel with HEK293 cell lysate or with 1 μg of recombinant poly-SUMO2 chains (Boston Biochem) in lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% NP-40, 10% glycerol, 20 mM NEM, protease inhibitors [Roche]).

    Techniques: Transfection, Plasmid Preparation, Positive Control, Construct, Purification, Incubation, Recombinant, In Vitro, Western Blot, Expressing, Ubiquitin Assay

    EhSir2a binds tubulin in E. histolytica and deacetylates mammalian tubulin A. EhSir2a-HH expressed in E. histolytica binds tubulin . The cellular extract of EhSir2a-HH transformant (Sir2a) was incubated with Ni-NTA agarose beads and the proteins bound to the beads (SB) were analyzed by western hybridization against anti-Eh β-tubulin and anti-HA. C denotes the cellular extract of control transformant and CB denotes proteins isolated on Ni-NTA beads added to control cell extracts. B. GST-EhSir2a expressed in E. coli . GST-EhSir2a and GST were expressed in BL21 DE3. The purified proteins are shown in a coomassie stained SDS-PAGE. The molecular weight marker is shown at the right. C. GST-EhSir2a binds to tubulin GST-EhSir2a bound to glutathione sepharose 4B beads was incubated with cellular extract of E. histolytica (HM1) and the bound proteins were analyzed. Western blot hybridization with anti-Eh ²-tubulin detects tubulin in the bead coated with GST-EhSir2a (SB) and not in control beads (CB). D. Tubulin is deacetylated by EhSir2a . Goat brain tubulin was incubated with GST alone (GST) and GST-Sir2a (Sir2a) and analyzed by western blot hybridization with anti-α-tubulin and anti-acetylated α-tubulin. E. Co-localization of EhSir2a on microtubules . EhSir2a transformants were fixed and stained with monoclonal anti-HA and polyclonal anti-Eh ²-tubulin. In the merged image, yellow colour shows the co-localization of EhSir2a with microtubules. The corresponding bright field image with nucleus (N) is shown. Bar represents 10 µm. F. Lower frequency of MT structures is due to over-expression of EhSir2a . MT structures were counted in stable EhSir2a transformants as described in the text. Bar diagram shows the distribution of MT structures in control and Sir2a transformants, ± S.D. (n=3, for each set 600 cells were taken).

    Journal: Cellular microbiology

    Article Title: Entamoeba histolytica sirtuin EhSir2a deacetylates tubulin and regulates the number of microtubular assemblies during the cell cycle

    doi: 10.1111/j.1462-5822.2010.01449.x

    Figure Lengend Snippet: EhSir2a binds tubulin in E. histolytica and deacetylates mammalian tubulin A. EhSir2a-HH expressed in E. histolytica binds tubulin . The cellular extract of EhSir2a-HH transformant (Sir2a) was incubated with Ni-NTA agarose beads and the proteins bound to the beads (SB) were analyzed by western hybridization against anti-Eh β-tubulin and anti-HA. C denotes the cellular extract of control transformant and CB denotes proteins isolated on Ni-NTA beads added to control cell extracts. B. GST-EhSir2a expressed in E. coli . GST-EhSir2a and GST were expressed in BL21 DE3. The purified proteins are shown in a coomassie stained SDS-PAGE. The molecular weight marker is shown at the right. C. GST-EhSir2a binds to tubulin GST-EhSir2a bound to glutathione sepharose 4B beads was incubated with cellular extract of E. histolytica (HM1) and the bound proteins were analyzed. Western blot hybridization with anti-Eh ²-tubulin detects tubulin in the bead coated with GST-EhSir2a (SB) and not in control beads (CB). D. Tubulin is deacetylated by EhSir2a . Goat brain tubulin was incubated with GST alone (GST) and GST-Sir2a (Sir2a) and analyzed by western blot hybridization with anti-α-tubulin and anti-acetylated α-tubulin. E. Co-localization of EhSir2a on microtubules . EhSir2a transformants were fixed and stained with monoclonal anti-HA and polyclonal anti-Eh ²-tubulin. In the merged image, yellow colour shows the co-localization of EhSir2a with microtubules. The corresponding bright field image with nucleus (N) is shown. Bar represents 10 µm. F. Lower frequency of MT structures is due to over-expression of EhSir2a . MT structures were counted in stable EhSir2a transformants as described in the text. Bar diagram shows the distribution of MT structures in control and Sir2a transformants, ± S.D. (n=3, for each set 600 cells were taken).

    Article Snippet: After centrifugation (23,500 g at 4 °C), the lysate was incubated with pre-equilibrated Glutathione-Sepharose 4B beads (Amersham Bioscience, Germany) for 2 h. GST-EhSir2a was eluted in a buffer containing 50 mM Tris-HCl pH 8.0, 5 mM MgCl2 and 10% glycerol in presence of 20 mM reduced glutathione.

    Techniques: Incubation, Western Blot, Hybridization, Isolation, Purification, Staining, SDS Page, Molecular Weight, Marker, Over Expression

    Western blots of RegA, PKAcat, and PKA-R in various cell lines. ( A ). ( B ) Wild-type and mutant strains expressing GST–FbxA:F-box/WD40 were lysed and the 10,000 × G supernatant was adsorbed to g–Sepharose. The beads were washed and the bound material was examined by Western blot analysis and probed with anti-RegA, Cul-1, and GST antibodies (see Materials and Methods). ( C ) Samples were taken and processed as described for A and probed with either anti-PKAcat, or anti-PKA-R antibodies, as indicated. The anti-PKAcat and anti-PKA-R antibodies were a generous gift of M. Veron (Institut Pasteur, Paris). ( D ) The experiment is the same as described in B except that it was performed using cells that were transformed with GST alone.

    Journal: Genes & Development

    Article Title: Regulated protein degradation controls PKA function and cell-type differentiation in Dictyostelium

    doi: 10.1101/gad.871101

    Figure Lengend Snippet: Western blots of RegA, PKAcat, and PKA-R in various cell lines. ( A ). ( B ) Wild-type and mutant strains expressing GST–FbxA:F-box/WD40 were lysed and the 10,000 × G supernatant was adsorbed to g–Sepharose. The beads were washed and the bound material was examined by Western blot analysis and probed with anti-RegA, Cul-1, and GST antibodies (see Materials and Methods). ( C ) Samples were taken and processed as described for A and probed with either anti-PKAcat, or anti-PKA-R antibodies, as indicated. The anti-PKAcat and anti-PKA-R antibodies were a generous gift of M. Veron (Institut Pasteur, Paris). ( D ) The experiment is the same as described in B except that it was performed using cells that were transformed with GST alone.

    Article Snippet: Samples were centrifuged at 15,000 rpm for 10 min. We incubated the supernatant with 80 μL of GST beads (glutathione–Sepharose 4B, purchased from Amersham Pharmacia Biotech) for 1 h at 4°C with gentle rocking.

    Techniques: Western Blot, Mutagenesis, Expressing, Transformation Assay

    The UAP1 strain under repressive growth conditions.A. Growth on solid MM supplemented with 0.1 M threonine and 0.01%, 0.03%, 0.06% or 0.1% glucose, using serial dilutions of 10 5 –10 2 conidia.B. Semi-quantitative RT-PCR to amplify the tbp and uap1 gene. The RT-PCR products were separated on agarose gel (inset) and the relative transcription level was calculated based on the band intensity.C. Enzyme activity of the UAP1 strain grown under suppression condition. Intracellular proteins were extracted from mycelium and the enzyme activity was detected by Biomol green assay coupled with pyrophosphatase.D. TEM spore and mycelia morphology of strains grown in solid and liquid MMTG medium at 37°C for 36 h. Conidia (upper panel) and mycelia (lower panel) were fixed and examined with an H-600 electron microscope (Hitachi). Bar = 0.5 μm.

    Journal: Molecular Microbiology

    Article Title: Genetic and structural validation of Aspergillus fumigatus UDP-N-acetylglucosamine pyrophosphorylase as an antifungal target

    doi: 10.1111/mmi.12290

    Figure Lengend Snippet: The UAP1 strain under repressive growth conditions.A. Growth on solid MM supplemented with 0.1 M threonine and 0.01%, 0.03%, 0.06% or 0.1% glucose, using serial dilutions of 10 5 –10 2 conidia.B. Semi-quantitative RT-PCR to amplify the tbp and uap1 gene. The RT-PCR products were separated on agarose gel (inset) and the relative transcription level was calculated based on the band intensity.C. Enzyme activity of the UAP1 strain grown under suppression condition. Intracellular proteins were extracted from mycelium and the enzyme activity was detected by Biomol green assay coupled with pyrophosphatase.D. TEM spore and mycelia morphology of strains grown in solid and liquid MMTG medium at 37°C for 36 h. Conidia (upper panel) and mycelia (lower panel) were fixed and examined with an H-600 electron microscope (Hitachi). Bar = 0.5 μm.

    Article Snippet: The cell lysate was centrifuged at 40 000 g for 30 min to remove cell debris and the supernatant was incubated with pre-washed glutathione sepharose beads (GE Healthcare) at 4°C on a rotating platform for 2 h. The GST-Af UAP1 fusion protein was isolated by centrifugation and washed with buffer (25 mM Tris-HCl, 150 mM NaCl, pH 7.5).

    Techniques: Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Activity Assay, Transmission Electron Microscopy, Microscopy

    Interaction between HIV nucleocapsid (NC) protein and Tat ( A ) Plasmid expressing NC and plasmid expressing Tat were transformed to AH109 and the yeast was grown on media lacking histidine. Murine p53 and SV40 large T antigens were used as a positive control. HIV Vpu was used as a negative control. The lower panel shows yeast colonies resulting from interaction of the two proteins. Plasmids pGBK-NC, pGAD-Tat and pGAD-Vpu were constructed and utilized. pGBKT7-53 (murine p53) and pGADT7-T (SV40 large T antigen) were used as positive controls. Yeast strain AH109, transformed only with pGAD-Tat or pGAD-Vpu were used as negative controls; ( B ) Purified recombinant GST-fused NC was used to pulldown BKH21 cell lysate expressing Tat. GST was used as the negative control (third lane). pcDNA/V5-Tat was transfected to BHK-21 cells. After 24 h, a RIPA lysis buffer was added and lysis was performed at 4 °C for 30 min. The pellet was discarded after centrifugation and the lysate was obtained. GST or GST-NC and 30 μL of glutathione sepharose 4B bead was added to the lysate and the mixture was incubated at 4 °C overnight. HNGT buffer was used for washing and bound proteins were eluted after boiling with a 4XSDS sample buffer; ( C ) Flag-tagged NC and V5-tagged Tat or V5-tagged Vpu were expressed in HEK293 cells. The lysate was subjected to an immunoprecipitation with the anti-Flag antibody and detected with the anti-V5 antibody in a western blot analysis. The anti-IgG antibody was used a as the negative control; ( D ) Flag-tagged NC (green) and V5-tagged Tat (red) were expressed in HEK293 cells and observed under a confocal laser scanning microscope. At least three experiments were performed with essentially the same results.

    Journal: Viruses

    Article Title: Induced Degradation of Tat by Nucleocapsid (NC) via the Proteasome Pathway and Its Effect on HIV Transcription

    doi: 10.3390/v5041143

    Figure Lengend Snippet: Interaction between HIV nucleocapsid (NC) protein and Tat ( A ) Plasmid expressing NC and plasmid expressing Tat were transformed to AH109 and the yeast was grown on media lacking histidine. Murine p53 and SV40 large T antigens were used as a positive control. HIV Vpu was used as a negative control. The lower panel shows yeast colonies resulting from interaction of the two proteins. Plasmids pGBK-NC, pGAD-Tat and pGAD-Vpu were constructed and utilized. pGBKT7-53 (murine p53) and pGADT7-T (SV40 large T antigen) were used as positive controls. Yeast strain AH109, transformed only with pGAD-Tat or pGAD-Vpu were used as negative controls; ( B ) Purified recombinant GST-fused NC was used to pulldown BKH21 cell lysate expressing Tat. GST was used as the negative control (third lane). pcDNA/V5-Tat was transfected to BHK-21 cells. After 24 h, a RIPA lysis buffer was added and lysis was performed at 4 °C for 30 min. The pellet was discarded after centrifugation and the lysate was obtained. GST or GST-NC and 30 μL of glutathione sepharose 4B bead was added to the lysate and the mixture was incubated at 4 °C overnight. HNGT buffer was used for washing and bound proteins were eluted after boiling with a 4XSDS sample buffer; ( C ) Flag-tagged NC and V5-tagged Tat or V5-tagged Vpu were expressed in HEK293 cells. The lysate was subjected to an immunoprecipitation with the anti-Flag antibody and detected with the anti-V5 antibody in a western blot analysis. The anti-IgG antibody was used a as the negative control; ( D ) Flag-tagged NC (green) and V5-tagged Tat (red) were expressed in HEK293 cells and observed under a confocal laser scanning microscope. At least three experiments were performed with essentially the same results.

    Article Snippet: Exactly 4 μg each of GST and GST-NC and 30 μL of glutathione sepharose 4B bead (Amersham Biosciences) were added to the lysate and the mixture was incubated at 4 °C overnight.

    Techniques: Plasmid Preparation, Expressing, Transformation Assay, Positive Control, Negative Control, Construct, Purification, Recombinant, Transfection, Lysis, Centrifugation, Incubation, Immunoprecipitation, Western Blot, Laser-Scanning Microscopy

    Brg1 interacts with the N-terminal and central parts of Daxx. (a) Human Daxx (SIM-SUMO interacting motif, DHB-Daxx homology bundle, SPE-Ser/Pro/Glu rich, SPT-Ser/Pro/Thr rich) were divided into N-terminal (amino acids 1–224), central (amino acids 225–503) and C-terminal (amino acids 504–740) fragments. (b) GST-tagged Daxx fragments GST-Daxx WT and GST tag alone were incubated with 35 S-labelled Brg1 together with Glutathion-Sepharose 4B beads. After the SDS-PAGE, GST-tagged proteins were visualised by Coomassie staining and 35 S-labelled Brg1 by autoradiography. (c) HEK293T cells were transfected with FLAG-Brg1 FL or empty vector together with empty vector or Myc-Daxx fragments and Myc-Daxx FL. The cell lysates were immunoprecipitated with either anti-Myc or anti-FLAG antibodies and analysed by Western blotting.

    Journal: Biochemistry and Biophysics Reports

    Article Title: Multifunctional adaptor protein Daxx interacts with chromatin-remodelling ATPase Brg1

    doi: 10.1016/j.bbrep.2015.12.012

    Figure Lengend Snippet: Brg1 interacts with the N-terminal and central parts of Daxx. (a) Human Daxx (SIM-SUMO interacting motif, DHB-Daxx homology bundle, SPE-Ser/Pro/Glu rich, SPT-Ser/Pro/Thr rich) were divided into N-terminal (amino acids 1–224), central (amino acids 225–503) and C-terminal (amino acids 504–740) fragments. (b) GST-tagged Daxx fragments GST-Daxx WT and GST tag alone were incubated with 35 S-labelled Brg1 together with Glutathion-Sepharose 4B beads. After the SDS-PAGE, GST-tagged proteins were visualised by Coomassie staining and 35 S-labelled Brg1 by autoradiography. (c) HEK293T cells were transfected with FLAG-Brg1 FL or empty vector together with empty vector or Myc-Daxx fragments and Myc-Daxx FL. The cell lysates were immunoprecipitated with either anti-Myc or anti-FLAG antibodies and analysed by Western blotting.

    Article Snippet: 2.6 Purification of recombinant protein and in vitro pulldown assay Combined His6+GST-tagged Daxx and its variants were produced in E. coli BL21 (DE3) from pET42b-based expression plasmids and purified with TALON (Clontech ) and Glutathion-Sepharose beads (GE Healthcare ).

    Techniques: Single-particle Tracking, Incubation, SDS Page, Staining, Autoradiography, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot

    Effects of the interaction between GlpK and the KPN00353 variants on 1,3-PD production. After the interaction of GST-tagged GlpK with wild-type or variant His-tagged KPN00353, the proteins captured on GSH-Sepharose beads were analyzed by Western blotting using either anti-GST antibody (α-GST) or anti-His antibody (α-His). From the Western blotting results, we defined the binding affinity of GlpK and wild-type KPN00353 (WT) to be 1+. The binding affinity of GlpK and the KPN00353 variant was comparable to that of GlpK and H65wt. H65wt, E. coli with pET30b::353WT; H65D, E. coli with pET30b::H65D; H65E, E. coli with pET30b::H65E; H65R, E. coli with pET30b::H65R; H65Q, E. coli with pET30b::H65Q; H110Q, E. coli with pET30b::H110Q. The 1,3-PD production of K. pneumoniae with pBAD33 (Vec) after incubation for 4 h was 0.186 g/L as 100%. The 1,3-PD production of K. pneumoniae with plasmids expressing wild-type or variant KPN00353 was compared to that of Vec. The residual glycerol in the medium was compared to the glycerol concentration in fresh MCM. H65wt, K. pneumoniae with pBAD33::Histaq353WT; H65D, K. pneumoniae with pBAD33::Histaq353H65D; H65E, K. pneumoniae with pBAD33::Histaq353H65E; H65R, K. pneumoniae with pBAD33::Histaq353H65R; H65Q, K. pneumoniae with pBAD33::Histaq353H65Q; H110Q, K. pneumoniae with pBAD33::Histaq353H110Q.

    Journal: Frontiers in Microbiology

    Article Title: The Negative Effects of KPN00353 on Glycerol Kinase and Microaerobic 1,3-Propanediol Production in Klebsiella pneumoniae

    doi: 10.3389/fmicb.2017.02441

    Figure Lengend Snippet: Effects of the interaction between GlpK and the KPN00353 variants on 1,3-PD production. After the interaction of GST-tagged GlpK with wild-type or variant His-tagged KPN00353, the proteins captured on GSH-Sepharose beads were analyzed by Western blotting using either anti-GST antibody (α-GST) or anti-His antibody (α-His). From the Western blotting results, we defined the binding affinity of GlpK and wild-type KPN00353 (WT) to be 1+. The binding affinity of GlpK and the KPN00353 variant was comparable to that of GlpK and H65wt. H65wt, E. coli with pET30b::353WT; H65D, E. coli with pET30b::H65D; H65E, E. coli with pET30b::H65E; H65R, E. coli with pET30b::H65R; H65Q, E. coli with pET30b::H65Q; H110Q, E. coli with pET30b::H110Q. The 1,3-PD production of K. pneumoniae with pBAD33 (Vec) after incubation for 4 h was 0.186 g/L as 100%. The 1,3-PD production of K. pneumoniae with plasmids expressing wild-type or variant KPN00353 was compared to that of Vec. The residual glycerol in the medium was compared to the glycerol concentration in fresh MCM. H65wt, K. pneumoniae with pBAD33::Histaq353WT; H65D, K. pneumoniae with pBAD33::Histaq353H65D; H65E, K. pneumoniae with pBAD33::Histaq353H65E; H65R, K. pneumoniae with pBAD33::Histaq353H65R; H65Q, K. pneumoniae with pBAD33::Histaq353H65Q; H110Q, K. pneumoniae with pBAD33::Histaq353H110Q.

    Article Snippet: Glutathione-sepharose (GSH) beads (50 μL; GE Healthcare, United Kingdom) were then added to the spent supernatant, and the mixture was incubated with mild shaking for 1 h at 4°C.

    Techniques: Variant Assay, Western Blot, Binding Assay, Incubation, Expressing, Concentration Assay

    Interaction between KPN00353 and GlpK. GST-tagged GlpK (GST-GlpK) was allowed to interact with His-tagged protein (lane 2, 3 in A,B ), and then the proteins captured on GSH-Sepharose beads were analyzed by (i) SDS-PAGE followed by Coomassie Blue staining and (ii) Western blotting using anti-His antibody. The purified proteins were analyzed simultaneously with the control (lane 4, 5, 6 in A,B ). The protein marker is shown in lane 1. His-353, His-tagged KPN00353; His-MrkD, His-tagged MrkD; GST, glutathione S-transferase.

    Journal: Frontiers in Microbiology

    Article Title: The Negative Effects of KPN00353 on Glycerol Kinase and Microaerobic 1,3-Propanediol Production in Klebsiella pneumoniae

    doi: 10.3389/fmicb.2017.02441

    Figure Lengend Snippet: Interaction between KPN00353 and GlpK. GST-tagged GlpK (GST-GlpK) was allowed to interact with His-tagged protein (lane 2, 3 in A,B ), and then the proteins captured on GSH-Sepharose beads were analyzed by (i) SDS-PAGE followed by Coomassie Blue staining and (ii) Western blotting using anti-His antibody. The purified proteins were analyzed simultaneously with the control (lane 4, 5, 6 in A,B ). The protein marker is shown in lane 1. His-353, His-tagged KPN00353; His-MrkD, His-tagged MrkD; GST, glutathione S-transferase.

    Article Snippet: Glutathione-sepharose (GSH) beads (50 μL; GE Healthcare, United Kingdom) were then added to the spent supernatant, and the mixture was incubated with mild shaking for 1 h at 4°C.

    Techniques: SDS Page, Staining, Western Blot, Purification, Marker

    High NaCl reduces binding of MKP-1 to p38 and has no significant effect on MKP-1 protein expression. ( A–C ) High NaCl reduces binding of MKP-1 to p38. Osmolality bathing mIMCD3 cells stably expressing GST, GST-MKP-1, or GST-p38 was increased to 550 mosmol/kg (NaCl added) for 30 min, then GST-MKP-1 or GST-p38 was pulled down with glutathione Sepharose beads, and the amount of accompanying native MKP-1 or p38 was measured by Western analysis (*, P

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

    Article Title: MKP-1 inhibits high NaCl-induced activation of p38 but does not inhibit the activation of TonEBP/OREBP: Opposite roles of p38α and p38δ

    doi: 10.1073/pnas.0801453105

    Figure Lengend Snippet: High NaCl reduces binding of MKP-1 to p38 and has no significant effect on MKP-1 protein expression. ( A–C ) High NaCl reduces binding of MKP-1 to p38. Osmolality bathing mIMCD3 cells stably expressing GST, GST-MKP-1, or GST-p38 was increased to 550 mosmol/kg (NaCl added) for 30 min, then GST-MKP-1 or GST-p38 was pulled down with glutathione Sepharose beads, and the amount of accompanying native MKP-1 or p38 was measured by Western analysis (*, P

    Article Snippet: Cell extracts were incubated with Glutathione Sepharose High Performance beads (Amersham Biosciences) at 4°C overnight with gentle shaking.

    Techniques: Binding Assay, Expressing, Stable Transfection, Western Blot

    Anti-AQP1 specificity of the identified autoantibodies. (A) Patient’s autoantibodies recognize the AQP1 moiety of AQP1-GST. Five sera that had tested positive for binding to the AQP1-GST fusion protein were preincubated with an excess of GST immobilized on Sepharose-glutathione beads, then were tested by RIPA using 125 I-streptavidin labeled AQP1-GST. (B) Binding of anti-AQP1 autoantibodies is specifically inhibited by an extract from AQP1-expressing HEK293 cells, but not control HEK293 cells. Four anti-AQP1-positive sera were preincubated with extracts prepared from either EGFP-transfected or AQP1-GFP-transfected HEK293 cells, then were tested by RIPA for binding to the commercial AQP1 preparation. (C) Binding of anti-AQP1 autoantibodies is specifically inhibited by yeast-expressed human AQP1. Four exclusively anti-AQP1-positive sera were preincubated with human AQP1 or AQP4 that had been expressed in yeast and purified or with BSA as control, then were tested by RIPA using 125 I-streptavidin-labeled commercial AQP1-GST fusion protein. (D) AQP1 autoantibody binding is independent of the source of AQP1. Both the commercial AQP1-GST fusion protein and the in house AQP1 purified from yeast were biotinylated, indirectly labeled by preincubation with 125 I-streptavidin, and used in the RIPA. Five anti-AQP1-positive sera and one serum sample from a healthy control (HC) were tested.

    Journal: PLoS ONE

    Article Title: Anti-Aquaporin-1 Autoantibodies in Patients with Neuromyelitis Optica Spectrum Disorders

    doi: 10.1371/journal.pone.0074773

    Figure Lengend Snippet: Anti-AQP1 specificity of the identified autoantibodies. (A) Patient’s autoantibodies recognize the AQP1 moiety of AQP1-GST. Five sera that had tested positive for binding to the AQP1-GST fusion protein were preincubated with an excess of GST immobilized on Sepharose-glutathione beads, then were tested by RIPA using 125 I-streptavidin labeled AQP1-GST. (B) Binding of anti-AQP1 autoantibodies is specifically inhibited by an extract from AQP1-expressing HEK293 cells, but not control HEK293 cells. Four anti-AQP1-positive sera were preincubated with extracts prepared from either EGFP-transfected or AQP1-GFP-transfected HEK293 cells, then were tested by RIPA for binding to the commercial AQP1 preparation. (C) Binding of anti-AQP1 autoantibodies is specifically inhibited by yeast-expressed human AQP1. Four exclusively anti-AQP1-positive sera were preincubated with human AQP1 or AQP4 that had been expressed in yeast and purified or with BSA as control, then were tested by RIPA using 125 I-streptavidin-labeled commercial AQP1-GST fusion protein. (D) AQP1 autoantibody binding is independent of the source of AQP1. Both the commercial AQP1-GST fusion protein and the in house AQP1 purified from yeast were biotinylated, indirectly labeled by preincubation with 125 I-streptavidin, and used in the RIPA. Five anti-AQP1-positive sera and one serum sample from a healthy control (HC) were tested.

    Article Snippet: To test whether the antibodies bound to the AQP1 moiety of the AQP1-GST fusion protein, 10 µl of sera positive for anti-AQP1 autoantibodies was preincubated for 3 h at 4°C with excess GST (1.6 µg) immobilized on Sepharose-glutathione beads (GE Healthcare), then 5 µl of the treated samples was tested in the RIPA using 125 I-streptavidin-labeled AQP1-GST.

    Techniques: Binding Assay, Labeling, Expressing, Transfection, Purification

    Identification of cell proteins interacting with the P150 PRR. (a) RUBV genome organization and location and sequence of the PRR. At the top is shown a diagram of the RUBV genome with the NS-ORF and SP-ORF shown as boxes containing the proteins that each encodes. An expanded version of P150 shows its four defined domains: MT, methyl/guanylyl transferase; Q, Q domain; X, X domain (ADP–ribose-binding domain); and P, protease. Below the P150 diagram is the sequence of the PRR, with its three predicted class II SH3-binding domains (underlined) and a poly-arginine cluster (shaded), mutation of which is lethal but which can be rescued by CP. Amino acid coordinates of P150, the domains within P150 and the PRR containing the PxxPxR motifs are shown. (b) GST–PRR pull-down of cell proteins. The P150 PRR and its mutant with motifs 1 and 2, both mutated from PxxPxR to AxxAxR, were cloned into the pEBG mammalian expression vector to produce GST–PRR/wt or GST–PRR/Mut1+2 fusion proteins, respectively, when expressed in transfected HEK293T cells. At 3 days post-transfection, the cells were lysed and the GST fusion proteins were captured on gluthathione–Sepharose 4B beads. Bound proteins were eluted and resolved by 6 (left gel) or 10 % (right gel) SDS-PAGE. Proteins that bound to the wt fusion protein but not to the mutant fusion protein (PRR1–PRR4) were excised and identified by trypsin digestion and mass spectroscopy. The determined identities and sizes of each of these proteins are indicated. (c) Confirmation of p32 binding to the PRR. HEK293T cells were transfected with the empty pEBG vector, GST–PRR/wt or GST–PRR/Mut1+2. At 3 days post-transfection, the cells were lysed and the GST or GST–PRR proteins and interacting cell proteins were isolated on glutathione–Sepharose 4B and resolved by 10 % SDS-PAGE followed by Western blotting with an anti-p32 antibody. The position of migration of p32 is indicated. (d) Determination of the motif to which p32 binds. Vero cells were mock transfected (lane 1) or transfected with a plasmid vector expressing an HA-tagged P150 Q domain (HA–P150Q) containing wt (lane 2), Mut1 (lane 3), Mut2 (lane 4) or Mut1+2 (lane 5) sequences. On day 1 post-transfection, the cells were lysed and the lysates immunoprecipitated (IP) with anti-p32 antibody. Following resolution by 10 % SDS-PAGE, Western blot (WB) analysis was performed with an anti-HA antibody. The upper panel (lysate) shows that all HA–P150Q constructs were expressed at similar levels, whilst the lower panel (immunoprecipitation with anti-p32 antibody) shows that wt, Mut1 and Mut2 HA–P150Q were co-immunoprecipitated with p32, whilst Mut1+2 HA–P150Q was not.

    Journal: The Journal of General Virology

    Article Title: Binding of cellular p32 protein to the rubella virus P150 replicase protein via PxxPxR motifs

    doi: 10.1099/vir.0.038901-0

    Figure Lengend Snippet: Identification of cell proteins interacting with the P150 PRR. (a) RUBV genome organization and location and sequence of the PRR. At the top is shown a diagram of the RUBV genome with the NS-ORF and SP-ORF shown as boxes containing the proteins that each encodes. An expanded version of P150 shows its four defined domains: MT, methyl/guanylyl transferase; Q, Q domain; X, X domain (ADP–ribose-binding domain); and P, protease. Below the P150 diagram is the sequence of the PRR, with its three predicted class II SH3-binding domains (underlined) and a poly-arginine cluster (shaded), mutation of which is lethal but which can be rescued by CP. Amino acid coordinates of P150, the domains within P150 and the PRR containing the PxxPxR motifs are shown. (b) GST–PRR pull-down of cell proteins. The P150 PRR and its mutant with motifs 1 and 2, both mutated from PxxPxR to AxxAxR, were cloned into the pEBG mammalian expression vector to produce GST–PRR/wt or GST–PRR/Mut1+2 fusion proteins, respectively, when expressed in transfected HEK293T cells. At 3 days post-transfection, the cells were lysed and the GST fusion proteins were captured on gluthathione–Sepharose 4B beads. Bound proteins were eluted and resolved by 6 (left gel) or 10 % (right gel) SDS-PAGE. Proteins that bound to the wt fusion protein but not to the mutant fusion protein (PRR1–PRR4) were excised and identified by trypsin digestion and mass spectroscopy. The determined identities and sizes of each of these proteins are indicated. (c) Confirmation of p32 binding to the PRR. HEK293T cells were transfected with the empty pEBG vector, GST–PRR/wt or GST–PRR/Mut1+2. At 3 days post-transfection, the cells were lysed and the GST or GST–PRR proteins and interacting cell proteins were isolated on glutathione–Sepharose 4B and resolved by 10 % SDS-PAGE followed by Western blotting with an anti-p32 antibody. The position of migration of p32 is indicated. (d) Determination of the motif to which p32 binds. Vero cells were mock transfected (lane 1) or transfected with a plasmid vector expressing an HA-tagged P150 Q domain (HA–P150Q) containing wt (lane 2), Mut1 (lane 3), Mut2 (lane 4) or Mut1+2 (lane 5) sequences. On day 1 post-transfection, the cells were lysed and the lysates immunoprecipitated (IP) with anti-p32 antibody. Following resolution by 10 % SDS-PAGE, Western blot (WB) analysis was performed with an anti-HA antibody. The upper panel (lysate) shows that all HA–P150Q constructs were expressed at similar levels, whilst the lower panel (immunoprecipitation with anti-p32 antibody) shows that wt, Mut1 and Mut2 HA–P150Q were co-immunoprecipitated with p32, whilst Mut1+2 HA–P150Q was not.

    Article Snippet: At 3 days post-transfection, the cells were washed twice with lysis wash buffer [50 mM Tris/HCl (pH 7.4), 1 % NP-40, 150 mM NaCl, 2 mM EDTA and 150 mM sodium salicylate] and lysed with 1 % NP-40 buffer [50 mM Tris/HCl (pH 7.4), 1 % NP-40, 150 mM NaCl and 2 mM EDTA], and GST fusion proteins were isolated from the lysate using gluthathione–Sepharose 4B beads (Amersham Pharmacia Biotech).

    Techniques: Sequencing, Binding Assay, Mutagenesis, Clone Assay, Expressing, Plasmid Preparation, Transfection, SDS Page, Mass Spectrometry, Isolation, Western Blot, Migration, Immunoprecipitation, Construct