Structured Review

GE Healthcare glutathione sepharose binding buffer
Crystal structure of CaM in complex with its AKAP79 binding site. a Cartoon representation showing one of the two copies (chains B and D) of AKAP79 peptide (orange) bound to CaM ( blue ) in the asymmetric unit. The C-lobe (lighter blue ) is in the open conformation with each of its two EF hands coordinating Ca 2+ (yellow). b Rotation of the complex through 90° highlighting the position of the four hydrophobic amino acids comprising the 1-4-7-8 motif. c Reduction in alphascreen signal between biotin-CaM and GST-AKAP79 (1–153) upon addition of 20-mer peptides derived from AKAP79 77–96 ( n = 4). The effects of point mutations within the disruptor peptide were compared. d Binding of purified full-length WT, Δ79–86 or W79A AKAP79 to CaM <t>sepharose.</t> Each AKAP79 variant was purified in complex with the D/D of RIIα. AKAP79 was released from the beads by incubation with EGTA, and detected by anti-AKAP79 IB. The experiment was performed in triplicate with each replicate leading to the same pattern of bands. e Limited Ramachandran plot showing dihedral angles for both copies of AKAP79 positions 80–85 in the asymmetric unit. Black triangles represent amino acids with angles characteristic of 3 10 helices; white diamonds are amino acids with α-helical geometry. f Representation of backbone H-bonds within the AKAP79 helix with distances shown in Å. The two α-helix-type bonds are shown by dotted lines; 3 10 -helical H-bonds as striped lines. The carbonyl group of S81 that does not H-bond to a backbone group is asterisked. g Rotation of the helix through 90°. The triangular backbone geometry of positions 83–86 is such that the side-chains of W79, L83 and T86 extend in the same direction. ** P
Glutathione Sepharose Binding Buffer, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Molecular basis of AKAP79 regulation by calmodulin"

Article Title: Molecular basis of AKAP79 regulation by calmodulin

Journal: Nature Communications

doi: 10.1038/s41467-017-01715-w

Crystal structure of CaM in complex with its AKAP79 binding site. a Cartoon representation showing one of the two copies (chains B and D) of AKAP79 peptide (orange) bound to CaM ( blue ) in the asymmetric unit. The C-lobe (lighter blue ) is in the open conformation with each of its two EF hands coordinating Ca 2+ (yellow). b Rotation of the complex through 90° highlighting the position of the four hydrophobic amino acids comprising the 1-4-7-8 motif. c Reduction in alphascreen signal between biotin-CaM and GST-AKAP79 (1–153) upon addition of 20-mer peptides derived from AKAP79 77–96 ( n = 4). The effects of point mutations within the disruptor peptide were compared. d Binding of purified full-length WT, Δ79–86 or W79A AKAP79 to CaM sepharose. Each AKAP79 variant was purified in complex with the D/D of RIIα. AKAP79 was released from the beads by incubation with EGTA, and detected by anti-AKAP79 IB. The experiment was performed in triplicate with each replicate leading to the same pattern of bands. e Limited Ramachandran plot showing dihedral angles for both copies of AKAP79 positions 80–85 in the asymmetric unit. Black triangles represent amino acids with angles characteristic of 3 10 helices; white diamonds are amino acids with α-helical geometry. f Representation of backbone H-bonds within the AKAP79 helix with distances shown in Å. The two α-helix-type bonds are shown by dotted lines; 3 10 -helical H-bonds as striped lines. The carbonyl group of S81 that does not H-bond to a backbone group is asterisked. g Rotation of the helix through 90°. The triangular backbone geometry of positions 83–86 is such that the side-chains of W79, L83 and T86 extend in the same direction. ** P
Figure Legend Snippet: Crystal structure of CaM in complex with its AKAP79 binding site. a Cartoon representation showing one of the two copies (chains B and D) of AKAP79 peptide (orange) bound to CaM ( blue ) in the asymmetric unit. The C-lobe (lighter blue ) is in the open conformation with each of its two EF hands coordinating Ca 2+ (yellow). b Rotation of the complex through 90° highlighting the position of the four hydrophobic amino acids comprising the 1-4-7-8 motif. c Reduction in alphascreen signal between biotin-CaM and GST-AKAP79 (1–153) upon addition of 20-mer peptides derived from AKAP79 77–96 ( n = 4). The effects of point mutations within the disruptor peptide were compared. d Binding of purified full-length WT, Δ79–86 or W79A AKAP79 to CaM sepharose. Each AKAP79 variant was purified in complex with the D/D of RIIα. AKAP79 was released from the beads by incubation with EGTA, and detected by anti-AKAP79 IB. The experiment was performed in triplicate with each replicate leading to the same pattern of bands. e Limited Ramachandran plot showing dihedral angles for both copies of AKAP79 positions 80–85 in the asymmetric unit. Black triangles represent amino acids with angles characteristic of 3 10 helices; white diamonds are amino acids with α-helical geometry. f Representation of backbone H-bonds within the AKAP79 helix with distances shown in Å. The two α-helix-type bonds are shown by dotted lines; 3 10 -helical H-bonds as striped lines. The carbonyl group of S81 that does not H-bond to a backbone group is asterisked. g Rotation of the helix through 90°. The triangular backbone geometry of positions 83–86 is such that the side-chains of W79, L83 and T86 extend in the same direction. ** P

Techniques Used: Chick Chorioallantoic Membrane Assay, Binding Assay, Amplified Luminescent Proximity Homogenous Assay, Derivative Assay, Purification, Variant Assay, Incubation

Delineation of key residues in AKAP79 required for CaM binding. a Pull-down of either WT, Δ33–48, Δ79–86, or Δ391–400 FLAG-tagged-AKAP79 (inputs shown in bottom panel) with either CaM sepharose (top panel) or cAMP agarose (middle panel). AKAP79 was detected by anti-FLAG immunoblotting. The experiment was performed in triplicate with each replicate producing the same pattern of bands. b Sequence LOGO for AKAP5 gene products aligned with predicted helical region. The cross-linking cluster between AKAP79 positions 90–99 and K94 in CaM is indicated along with the boundaries of peptides used in the following panels. c – e Determination of inhibitory constants for the peptides outlined in ( b ) in disrupting interaction between biotin-CaM and GST-AKAP79 (1–153) detected using the alphascreen assay ( n = 4 for all data points). K i constants were determined for the 9-mer and 11-mer peptides c , 16-mer and 20-mer peptides d , and for either WT or L101A 26-mer peptides e .***P
Figure Legend Snippet: Delineation of key residues in AKAP79 required for CaM binding. a Pull-down of either WT, Δ33–48, Δ79–86, or Δ391–400 FLAG-tagged-AKAP79 (inputs shown in bottom panel) with either CaM sepharose (top panel) or cAMP agarose (middle panel). AKAP79 was detected by anti-FLAG immunoblotting. The experiment was performed in triplicate with each replicate producing the same pattern of bands. b Sequence LOGO for AKAP5 gene products aligned with predicted helical region. The cross-linking cluster between AKAP79 positions 90–99 and K94 in CaM is indicated along with the boundaries of peptides used in the following panels. c – e Determination of inhibitory constants for the peptides outlined in ( b ) in disrupting interaction between biotin-CaM and GST-AKAP79 (1–153) detected using the alphascreen assay ( n = 4 for all data points). K i constants were determined for the 9-mer and 11-mer peptides c , 16-mer and 20-mer peptides d , and for either WT or L101A 26-mer peptides e .***P

Techniques Used: Chick Chorioallantoic Membrane Assay, Binding Assay, Sequencing, Amplified Luminescent Proximity Homogenous Assay

Related Articles

Incubation:

Article Title: Molecular basis of AKAP79 regulation by calmodulin
Article Snippet: .. The eluted protein was buffer exchanged into glutathione sepharose binding buffer (25 mM Tris pH 7.4, 500 mM NaCl, 2 mM DTT, 1 mM EDTA, 1 mM Benzamidine) using a Sephadex G-25 column, before 3 h incubation with glutathione sepharose 4B (GE Life Sciences). .. AKAP79-D/D was eluted by overnight incubation with PreScission protease (GE Life Sciences).

Binding Assay:

Article Title: Molecular basis of AKAP79 regulation by calmodulin
Article Snippet: .. The eluted protein was buffer exchanged into glutathione sepharose binding buffer (25 mM Tris pH 7.4, 500 mM NaCl, 2 mM DTT, 1 mM EDTA, 1 mM Benzamidine) using a Sephadex G-25 column, before 3 h incubation with glutathione sepharose 4B (GE Life Sciences). .. AKAP79-D/D was eluted by overnight incubation with PreScission protease (GE Life Sciences).

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  • 91
    GE Healthcare glutathione sepharose binding buffer
    Crystal structure of CaM in complex with its AKAP79 binding site. a Cartoon representation showing one of the two copies (chains B and D) of AKAP79 peptide (orange) bound to CaM ( blue ) in the asymmetric unit. The C-lobe (lighter blue ) is in the open conformation with each of its two EF hands coordinating Ca 2+ (yellow). b Rotation of the complex through 90° highlighting the position of the four hydrophobic amino acids comprising the 1-4-7-8 motif. c Reduction in alphascreen signal between biotin-CaM and GST-AKAP79 (1–153) upon addition of 20-mer peptides derived from AKAP79 77–96 ( n = 4). The effects of point mutations within the disruptor peptide were compared. d Binding of purified full-length WT, Δ79–86 or W79A AKAP79 to CaM <t>sepharose.</t> Each AKAP79 variant was purified in complex with the D/D of RIIα. AKAP79 was released from the beads by incubation with EGTA, and detected by anti-AKAP79 IB. The experiment was performed in triplicate with each replicate leading to the same pattern of bands. e Limited Ramachandran plot showing dihedral angles for both copies of AKAP79 positions 80–85 in the asymmetric unit. Black triangles represent amino acids with angles characteristic of 3 10 helices; white diamonds are amino acids with α-helical geometry. f Representation of backbone H-bonds within the AKAP79 helix with distances shown in Å. The two α-helix-type bonds are shown by dotted lines; 3 10 -helical H-bonds as striped lines. The carbonyl group of S81 that does not H-bond to a backbone group is asterisked. g Rotation of the helix through 90°. The triangular backbone geometry of positions 83–86 is such that the side-chains of W79, L83 and T86 extend in the same direction. ** P
    Glutathione Sepharose Binding Buffer, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GE Healthcare glutathione sepharose high performance media
    Residues L191 and V263 in the p85α BH domain are important for Rab5 binding. ( a ) The BH domain of human p85α with the proposed G protein binding (i.e. Rab5) residues indicated. Residues with little or no effect on Rab5 binding are shown in grey (K187, I267 [L267 in bovine p85α], M271 [L271 in bovine p85α]), with catalytically important R151 in orange. Residues important for both Rab5 binding and catalytic activity are shown in red (L191, V263, R274). Residues that help mediate BH–BH domain dimerization within the crystal structure are shown in M176 (purple) from one BH domain fitting into a hydrophobic pocket containing L161, I177 and V181 (pink) on the other BH domain 29 . ( b ) Pull-down assay with GST and GST-Rab5 mutants immobilized on glutathione <t>Sepharose</t> beads and loaded with the indicated nucleotide. Binding of purified p85α wild type or mutant protein was detected using an immunoblot analysis. The input lanes contain 0.4% of the purified p85α protein used in the pull-down assay. Full-length images of the cropped blots are in Supplementary Fig. S6 . ( c ) The ability of each p85α mutant protein to regulate Rab5 GTPase activity was determined using a Rab5 GAP assay. Mean ± SEM from three independent experiments. *** P
    Glutathione Sepharose High Performance Media, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    GE Healthcare glutathione sepharose resin
    Arg-1261 is not necessary for larger truncations of Vps15 to bind to Gpa1 (A) Detergent-solubilized extracts (Total) from cells expressing the indicated Myc fusion proteins and Gpa1 or Gpa2 fused to GST were incubated with <t>glutathione-Sepharose</t> resin, eluted with glutathione (GST PD), resolved by 10% SDS-PAGE, and analyzed by immunoblotting with antibodies against Myc, GST, Ste4 and G6PDH (Load control). (B) Detergent-solubilized extracts from cells expressing the indicated Myc fusion proteins and Gpa1 fused to GST were lysed in the presence of either GDP or GDP-AlF 4 - , incubated with glutathione-Sepharose resin, eluted with glutathione, and analyzed by immunoblotting with antibodies against Myc, GST, Ste 4, and G6PDH. PD, pulldown. GST, glutathione S-transferase. RA, Arg-1261 to Ala. RK, Arg-1261 to Lys.
    Glutathione Sepharose Resin, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 94/100, based on 63 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    GE Healthcare glutathione sepharose 4b beads
    Arg-1261 is not necessary for larger truncations of Vps15 to bind to Gpa1 (A) Detergent-solubilized extracts (Total) from cells expressing the indicated Myc fusion proteins and Gpa1 or Gpa2 fused to GST were incubated with <t>glutathione-Sepharose</t> resin, eluted with glutathione (GST PD), resolved by 10% SDS-PAGE, and analyzed by immunoblotting with antibodies against Myc, GST, Ste4 and G6PDH (Load control). (B) Detergent-solubilized extracts from cells expressing the indicated Myc fusion proteins and Gpa1 fused to GST were lysed in the presence of either GDP or GDP-AlF 4 - , incubated with glutathione-Sepharose resin, eluted with glutathione, and analyzed by immunoblotting with antibodies against Myc, GST, Ste 4, and G6PDH. PD, pulldown. GST, glutathione S-transferase. RA, Arg-1261 to Ala. RK, Arg-1261 to Lys.
    Glutathione Sepharose 4b Beads, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 91/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Crystal structure of CaM in complex with its AKAP79 binding site. a Cartoon representation showing one of the two copies (chains B and D) of AKAP79 peptide (orange) bound to CaM ( blue ) in the asymmetric unit. The C-lobe (lighter blue ) is in the open conformation with each of its two EF hands coordinating Ca 2+ (yellow). b Rotation of the complex through 90° highlighting the position of the four hydrophobic amino acids comprising the 1-4-7-8 motif. c Reduction in alphascreen signal between biotin-CaM and GST-AKAP79 (1–153) upon addition of 20-mer peptides derived from AKAP79 77–96 ( n = 4). The effects of point mutations within the disruptor peptide were compared. d Binding of purified full-length WT, Δ79–86 or W79A AKAP79 to CaM sepharose. Each AKAP79 variant was purified in complex with the D/D of RIIα. AKAP79 was released from the beads by incubation with EGTA, and detected by anti-AKAP79 IB. The experiment was performed in triplicate with each replicate leading to the same pattern of bands. e Limited Ramachandran plot showing dihedral angles for both copies of AKAP79 positions 80–85 in the asymmetric unit. Black triangles represent amino acids with angles characteristic of 3 10 helices; white diamonds are amino acids with α-helical geometry. f Representation of backbone H-bonds within the AKAP79 helix with distances shown in Å. The two α-helix-type bonds are shown by dotted lines; 3 10 -helical H-bonds as striped lines. The carbonyl group of S81 that does not H-bond to a backbone group is asterisked. g Rotation of the helix through 90°. The triangular backbone geometry of positions 83–86 is such that the side-chains of W79, L83 and T86 extend in the same direction. ** P

    Journal: Nature Communications

    Article Title: Molecular basis of AKAP79 regulation by calmodulin

    doi: 10.1038/s41467-017-01715-w

    Figure Lengend Snippet: Crystal structure of CaM in complex with its AKAP79 binding site. a Cartoon representation showing one of the two copies (chains B and D) of AKAP79 peptide (orange) bound to CaM ( blue ) in the asymmetric unit. The C-lobe (lighter blue ) is in the open conformation with each of its two EF hands coordinating Ca 2+ (yellow). b Rotation of the complex through 90° highlighting the position of the four hydrophobic amino acids comprising the 1-4-7-8 motif. c Reduction in alphascreen signal between biotin-CaM and GST-AKAP79 (1–153) upon addition of 20-mer peptides derived from AKAP79 77–96 ( n = 4). The effects of point mutations within the disruptor peptide were compared. d Binding of purified full-length WT, Δ79–86 or W79A AKAP79 to CaM sepharose. Each AKAP79 variant was purified in complex with the D/D of RIIα. AKAP79 was released from the beads by incubation with EGTA, and detected by anti-AKAP79 IB. The experiment was performed in triplicate with each replicate leading to the same pattern of bands. e Limited Ramachandran plot showing dihedral angles for both copies of AKAP79 positions 80–85 in the asymmetric unit. Black triangles represent amino acids with angles characteristic of 3 10 helices; white diamonds are amino acids with α-helical geometry. f Representation of backbone H-bonds within the AKAP79 helix with distances shown in Å. The two α-helix-type bonds are shown by dotted lines; 3 10 -helical H-bonds as striped lines. The carbonyl group of S81 that does not H-bond to a backbone group is asterisked. g Rotation of the helix through 90°. The triangular backbone geometry of positions 83–86 is such that the side-chains of W79, L83 and T86 extend in the same direction. ** P

    Article Snippet: The eluted protein was buffer exchanged into glutathione sepharose binding buffer (25 mM Tris pH 7.4, 500 mM NaCl, 2 mM DTT, 1 mM EDTA, 1 mM Benzamidine) using a Sephadex G-25 column, before 3 h incubation with glutathione sepharose 4B (GE Life Sciences).

    Techniques: Chick Chorioallantoic Membrane Assay, Binding Assay, Amplified Luminescent Proximity Homogenous Assay, Derivative Assay, Purification, Variant Assay, Incubation

    Delineation of key residues in AKAP79 required for CaM binding. a Pull-down of either WT, Δ33–48, Δ79–86, or Δ391–400 FLAG-tagged-AKAP79 (inputs shown in bottom panel) with either CaM sepharose (top panel) or cAMP agarose (middle panel). AKAP79 was detected by anti-FLAG immunoblotting. The experiment was performed in triplicate with each replicate producing the same pattern of bands. b Sequence LOGO for AKAP5 gene products aligned with predicted helical region. The cross-linking cluster between AKAP79 positions 90–99 and K94 in CaM is indicated along with the boundaries of peptides used in the following panels. c – e Determination of inhibitory constants for the peptides outlined in ( b ) in disrupting interaction between biotin-CaM and GST-AKAP79 (1–153) detected using the alphascreen assay ( n = 4 for all data points). K i constants were determined for the 9-mer and 11-mer peptides c , 16-mer and 20-mer peptides d , and for either WT or L101A 26-mer peptides e .***P

    Journal: Nature Communications

    Article Title: Molecular basis of AKAP79 regulation by calmodulin

    doi: 10.1038/s41467-017-01715-w

    Figure Lengend Snippet: Delineation of key residues in AKAP79 required for CaM binding. a Pull-down of either WT, Δ33–48, Δ79–86, or Δ391–400 FLAG-tagged-AKAP79 (inputs shown in bottom panel) with either CaM sepharose (top panel) or cAMP agarose (middle panel). AKAP79 was detected by anti-FLAG immunoblotting. The experiment was performed in triplicate with each replicate producing the same pattern of bands. b Sequence LOGO for AKAP5 gene products aligned with predicted helical region. The cross-linking cluster between AKAP79 positions 90–99 and K94 in CaM is indicated along with the boundaries of peptides used in the following panels. c – e Determination of inhibitory constants for the peptides outlined in ( b ) in disrupting interaction between biotin-CaM and GST-AKAP79 (1–153) detected using the alphascreen assay ( n = 4 for all data points). K i constants were determined for the 9-mer and 11-mer peptides c , 16-mer and 20-mer peptides d , and for either WT or L101A 26-mer peptides e .***P

    Article Snippet: The eluted protein was buffer exchanged into glutathione sepharose binding buffer (25 mM Tris pH 7.4, 500 mM NaCl, 2 mM DTT, 1 mM EDTA, 1 mM Benzamidine) using a Sephadex G-25 column, before 3 h incubation with glutathione sepharose 4B (GE Life Sciences).

    Techniques: Chick Chorioallantoic Membrane Assay, Binding Assay, Sequencing, Amplified Luminescent Proximity Homogenous Assay

    Residues L191 and V263 in the p85α BH domain are important for Rab5 binding. ( a ) The BH domain of human p85α with the proposed G protein binding (i.e. Rab5) residues indicated. Residues with little or no effect on Rab5 binding are shown in grey (K187, I267 [L267 in bovine p85α], M271 [L271 in bovine p85α]), with catalytically important R151 in orange. Residues important for both Rab5 binding and catalytic activity are shown in red (L191, V263, R274). Residues that help mediate BH–BH domain dimerization within the crystal structure are shown in M176 (purple) from one BH domain fitting into a hydrophobic pocket containing L161, I177 and V181 (pink) on the other BH domain 29 . ( b ) Pull-down assay with GST and GST-Rab5 mutants immobilized on glutathione Sepharose beads and loaded with the indicated nucleotide. Binding of purified p85α wild type or mutant protein was detected using an immunoblot analysis. The input lanes contain 0.4% of the purified p85α protein used in the pull-down assay. Full-length images of the cropped blots are in Supplementary Fig. S6 . ( c ) The ability of each p85α mutant protein to regulate Rab5 GTPase activity was determined using a Rab5 GAP assay. Mean ± SEM from three independent experiments. *** P

    Journal: Scientific Reports

    Article Title: Patient-derived mutations within the N-terminal domains of p85α impact PTEN or Rab5 binding and regulation

    doi: 10.1038/s41598-018-25487-5

    Figure Lengend Snippet: Residues L191 and V263 in the p85α BH domain are important for Rab5 binding. ( a ) The BH domain of human p85α with the proposed G protein binding (i.e. Rab5) residues indicated. Residues with little or no effect on Rab5 binding are shown in grey (K187, I267 [L267 in bovine p85α], M271 [L271 in bovine p85α]), with catalytically important R151 in orange. Residues important for both Rab5 binding and catalytic activity are shown in red (L191, V263, R274). Residues that help mediate BH–BH domain dimerization within the crystal structure are shown in M176 (purple) from one BH domain fitting into a hydrophobic pocket containing L161, I177 and V181 (pink) on the other BH domain 29 . ( b ) Pull-down assay with GST and GST-Rab5 mutants immobilized on glutathione Sepharose beads and loaded with the indicated nucleotide. Binding of purified p85α wild type or mutant protein was detected using an immunoblot analysis. The input lanes contain 0.4% of the purified p85α protein used in the pull-down assay. Full-length images of the cropped blots are in Supplementary Fig. S6 . ( c ) The ability of each p85α mutant protein to regulate Rab5 GTPase activity was determined using a Rab5 GAP assay. Mean ± SEM from three independent experiments. *** P

    Article Snippet: GST-p85α protein fragments in the supernatant were incubated with glutathione Sepharose high performance media (GE Healthcare, column bed height 8.0 cm, column bed diameter 1.6 cm, column volume 16 mL) using an ÄKTA Purifier system.

    Techniques: Binding Assay, Protein Binding, Activity Assay, Pull Down Assay, Purification, Mutagenesis, GAP Assay

    Binding and regulation of Rab5 by p85α patient-derived SH3 and BH domain mutants. ( a ) Pull-down assay with GST and GST-Rab5 mutants immobilized on glutathione Sepharose beads and loaded with the indicated nucleotide. GTPγS is a non-hydrolyzable analogue of GTP. Binding of purified p85α wild type or mutant protein was detected using an immunoblot analysis. Input of p85α is 4% of the amount used in the pull-down binding experiments. Data representative of at least 4 independent experiments. Full-length images of the cropped blots are in Supplementary Fig. S5 . ( b ) Quantification of binding assay results from panel a. Mean ± SEM. *** P

    Journal: Scientific Reports

    Article Title: Patient-derived mutations within the N-terminal domains of p85α impact PTEN or Rab5 binding and regulation

    doi: 10.1038/s41598-018-25487-5

    Figure Lengend Snippet: Binding and regulation of Rab5 by p85α patient-derived SH3 and BH domain mutants. ( a ) Pull-down assay with GST and GST-Rab5 mutants immobilized on glutathione Sepharose beads and loaded with the indicated nucleotide. GTPγS is a non-hydrolyzable analogue of GTP. Binding of purified p85α wild type or mutant protein was detected using an immunoblot analysis. Input of p85α is 4% of the amount used in the pull-down binding experiments. Data representative of at least 4 independent experiments. Full-length images of the cropped blots are in Supplementary Fig. S5 . ( b ) Quantification of binding assay results from panel a. Mean ± SEM. *** P

    Article Snippet: GST-p85α protein fragments in the supernatant were incubated with glutathione Sepharose high performance media (GE Healthcare, column bed height 8.0 cm, column bed diameter 1.6 cm, column volume 16 mL) using an ÄKTA Purifier system.

    Techniques: Binding Assay, Derivative Assay, Pull Down Assay, Purification, Mutagenesis

    Arg-1261 is not necessary for larger truncations of Vps15 to bind to Gpa1 (A) Detergent-solubilized extracts (Total) from cells expressing the indicated Myc fusion proteins and Gpa1 or Gpa2 fused to GST were incubated with glutathione-Sepharose resin, eluted with glutathione (GST PD), resolved by 10% SDS-PAGE, and analyzed by immunoblotting with antibodies against Myc, GST, Ste4 and G6PDH (Load control). (B) Detergent-solubilized extracts from cells expressing the indicated Myc fusion proteins and Gpa1 fused to GST were lysed in the presence of either GDP or GDP-AlF 4 - , incubated with glutathione-Sepharose resin, eluted with glutathione, and analyzed by immunoblotting with antibodies against Myc, GST, Ste 4, and G6PDH. PD, pulldown. GST, glutathione S-transferase. RA, Arg-1261 to Ala. RK, Arg-1261 to Lys.

    Journal: Biochemistry

    Article Title: Structure and Function of Vps15 in the Endosomal G Protein Signaling Pathway †

    doi: 10.1021/bi900621w

    Figure Lengend Snippet: Arg-1261 is not necessary for larger truncations of Vps15 to bind to Gpa1 (A) Detergent-solubilized extracts (Total) from cells expressing the indicated Myc fusion proteins and Gpa1 or Gpa2 fused to GST were incubated with glutathione-Sepharose resin, eluted with glutathione (GST PD), resolved by 10% SDS-PAGE, and analyzed by immunoblotting with antibodies against Myc, GST, Ste4 and G6PDH (Load control). (B) Detergent-solubilized extracts from cells expressing the indicated Myc fusion proteins and Gpa1 fused to GST were lysed in the presence of either GDP or GDP-AlF 4 - , incubated with glutathione-Sepharose resin, eluted with glutathione, and analyzed by immunoblotting with antibodies against Myc, GST, Ste 4, and G6PDH. PD, pulldown. GST, glutathione S-transferase. RA, Arg-1261 to Ala. RK, Arg-1261 to Lys.

    Article Snippet: For determination of nucleotide specificity of binding, either 5 mM MgCl2 and 10 μM GDP or 10 mM NaF, 5 mM MgCl2 , 10 μM GDP, and 30 μM AlCl3 (GDP-AlF4 - ) was added to buffer F. Supernatants were mixed with 40 μl of glutathione-Sepharose resin (GE Healthcare) equilibrated with buffer F. The glutathione-Sepharose resin was incubated with the lysate and washed in a manner similar to that described above for the immunoprecipitation of Flag-tagged proteins.

    Techniques: Expressing, Incubation, SDS Page

    Arg-1261 is necessary for the WD domain of Vps15 to bind efficiently to Gpa1 Detergent-solubilized extracts (Total) from cells expressing the indicated Myc fusion proteins and Gpa1 or Gpa2 fused to GST were incubated with glutathione-Sepharose resin, eluted with glutathione (GST PD, pulldown), resolved by 10% SDS-PAGE, and analyzed by immunoblotting with antibodies against GST, Myc and G6PDH (Load control). PD, pulldown. GST, glutathione S-transferase. WD, WD domain. RA, Arg-1261 to Ala. RK, Arg-1261 to Lys.

    Journal: Biochemistry

    Article Title: Structure and Function of Vps15 in the Endosomal G Protein Signaling Pathway †

    doi: 10.1021/bi900621w

    Figure Lengend Snippet: Arg-1261 is necessary for the WD domain of Vps15 to bind efficiently to Gpa1 Detergent-solubilized extracts (Total) from cells expressing the indicated Myc fusion proteins and Gpa1 or Gpa2 fused to GST were incubated with glutathione-Sepharose resin, eluted with glutathione (GST PD, pulldown), resolved by 10% SDS-PAGE, and analyzed by immunoblotting with antibodies against GST, Myc and G6PDH (Load control). PD, pulldown. GST, glutathione S-transferase. WD, WD domain. RA, Arg-1261 to Ala. RK, Arg-1261 to Lys.

    Article Snippet: For determination of nucleotide specificity of binding, either 5 mM MgCl2 and 10 μM GDP or 10 mM NaF, 5 mM MgCl2 , 10 μM GDP, and 30 μM AlCl3 (GDP-AlF4 - ) was added to buffer F. Supernatants were mixed with 40 μl of glutathione-Sepharose resin (GE Healthcare) equilibrated with buffer F. The glutathione-Sepharose resin was incubated with the lysate and washed in a manner similar to that described above for the immunoprecipitation of Flag-tagged proteins.

    Techniques: Expressing, Incubation, SDS Page