gst exo84 protein  (Echelon Biosciences)


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    Echelon Biosciences gst exo84 protein
    Gst Exo84 Protein, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    gst exo84 protein  (Echelon Biosciences)


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    Echelon Biosciences gst exo84 protein
    Domain organization and Cdk1 target sites of Sc <t>Exo84</t> and Ca Exo84. (A) Domain organization and Cdk1 target sites. The VPS51 domain was identified in both CaExo84 and ScExo84 using the Pfam database ( Punta et al. , 2012 ). The PH domain and the C-terminal interaction domain were identified by threading as described in Materials and Methods . Studied sites refer to this article for Ca Exo84 and Luo et al. (2013) for Sc Exo84. The folding structure of the PH domain predicted by threading is shown in purple. Phosphorylation of potential Cdk1 target sites is shown by the yellow spheres. S284 lies within a protruding loop of the PH domain, and S256 and T488 are located at the boundaries of the PH domain. (B) Clustal alignments of sequences surrounding full Cdk1 sites in Ca Exo84. Color code follows standard Clustal X code: amino acids with similar properties are coded with the same color; the depth of the color denotes the degree of similarity, so that identical residues have the deepest color.
    Gst Exo84 Protein, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gst exo84 protein/product/Echelon Biosciences
    Average 86 stars, based on 1 article reviews
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    gst exo84 protein - by Bioz Stars, 2023-01
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    1) Product Images from "In Candida albicans , phosphorylation of Exo84 by Cdk1-Hgc1 is necessary for efficient hyphal extension"

    Article Title: In Candida albicans , phosphorylation of Exo84 by Cdk1-Hgc1 is necessary for efficient hyphal extension

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E13-11-0688

    Domain organization and Cdk1 target sites of Sc Exo84 and Ca Exo84. (A) Domain organization and Cdk1 target sites. The VPS51 domain was identified in both CaExo84 and ScExo84 using the Pfam database ( Punta et al. , 2012 ). The PH domain and the C-terminal interaction domain were identified by threading as described in Materials and Methods . Studied sites refer to this article for Ca Exo84 and Luo et al. (2013) for Sc Exo84. The folding structure of the PH domain predicted by threading is shown in purple. Phosphorylation of potential Cdk1 target sites is shown by the yellow spheres. S284 lies within a protruding loop of the PH domain, and S256 and T488 are located at the boundaries of the PH domain. (B) Clustal alignments of sequences surrounding full Cdk1 sites in Ca Exo84. Color code follows standard Clustal X code: amino acids with similar properties are coded with the same color; the depth of the color denotes the degree of similarity, so that identical residues have the deepest color.
    Figure Legend Snippet: Domain organization and Cdk1 target sites of Sc Exo84 and Ca Exo84. (A) Domain organization and Cdk1 target sites. The VPS51 domain was identified in both CaExo84 and ScExo84 using the Pfam database ( Punta et al. , 2012 ). The PH domain and the C-terminal interaction domain were identified by threading as described in Materials and Methods . Studied sites refer to this article for Ca Exo84 and Luo et al. (2013) for Sc Exo84. The folding structure of the PH domain predicted by threading is shown in purple. Phosphorylation of potential Cdk1 target sites is shown by the yellow spheres. S284 lies within a protruding loop of the PH domain, and S256 and T488 are located at the boundaries of the PH domain. (B) Clustal alignments of sequences surrounding full Cdk1 sites in Ca Exo84. Color code follows standard Clustal X code: amino acids with similar properties are coded with the same color; the depth of the color denotes the degree of similarity, so that identical residues have the deepest color.

    Techniques Used:

    Exo84 is phosphorylated by Cdk1. (A) Western blot using anti-GFP antisera of Exo84-YFP from yeast and hyphae as indicated. Numbers indicate time in minutes since unbudded stationary-phase cells were induced to form hyphae. λPP, λ-phosphatase–treated sample. (B) 2D gels of Exo84-YFP from lysates of wild-type (WT) exo 84 mutant strains as indicated and Δhgc 1 strains from WT unbudded stationary-phase cells (WT H 0 min) and from hyphae induced from WT cells after 30, 60, and 90 min and Δhgc 1 after 30 and 90 min as indicated. The stationary-phase cells were also reinoculated into yeast growth medium and lysates prepared after 3 h when the cells were in exponential growth (WT Yeast). (C) Exo84 extracted from Δhgc 1 cells is less phosphorylated, shown by the absence of the slower-migrating smear present in Exo84 extracted from wild-type cells. (D) Exo84 is still phosphorylated in cells depleted of Clb2 and Clb4. (E) Phosphorylation of Exo84 is inhibited by 1NM-PP1 when the strain carries the cdk 1 - 1 as allele. (F) Recombinant GST-Exo84 but not recombinant GST-Exo84-3A is phosphorylated in vitro by Cdk1. The indicated recombinant forms of GST-Exo-84 or GST alone were incubated in kinase reaction buffer with immunoprecipitated Cdk1-HA. The products were analyzed on a Western blot using the anti–pS-Cdk1 antibody. (G) The in vitro kinase reaction is inhibited by 1NM PP1 when the purified kinase carries the analogue-sensitive cdk1-1as allele.
    Figure Legend Snippet: Exo84 is phosphorylated by Cdk1. (A) Western blot using anti-GFP antisera of Exo84-YFP from yeast and hyphae as indicated. Numbers indicate time in minutes since unbudded stationary-phase cells were induced to form hyphae. λPP, λ-phosphatase–treated sample. (B) 2D gels of Exo84-YFP from lysates of wild-type (WT) exo 84 mutant strains as indicated and Δhgc 1 strains from WT unbudded stationary-phase cells (WT H 0 min) and from hyphae induced from WT cells after 30, 60, and 90 min and Δhgc 1 after 30 and 90 min as indicated. The stationary-phase cells were also reinoculated into yeast growth medium and lysates prepared after 3 h when the cells were in exponential growth (WT Yeast). (C) Exo84 extracted from Δhgc 1 cells is less phosphorylated, shown by the absence of the slower-migrating smear present in Exo84 extracted from wild-type cells. (D) Exo84 is still phosphorylated in cells depleted of Clb2 and Clb4. (E) Phosphorylation of Exo84 is inhibited by 1NM-PP1 when the strain carries the cdk 1 - 1 as allele. (F) Recombinant GST-Exo84 but not recombinant GST-Exo84-3A is phosphorylated in vitro by Cdk1. The indicated recombinant forms of GST-Exo-84 or GST alone were incubated in kinase reaction buffer with immunoprecipitated Cdk1-HA. The products were analyzed on a Western blot using the anti–pS-Cdk1 antibody. (G) The in vitro kinase reaction is inhibited by 1NM PP1 when the purified kinase carries the analogue-sensitive cdk1-1as allele.

    Techniques Used: Western Blot, Mutagenesis, Recombinant, In Vitro, Incubation, Immunoprecipitation, Purification

    Phosphorylation of Exo84-YFP detected by the anti–pS-Cdk1 antibody is reduced in Exo84-2A or in cells lacking Hgc1. (A) Membranes were stripped and reprobed with an anti-GFP antibody as a loading control. (B) Ratios of the wild-type to mutant intensities of the anti–pS-Cdk1 signals normalized by the GFP loading controls.
    Figure Legend Snippet: Phosphorylation of Exo84-YFP detected by the anti–pS-Cdk1 antibody is reduced in Exo84-2A or in cells lacking Hgc1. (A) Membranes were stripped and reprobed with an anti-GFP antibody as a loading control. (B) Ratios of the wild-type to mutant intensities of the anti–pS-Cdk1 signals normalized by the GFP loading controls.

    Techniques Used: Mutagenesis

    Effect of Exo84 phosphoacceptor-site mutations on the localization of Exo84-YFP in hyphal cells. Individual cells of the indicated strains were grown on agar pads containing minimal medium as described previously ( Bishop et al. , 2010 ), and at 3-min intervals each cell was examined to determine whether Exo84-YFP had located to the septum. (A) Appearance of cells where Exo84 is present at the tip and septum. The image is a merge image of the YFP fluorescence and the differential interference contrast (DIC) images, where the contrast of the DIC images has been increased to generate a cell outline. (B) The percentage of cells showing Exo84 at the septum plotted against time after hyphal induction. At least 10 cells were examined simultaneously for each strain. (C) Cumulative total of cells where Exo84 appeared at the septum after 150 min. (D) Length of the hypha when Exo84 first appeared at the septum for each strain (horizontal hatch) and the distance of the septum from the bud neck (hatched bars). The relevance of the Δ cho1 mutant is described in the section Phosphorylation of Exo84 may affect phospholipid binding .
    Figure Legend Snippet: Effect of Exo84 phosphoacceptor-site mutations on the localization of Exo84-YFP in hyphal cells. Individual cells of the indicated strains were grown on agar pads containing minimal medium as described previously ( Bishop et al. , 2010 ), and at 3-min intervals each cell was examined to determine whether Exo84-YFP had located to the septum. (A) Appearance of cells where Exo84 is present at the tip and septum. The image is a merge image of the YFP fluorescence and the differential interference contrast (DIC) images, where the contrast of the DIC images has been increased to generate a cell outline. (B) The percentage of cells showing Exo84 at the septum plotted against time after hyphal induction. At least 10 cells were examined simultaneously for each strain. (C) Cumulative total of cells where Exo84 appeared at the septum after 150 min. (D) Length of the hypha when Exo84 first appeared at the septum for each strain (horizontal hatch) and the distance of the septum from the bud neck (hatched bars). The relevance of the Δ cho1 mutant is described in the section Phosphorylation of Exo84 may affect phospholipid binding .

    Techniques Used: Fluorescence, Mutagenesis, Binding Assay

    Exo84 phospho-site mutations do not disrupt the association with Sec10. (A) Reciprocal immunoprecipitation of Exo84-YFP with Sec10-HA. (B) Immunoprecipitation of Sec10-HA with Exo84-YFP carrying the indicated mutations. Controls in A show that the anti-GFP and anti-HA antibodies do not show nonspecific interactions.
    Figure Legend Snippet: Exo84 phospho-site mutations do not disrupt the association with Sec10. (A) Reciprocal immunoprecipitation of Exo84-YFP with Sec10-HA. (B) Immunoprecipitation of Sec10-HA with Exo84-YFP carrying the indicated mutations. Controls in A show that the anti-GFP and anti-HA antibodies do not show nonspecific interactions.

    Techniques Used: Immunoprecipitation

    Exo84 interacts with phosphatidylserine. (A) Binding of Exo84 to phospholipids. We incubated 2 μg of recombinant GST-Exo84 and 8 μg of GST-Exo84-3E with the phospholipid test strip. The phospholipids on the PIP strip are organized in two columns. Left column is labeled to the left of the Exo84 strip, and right column is labeled to the right of the Exo84-3E strip. LPA, lysophosphatidic acid; LPC, lysophosphocholine; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; PtpIns, phosphoinositol; SIP, sphingosine-1-phosphate. (B) Profile of Exo84 and Exo84-3E binding to PA and PS on a PIP strip with the background subtracted. Units on the ordinate are arbitrary units. (C) Exo84-YFP and Sec3-YFP distribution in heterozygous CHO 1 /Δcho 1 and Δcho yeast and hyphal cells as indicated. Three-dimensional quantitation of fluorescence intensity is presented alongside each fluorescence image, which has been color inverted for clarity.
    Figure Legend Snippet: Exo84 interacts with phosphatidylserine. (A) Binding of Exo84 to phospholipids. We incubated 2 μg of recombinant GST-Exo84 and 8 μg of GST-Exo84-3E with the phospholipid test strip. The phospholipids on the PIP strip are organized in two columns. Left column is labeled to the left of the Exo84 strip, and right column is labeled to the right of the Exo84-3E strip. LPA, lysophosphatidic acid; LPC, lysophosphocholine; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; PtpIns, phosphoinositol; SIP, sphingosine-1-phosphate. (B) Profile of Exo84 and Exo84-3E binding to PA and PS on a PIP strip with the background subtracted. Units on the ordinate are arbitrary units. (C) Exo84-YFP and Sec3-YFP distribution in heterozygous CHO 1 /Δcho 1 and Δcho yeast and hyphal cells as indicated. Three-dimensional quantitation of fluorescence intensity is presented alongside each fluorescence image, which has been color inverted for clarity.

    Techniques Used: Binding Assay, Incubation, Recombinant, Stripping Membranes, Labeling, Quantitation Assay, Fluorescence

    Nonphosphorylatable substitutions reduce Exo84-YFP recovery after photobleaching. The indicated strains were grown as hyphae on agar pads in a DeltaVision microscope (TA, Exo84-EXO84 T488A). The tips were bleached using a laser. Images were recorded prebleach, postbleach, and postbleach plus 30 s. Exo84-YFP fluorescence was quantified as the fractional area of the tip above background fluorescence intensity. Recovery was defined as ( I 30 − I b )/( I 0 − I b ) × 100%, where I 0 , I b , and I 30 are the values of Exo84-YFP fluorescence prebleach, postbleach, and 30-s postbleach, respectively. ns, nonsignificant ( p > 0.05).
    Figure Legend Snippet: Nonphosphorylatable substitutions reduce Exo84-YFP recovery after photobleaching. The indicated strains were grown as hyphae on agar pads in a DeltaVision microscope (TA, Exo84-EXO84 T488A). The tips were bleached using a laser. Images were recorded prebleach, postbleach, and postbleach plus 30 s. Exo84-YFP fluorescence was quantified as the fractional area of the tip above background fluorescence intensity. Recovery was defined as ( I 30 − I b )/( I 0 − I b ) × 100%, where I 0 , I b , and I 30 are the values of Exo84-YFP fluorescence prebleach, postbleach, and 30-s postbleach, respectively. ns, nonsignificant ( p > 0.05).

    Techniques Used: Microscopy, Fluorescence

    gst fusion protein  (Echelon Biosciences)


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    Gst Fusion Protein, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Echelon Biosciences gst fusion protein
    Gst Fusion Protein, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Gst Fusion Protein, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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