α-galactosidase Search Results


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  • 94
    Millipore α galactosidase
    Treatment of CR-WT and CR-GLA cells with recombinant human <t>α–galactosidase</t> A and eliglustat A. CR-WT and CR-GLA cells were seeded in a 24-well plate. Next day, vehicle (25 mM Tris and 150 mM NaCl, pH 7.5) or recombinant human GLA (10 μg/mL, α-Gal A) was added to each well. After 2 days of the treatment, the medium was replaced with 2% serum medium containing vehicle or α-Gal A and incubated for 3 days. VWF levels were determined by ELISA and expressed as fold changes with respect to CR-WT vehicle (n=6–10 per group). p=0.13 between vehicle- and α-Gal A-treated CR-GLA; *p
    α Galactosidase, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 183 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    ProZyme α galactosidase
    Terminal α-galactose determined in complex type N-glycans of B7-H3 from Ca9-22. ( A ) The glycoforms of B7H3 with or without <t>α-galactosidase</t> treatment. To identify the presence of terminal α-galactose, we treated the full-length B7-H3 protein derived from Ca9-22 with α-galactosidase, which cleaves terminal α1,3/4/6-linked galactose. The result showed that after treatment with α-galactosidase, all of the additional terminal galactose(s) (yellow circles) speculated to appear on specified complex-type N-glycans were removed. The blue frames show the representative glycan structures before α-galactosidase treatment and red frames show the representative glycan structures after α-galactosidase treatment. ( B ) Comparison of the glycoforms with extra hexose(s) before and after α-galactosidase treatment. The percentages of glycoforms were calculated according to A .
    α Galactosidase, supplied by ProZyme, used in various techniques. Bioz Stars score: 90/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    TaKaRa x α galactosidase
    GTF2IRD1 binds to the E3 SUMO ligase PIASxβ and the E2 SUMO conjugating enzyme UBC9 in yeast assays. (A) Schematic diagram of human GTF2IRD1 protein structure and its various domains. The peptide regions used to map protein binding sites are indicated by the annotated thick lines above and the C-terminal truncations (TR1–3) are indicated below. The domains (from left to right) include a leucine zipper (LZ, purple), the repeat domains (RD1–5, gold), SUMO attachment sites (SUMO1 2, black), nuclear localization signal (blue), polyserine tract (green) and the conserved C-terminal domain (maroon). (B) Yeast 2-hybrid assays confirming and mapping the GTF2IRD1 interaction with PIASxβ. Double transformations were performed using pGADT7-PIASxβ plus the control empty pGBKT7 bait plasmid (CTR), pGBKT7-GTF2IRD1 full-length (FL), or the peptide regions indicated in A or the C-terminal truncation fragments (TR1–3). Positive interactions are indicated by survival on QDO plates and blue <t>α-galactosidase</t> staining. Survival on DDO plates was tested for all double transformants to ensure the presence of bait and prey plasmids in the yeast host (C) A similar assay testing the binding of GTF2IRD1 to UBC9 and mapping the interaction sites.
    X α Galactosidase, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore α galactosidase b
    Genetic organization of <t>α-galactosidase</t> genes in LAB. The genes are indicated by polygons of the same size regardless of their length. White, carbohydrate degradation enzymes; black, sugar transporters; gray, transcriptional regulators. The following genes code for the indicated proteins: aga , melA α-galactosidase; galK , galactokinase; galT , galactose 1-phosphate uridylyltransferase; galM , mutarotase; bgaB , bgaC , lacL , and lacM β-galactosidase; scrB , sucrose phosphorylase; scrK , fructokinase; gftA , sucrose 6-phosphate hydrolase; dexB , dextran glucosidase; galR , scrR , rafR , rafS , msmR , and orf2 transcriptional regulators; orf3 and orf4 , putative PTS EIIB and EIIC domains; scrA , PTS EII Suc ; rafP (also named lacS2 in Lactobacillus plantarum WCFS1), galactoside-pentose-hexuronide transporter; msmEFGK and rafEFG , ABC transporters; rafX , unknown. GB#, GenBank accession number.
    α Galactosidase B, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    ProZyme coffee bean α galactosidase
    Genetic organization of <t>α-galactosidase</t> genes in LAB. The genes are indicated by polygons of the same size regardless of their length. White, carbohydrate degradation enzymes; black, sugar transporters; gray, transcriptional regulators. The following genes code for the indicated proteins: aga , melA α-galactosidase; galK , galactokinase; galT , galactose 1-phosphate uridylyltransferase; galM , mutarotase; bgaB , bgaC , lacL , and lacM β-galactosidase; scrB , sucrose phosphorylase; scrK , fructokinase; gftA , sucrose 6-phosphate hydrolase; dexB , dextran glucosidase; galR , scrR , rafR , rafS , msmR , and orf2 transcriptional regulators; orf3 and orf4 , putative PTS EIIB and EIIC domains; scrA , PTS EII Suc ; rafP (also named lacS2 in Lactobacillus plantarum WCFS1), galactoside-pentose-hexuronide transporter; msmEFGK and rafEFG , ABC transporters; rafX , unknown. GB#, GenBank accession number.
    Coffee Bean α Galactosidase, supplied by ProZyme, used in various techniques. Bioz Stars score: 90/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Megazyme guar α galactosidase
    Genetic organization of <t>α-galactosidase</t> genes in LAB. The genes are indicated by polygons of the same size regardless of their length. White, carbohydrate degradation enzymes; black, sugar transporters; gray, transcriptional regulators. The following genes code for the indicated proteins: aga , melA α-galactosidase; galK , galactokinase; galT , galactose 1-phosphate uridylyltransferase; galM , mutarotase; bgaB , bgaC , lacL , and lacM β-galactosidase; scrB , sucrose phosphorylase; scrK , fructokinase; gftA , sucrose 6-phosphate hydrolase; dexB , dextran glucosidase; galR , scrR , rafR , rafS , msmR , and orf2 transcriptional regulators; orf3 and orf4 , putative PTS EIIB and EIIC domains; scrA , PTS EII Suc ; rafP (also named lacS2 in Lactobacillus plantarum WCFS1), galactoside-pentose-hexuronide transporter; msmEFGK and rafEFG , ABC transporters; rafX , unknown. GB#, GenBank accession number.
    Guar α Galactosidase, supplied by Megazyme, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Shire Plc recombinant α galactosidase a
    Effects of EtDO-P4 and α-galactosidase A (α-Gal A) treatments on CAMs expression
    Recombinant α Galactosidase A, supplied by Shire Plc, used in various techniques. Bioz Stars score: 88/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Valiant α galactosidase
    Effects of EtDO-P4 and α-galactosidase A (α-Gal A) treatments on CAMs expression
    α Galactosidase, supplied by Valiant, used in various techniques. Bioz Stars score: 90/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    TaKaRa α galactosidase staining
    Effects of EtDO-P4 and α-galactosidase A (α-Gal A) treatments on CAMs expression
    α Galactosidase Staining, supplied by TaKaRa, 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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    90
    Boehringer Mannheim α galactosidase
    Presence of Galα1-3Gal in immune complexes in the blood of baboons, after pulmonary xenotransplantation. Immune complexes in the serum taken from baboon blood after a pulmonary xenotransplantation were diluted 5:1 with 12% PEG 8000 and 60 mmol/L EDTA in VBS for 16 hours at 4°C to precipitate immune complexes. The solution was centrifuged at 2,000 × g for 20 minutes at 4°C to pellet immune complexes. Precipitates were resuspended in digestion buffer (100 mmol/L NaCl and 50 mmol/L sodium acetate). Samples were incubated with (+) or without (−) <t>α-galactosidase</t> (1 U/ml) for 5 hours at 37°C. Proteins were precipitated with ethanol, reduced, separated by SDS-PAGE (7.5% PA), and transferred to PVDF. The blots were reacted with human serum as a source of xenoreactive IgM and then with anti-human IgM. Lane 1 , α-galactosidase digested proteins targeted by human IgM, Lane 2 , control proteins targeted by human IgM. Digestion of samples with α-galactosidase abrogates the binding of human xenoreactive IgM.
    α Galactosidase, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 90/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Megazyme α galactosidase
    A, HPLC analysis of A. thaliana and C. reinhardtii peptidyl galactosyltransferase activity. P100 fractions were prepared from A. thaliana cells (Col-0), C. reinhardtii CC-125 cells, and C. reinhardtii cell wall-less mutant CC-503 cells. The P100 fraction (100 μg for A. thaliana or 10 μg for C. reinhardtii ) was incubated with the assay mixture described under “Experimental Procedures” containing AtEXT peptide as an acceptor at 30 °C for 10 h ( A. thaliana ) or 3 h ( C. reinhardtii ) and then analyzed by HPLC. The acceptor peptide (AtEXT) was eluted at 18.8 min, and three enzymatic reaction products were detected, indicated as products 1, 2, and 3, at 15.2, 16.1, and 13.7 min, respectively (these are indicated with vertical broken lines in A–D ). B–D, HPLC analysis of products after α- and β-galactosidase ( GSD ) treatment. B shows product 1 before and after galactosidase treatment, and C shows product 2 before and after treatment. D shows product 3 before and after treatment. α→β- GSD indicates product 3 treated with β- GSD after <t>α-galactosidase</t> treatment. β→α- GSD indicates product 3 treated with α-galactosidase after β-galactosidase treatment. The sequence of AtEXT peptide was FITC-Ahx-VYKSOOOOV-NH 2 . O indicates hydroxyproline. E, P100 fraction (10 μg) of C. reinhardtii CC-503 cells was incubated in an assay mixture containing VYKAOOOOV peptide as an acceptor at 30 °C for 3 h and then analyzed by HPLC. The product was collected and treated with β-galactosidase. F, ), and analyzed by HPLC using a TSKgel sugar AXI column. PA-galactose was detected at 97 min, whereas PA-glucose and PA-mannose were detected at 55 and 62 min, respectively (data not shown).
    α Galactosidase, supplied by Megazyme, used in various techniques. Bioz Stars score: 90/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Genecopoeia α galactosidase gla genes
    Targeted integration of the human coagulation factor IX and <t>α-galactosidase</t> genes by enhanced ZFRs. (a) Bulk PCR analysis of HEK293 cells transfected with eZFRs targeting human chromosome 4 and donor plasmids harboring either the human coagulation factor IX (FIX) or α-galactosidase genes <t>(GLA).</t> Integration was evaluated in the forward and reverse orientations. GAPDH indicates PCR control. DO indicates donor only (no eZFRs). Genome-wide integration rates indicated beneath each lane. (b) Clonal analysis of puromycin-resistant cells transfected with eZFRs and donor plasmids containing the FIX or GLA genes.
    α Galactosidase Gla Genes, supplied by Genecopoeia, used in various techniques. Bioz Stars score: 89/100, based on 63 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Amano Enzyme Inc α galactosidase
    Targeted integration of the human coagulation factor IX and <t>α-galactosidase</t> genes by enhanced ZFRs. (a) Bulk PCR analysis of HEK293 cells transfected with eZFRs targeting human chromosome 4 and donor plasmids harboring either the human coagulation factor IX (FIX) or α-galactosidase genes <t>(GLA).</t> Integration was evaluated in the forward and reverse orientations. GAPDH indicates PCR control. DO indicates donor only (no eZFRs). Genome-wide integration rates indicated beneath each lane. (b) Clonal analysis of puromycin-resistant cells transfected with eZFRs and donor plasmids containing the FIX or GLA genes.
    α Galactosidase, supplied by Amano Enzyme Inc, used in various techniques. Bioz Stars score: 93/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Genzyme α galactosidase
    Targeted integration of the human coagulation factor IX and <t>α-galactosidase</t> genes by enhanced ZFRs. (a) Bulk PCR analysis of HEK293 cells transfected with eZFRs targeting human chromosome 4 and donor plasmids harboring either the human coagulation factor IX (FIX) or α-galactosidase genes <t>(GLA).</t> Integration was evaluated in the forward and reverse orientations. GAPDH indicates PCR control. DO indicates donor only (no eZFRs). Genome-wide integration rates indicated beneath each lane. (b) Clonal analysis of puromycin-resistant cells transfected with eZFRs and donor plasmids containing the FIX or GLA genes.
    α Galactosidase, supplied by Genzyme, used in various techniques. Bioz Stars score: 90/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Genzyme recombinant α galactosidase gla
    (A) Schematic representation of processing of human <t>α‐galactosidase</t> <t>GLA</t> by GH99 endomannosidase. GLA is pre‐labeled by fluorescent TB340, and contains high‐mannose N‐glycans which can be truncated by endomannosidase, resulting in a decrease in GLA molecular weight. Activity‐based labeling of endomannosidase by spiro‐epoxyglycosides 4 or 5 (prior to incubation with GLA) blocks its activity, and is therefore unable to process GLA. (B) Bt GH99 wild‐type demannosylates GLA, causing a shift in molecular weight for the protein bands. Pre‐labeling Bt GH99 wild‐type with 4 or 5 abrogates GLA demannosylation. Endo‐H cleaves high‐mannose structures, PNGase‐F cleaves full N‐linked glycan (leaving Asp‐GlcNAc). (C) Bx GH99 wild‐type demannosylates GLA, while Bx GH99 pre‐labeled with 4 or 5 is unable to do so. Bx GH99 active‐site mutants E333Q and E3336Q are unable to process GLA. (D) Fluorescent labeling of Bt GH99 (top) and Bx GH99 (bottom) by 4 or 5 competed by different concentrations of 17 , 18 , ManIFG ( 2 ) and yeast mannan. The marker is annotated with an asterisk (*).
    Recombinant α Galactosidase Gla, supplied by Genzyme, used in various techniques. Bioz Stars score: 92/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Shire Plc α galactosidase a
    (A) Schematic representation of processing of human <t>α‐galactosidase</t> <t>GLA</t> by GH99 endomannosidase. GLA is pre‐labeled by fluorescent TB340, and contains high‐mannose N‐glycans which can be truncated by endomannosidase, resulting in a decrease in GLA molecular weight. Activity‐based labeling of endomannosidase by spiro‐epoxyglycosides 4 or 5 (prior to incubation with GLA) blocks its activity, and is therefore unable to process GLA. (B) Bt GH99 wild‐type demannosylates GLA, causing a shift in molecular weight for the protein bands. Pre‐labeling Bt GH99 wild‐type with 4 or 5 abrogates GLA demannosylation. Endo‐H cleaves high‐mannose structures, PNGase‐F cleaves full N‐linked glycan (leaving Asp‐GlcNAc). (C) Bx GH99 wild‐type demannosylates GLA, while Bx GH99 pre‐labeled with 4 or 5 is unable to do so. Bx GH99 active‐site mutants E333Q and E3336Q are unable to process GLA. (D) Fluorescent labeling of Bt GH99 (top) and Bx GH99 (bottom) by 4 or 5 competed by different concentrations of 17 , 18 , ManIFG ( 2 ) and yeast mannan. The marker is annotated with an asterisk (*).
    α Galactosidase A, supplied by Shire Plc, used in various techniques. Bioz Stars score: 88/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Treatment of CR-WT and CR-GLA cells with recombinant human α–galactosidase A and eliglustat A. CR-WT and CR-GLA cells were seeded in a 24-well plate. Next day, vehicle (25 mM Tris and 150 mM NaCl, pH 7.5) or recombinant human GLA (10 μg/mL, α-Gal A) was added to each well. After 2 days of the treatment, the medium was replaced with 2% serum medium containing vehicle or α-Gal A and incubated for 3 days. VWF levels were determined by ELISA and expressed as fold changes with respect to CR-WT vehicle (n=6–10 per group). p=0.13 between vehicle- and α-Gal A-treated CR-GLA; *p

    Journal: Kidney international

    Article Title: α-galactosidase A deficiency promotes von Willebrand factor secretion in models of Fabry disease

    doi: 10.1016/j.kint.2018.08.033

    Figure Lengend Snippet: Treatment of CR-WT and CR-GLA cells with recombinant human α–galactosidase A and eliglustat A. CR-WT and CR-GLA cells were seeded in a 24-well plate. Next day, vehicle (25 mM Tris and 150 mM NaCl, pH 7.5) or recombinant human GLA (10 μg/mL, α-Gal A) was added to each well. After 2 days of the treatment, the medium was replaced with 2% serum medium containing vehicle or α-Gal A and incubated for 3 days. VWF levels were determined by ELISA and expressed as fold changes with respect to CR-WT vehicle (n=6–10 per group). p=0.13 between vehicle- and α-Gal A-treated CR-GLA; *p

    Article Snippet: GLA activity in the cell lysates were calculated using α-galactosidase (Sigma) as reference, which hydrolyzes 1.0 μmol of substrate to p -nitrophenol and D-galactose per minute.

    Techniques: Recombinant, Incubation, Enzyme-linked Immunosorbent Assay

    (A) Gal expression on untreated and α-galactosidase-treated WT porcine aortic endothelial cells (pAECs) was measured by flow cytometry. The mean fluorescence (MFI) was reduced from 670 (untreated) by 28% to 480 by a treatment of 4 μl/10 6 cells α-galactosidase and by 44% to 373 at 8 μl/10 6 cells. For reference, the average MFI from all GTKO pAECs was five (not shown), and the MFI of unstained cells was four. (B) Human peripheral blood mononuclear cell (PBMC) proliferation was reduced following treatments of WT pAECs with α-galactosidase. In MLR, WT pAECs treated with α-galactosidase at 4 and 8 μl/10 6 reduced Gal expression by 28 and 44% and stimulated 61 and 77% less human PBMC proliferation, respectively, than those untreated. PAEC viability was confirmed before MLR and equal numbers of stimulators and responders were used in each study. 3 H incorporation values are presented as counts per minute. Data represent the mean plus or minus standard error of the mean (±SEM) and are representative of three different experiments.

    Journal: Xenotransplantation

    Article Title: The effect of Gal expression on pig cells on the human T-cell xenoresponse

    doi: 10.1111/j.1399-3089.2011.00691.x

    Figure Lengend Snippet: (A) Gal expression on untreated and α-galactosidase-treated WT porcine aortic endothelial cells (pAECs) was measured by flow cytometry. The mean fluorescence (MFI) was reduced from 670 (untreated) by 28% to 480 by a treatment of 4 μl/10 6 cells α-galactosidase and by 44% to 373 at 8 μl/10 6 cells. For reference, the average MFI from all GTKO pAECs was five (not shown), and the MFI of unstained cells was four. (B) Human peripheral blood mononuclear cell (PBMC) proliferation was reduced following treatments of WT pAECs with α-galactosidase. In MLR, WT pAECs treated with α-galactosidase at 4 and 8 μl/10 6 reduced Gal expression by 28 and 44% and stimulated 61 and 77% less human PBMC proliferation, respectively, than those untreated. PAEC viability was confirmed before MLR and equal numbers of stimulators and responders were used in each study. 3 H incorporation values are presented as counts per minute. Data represent the mean plus or minus standard error of the mean (±SEM) and are representative of three different experiments.

    Article Snippet: Gal expression on WT pAECs was reduced by incubation with green coffee bean α-galactosidase (Sigma-Aldrich).

    Techniques: Expressing, Flow Cytometry, Cytometry, Fluorescence

    Terminal α-galactose determined in complex type N-glycans of B7-H3 from Ca9-22. ( A ) The glycoforms of B7H3 with or without α-galactosidase treatment. To identify the presence of terminal α-galactose, we treated the full-length B7-H3 protein derived from Ca9-22 with α-galactosidase, which cleaves terminal α1,3/4/6-linked galactose. The result showed that after treatment with α-galactosidase, all of the additional terminal galactose(s) (yellow circles) speculated to appear on specified complex-type N-glycans were removed. The blue frames show the representative glycan structures before α-galactosidase treatment and red frames show the representative glycan structures after α-galactosidase treatment. ( B ) Comparison of the glycoforms with extra hexose(s) before and after α-galactosidase treatment. The percentages of glycoforms were calculated according to A .

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

    Article Title: Glycoprotein B7-H3 overexpression and aberrant glycosylation in oral cancer and immune response

    doi: 10.1073/pnas.1516991112

    Figure Lengend Snippet: Terminal α-galactose determined in complex type N-glycans of B7-H3 from Ca9-22. ( A ) The glycoforms of B7H3 with or without α-galactosidase treatment. To identify the presence of terminal α-galactose, we treated the full-length B7-H3 protein derived from Ca9-22 with α-galactosidase, which cleaves terminal α1,3/4/6-linked galactose. The result showed that after treatment with α-galactosidase, all of the additional terminal galactose(s) (yellow circles) speculated to appear on specified complex-type N-glycans were removed. The blue frames show the representative glycan structures before α-galactosidase treatment and red frames show the representative glycan structures after α-galactosidase treatment. ( B ) Comparison of the glycoforms with extra hexose(s) before and after α-galactosidase treatment. The percentages of glycoforms were calculated according to A .

    Article Snippet: To detect the presence of terminal α-galactose, full-length B7-H3 glycopeptide samples were treated with α-galactosidase (Prozyme) at 37 °C overnight in a 10-μL reaction under the conditions suggested by the vender.

    Techniques: Derivative Assay

    GTF2IRD1 binds to the E3 SUMO ligase PIASxβ and the E2 SUMO conjugating enzyme UBC9 in yeast assays. (A) Schematic diagram of human GTF2IRD1 protein structure and its various domains. The peptide regions used to map protein binding sites are indicated by the annotated thick lines above and the C-terminal truncations (TR1–3) are indicated below. The domains (from left to right) include a leucine zipper (LZ, purple), the repeat domains (RD1–5, gold), SUMO attachment sites (SUMO1 2, black), nuclear localization signal (blue), polyserine tract (green) and the conserved C-terminal domain (maroon). (B) Yeast 2-hybrid assays confirming and mapping the GTF2IRD1 interaction with PIASxβ. Double transformations were performed using pGADT7-PIASxβ plus the control empty pGBKT7 bait plasmid (CTR), pGBKT7-GTF2IRD1 full-length (FL), or the peptide regions indicated in A or the C-terminal truncation fragments (TR1–3). Positive interactions are indicated by survival on QDO plates and blue α-galactosidase staining. Survival on DDO plates was tested for all double transformants to ensure the presence of bait and prey plasmids in the yeast host (C) A similar assay testing the binding of GTF2IRD1 to UBC9 and mapping the interaction sites.

    Journal: PLoS ONE

    Article Title: SUMOylation of GTF2IRD1 Regulates Protein Partner Interactions and Ubiquitin-Mediated Degradation

    doi: 10.1371/journal.pone.0049283

    Figure Lengend Snippet: GTF2IRD1 binds to the E3 SUMO ligase PIASxβ and the E2 SUMO conjugating enzyme UBC9 in yeast assays. (A) Schematic diagram of human GTF2IRD1 protein structure and its various domains. The peptide regions used to map protein binding sites are indicated by the annotated thick lines above and the C-terminal truncations (TR1–3) are indicated below. The domains (from left to right) include a leucine zipper (LZ, purple), the repeat domains (RD1–5, gold), SUMO attachment sites (SUMO1 2, black), nuclear localization signal (blue), polyserine tract (green) and the conserved C-terminal domain (maroon). (B) Yeast 2-hybrid assays confirming and mapping the GTF2IRD1 interaction with PIASxβ. Double transformations were performed using pGADT7-PIASxβ plus the control empty pGBKT7 bait plasmid (CTR), pGBKT7-GTF2IRD1 full-length (FL), or the peptide regions indicated in A or the C-terminal truncation fragments (TR1–3). Positive interactions are indicated by survival on QDO plates and blue α-galactosidase staining. Survival on DDO plates was tested for all double transformants to ensure the presence of bait and prey plasmids in the yeast host (C) A similar assay testing the binding of GTF2IRD1 to UBC9 and mapping the interaction sites.

    Article Snippet: Resistant colonies were re-spotted onto quadruple dropout (QDO) Trp/Leu/His/Ade-deficient SD plates containing x-α-galactosidase (Clontech) to test for evidence of interaction by activation of the HIS3 , ADE2 and MEL1 selection markers.

    Techniques: Protein Binding, Plasmid Preparation, Staining, Binding Assay

    Characterization of a novel interaction between GTF2IRD1 and ZMYM5. (A) Yeast 2-hybrid assays showing relative survival and α-galactosidase activation of yeast colonies on QDO and DDO plates. The yeast cells were co-transformed with pGADT7-ZMYM5 and a variety of bait plasmids including an empty pGBKT7 vector control (CTR), full-length GTF2IRD1 and the individual domains of GTF2IRD1. (B) Schematic diagram of ZMYM5 showing the position of 2 SUMO-interacting motifs (SIMs) and the MYM-type zinc finger domains (M). The black bars below indicate the extent of the ORF found in the ZMYM5 clone isolated in the original yeast 2-hybrid screen (Y2H CDS) and the truncated forms (TR99–TR486) generated to map the interaction domain. (C) Yeast 2-hybrid analysis of GTF2IRD1 interaction domain in ZMYM5 using the truncation series (TR99–TR486). (D) Immunofluorescence analysis of COS-7 cells transfected with pEGFP-ZMYM5 and pMyc-GTF2IRD1 showing extensive colocalization in the nucleus as illustrated by the overlay of GFP, anti-Myc immunofluorescence and DAPI (MERGE). (E) Western blot analysis of protein extracts from HEK293 cells co-transfected with pEGFP or pEGFP-ZMYM5 and pMyc-GTF2IRD1 plasmids. Whole cell extracts (INPUT) or proteins immunoprecipitated using anti-GFP antibody (IP) were immunoblotted (IB) with anti-Myc and anti-GFP antibodies. GFP-ZMYM5 and GFP were detected at approximately 100 kDa and 25 kDa respectively. Asterisks indicate IgG heavy and light chains.

    Journal: PLoS ONE

    Article Title: SUMOylation of GTF2IRD1 Regulates Protein Partner Interactions and Ubiquitin-Mediated Degradation

    doi: 10.1371/journal.pone.0049283

    Figure Lengend Snippet: Characterization of a novel interaction between GTF2IRD1 and ZMYM5. (A) Yeast 2-hybrid assays showing relative survival and α-galactosidase activation of yeast colonies on QDO and DDO plates. The yeast cells were co-transformed with pGADT7-ZMYM5 and a variety of bait plasmids including an empty pGBKT7 vector control (CTR), full-length GTF2IRD1 and the individual domains of GTF2IRD1. (B) Schematic diagram of ZMYM5 showing the position of 2 SUMO-interacting motifs (SIMs) and the MYM-type zinc finger domains (M). The black bars below indicate the extent of the ORF found in the ZMYM5 clone isolated in the original yeast 2-hybrid screen (Y2H CDS) and the truncated forms (TR99–TR486) generated to map the interaction domain. (C) Yeast 2-hybrid analysis of GTF2IRD1 interaction domain in ZMYM5 using the truncation series (TR99–TR486). (D) Immunofluorescence analysis of COS-7 cells transfected with pEGFP-ZMYM5 and pMyc-GTF2IRD1 showing extensive colocalization in the nucleus as illustrated by the overlay of GFP, anti-Myc immunofluorescence and DAPI (MERGE). (E) Western blot analysis of protein extracts from HEK293 cells co-transfected with pEGFP or pEGFP-ZMYM5 and pMyc-GTF2IRD1 plasmids. Whole cell extracts (INPUT) or proteins immunoprecipitated using anti-GFP antibody (IP) were immunoblotted (IB) with anti-Myc and anti-GFP antibodies. GFP-ZMYM5 and GFP were detected at approximately 100 kDa and 25 kDa respectively. Asterisks indicate IgG heavy and light chains.

    Article Snippet: Resistant colonies were re-spotted onto quadruple dropout (QDO) Trp/Leu/His/Ade-deficient SD plates containing x-α-galactosidase (Clontech) to test for evidence of interaction by activation of the HIS3 , ADE2 and MEL1 selection markers.

    Techniques: Activation Assay, Transformation Assay, Plasmid Preparation, Isolation, Generated, Immunofluorescence, Transfection, Western Blot, Immunoprecipitation

    Yeast two-hybrid based validation of positive interactions obtained from BiFC analysis. (A) The Y2H assays where M29 is cloned in bait vector and the cds of all other positive interactors are cloned in the prey vector. Binary interactions between bait and prey proteins were observed by mating and subsequent growth on –LT, quadruple drop out (QDO) and QDO+X-alpha-Gal (XαG). (B) α-Galactosidase quantitative assay for monitoring catalytic activity of the endogenous MELI reporter gene in liquid media, which confirms the positive interaction between bait and prey proteins and successful activation of Gal4 responsive promoter. The αGalactosidase activity is indicative of relative strengths of the interactions.

    Journal: Journal of Experimental Botany

    Article Title: Post-translational regulation of rice MADS29 function: homodimerization or binary interactions with other seed-expressed MADS proteins modulate its translocation into the nucleus

    doi: 10.1093/jxb/eru296

    Figure Lengend Snippet: Yeast two-hybrid based validation of positive interactions obtained from BiFC analysis. (A) The Y2H assays where M29 is cloned in bait vector and the cds of all other positive interactors are cloned in the prey vector. Binary interactions between bait and prey proteins were observed by mating and subsequent growth on –LT, quadruple drop out (QDO) and QDO+X-alpha-Gal (XαG). (B) α-Galactosidase quantitative assay for monitoring catalytic activity of the endogenous MELI reporter gene in liquid media, which confirms the positive interaction between bait and prey proteins and successful activation of Gal4 responsive promoter. The αGalactosidase activity is indicative of relative strengths of the interactions.

    Article Snippet: The α-galactosidase units were calculated based on manufacturer’s protocol (Clontech).

    Techniques: Bimolecular Fluorescence Complementation Assay, Clone Assay, Plasmid Preparation, Activity Assay, Activation Assay

    Genetic organization of α-galactosidase genes in LAB. The genes are indicated by polygons of the same size regardless of their length. White, carbohydrate degradation enzymes; black, sugar transporters; gray, transcriptional regulators. The following genes code for the indicated proteins: aga , melA α-galactosidase; galK , galactokinase; galT , galactose 1-phosphate uridylyltransferase; galM , mutarotase; bgaB , bgaC , lacL , and lacM β-galactosidase; scrB , sucrose phosphorylase; scrK , fructokinase; gftA , sucrose 6-phosphate hydrolase; dexB , dextran glucosidase; galR , scrR , rafR , rafS , msmR , and orf2 transcriptional regulators; orf3 and orf4 , putative PTS EIIB and EIIC domains; scrA , PTS EII Suc ; rafP (also named lacS2 in Lactobacillus plantarum WCFS1), galactoside-pentose-hexuronide transporter; msmEFGK and rafEFG , ABC transporters; rafX , unknown. GB#, GenBank accession number.

    Journal: Applied and Environmental Microbiology

    Article Title: Characterization of Genes Involved in the Metabolism of ?-Galactosides by Lactococcus raffinolactis

    doi: 10.1128/AEM.69.7.4049-4056.2003

    Figure Lengend Snippet: Genetic organization of α-galactosidase genes in LAB. The genes are indicated by polygons of the same size regardless of their length. White, carbohydrate degradation enzymes; black, sugar transporters; gray, transcriptional regulators. The following genes code for the indicated proteins: aga , melA α-galactosidase; galK , galactokinase; galT , galactose 1-phosphate uridylyltransferase; galM , mutarotase; bgaB , bgaC , lacL , and lacM β-galactosidase; scrB , sucrose phosphorylase; scrK , fructokinase; gftA , sucrose 6-phosphate hydrolase; dexB , dextran glucosidase; galR , scrR , rafR , rafS , msmR , and orf2 transcriptional regulators; orf3 and orf4 , putative PTS EIIB and EIIC domains; scrA , PTS EII Suc ; rafP (also named lacS2 in Lactobacillus plantarum WCFS1), galactoside-pentose-hexuronide transporter; msmEFGK and rafEFG , ABC transporters; rafX , unknown. GB#, GenBank accession number.

    Article Snippet: The α-galactosidase activity was assayed at 30°C and at pH 7.0 with p -nitrophenyl-α- d -galactopyranoside (Sigma) as the substrate ( ).

    Techniques:

    Anti-NGP5B antibody response is specific against terminal α-Gal residues. (A-B) Chemiluminescent ELISA reactivity of mouse serum pools obtained at Boost 3 (n = 6) and endpoint (n = 3) from α1,3GalT-KO mice vaccinated with NGP5B, NGP5B+CpG, CpG, or PBS. Immunized groups are indicated in the legend and antigens on the microplate are shown in the Y-axis. (C) Chemiluminescent ELISA reactivity of mouse serum obtained at Boost 2. NGP5B (125 ng/well) was treated or not with green-coffee bean α-galactosidase. One-way ANOVA (compared with untreated sample): (**), P

    Journal: PLoS Neglected Tropical Diseases

    Article Title: An α-Gal-containing neoglycoprotein-based vaccine partially protects against murine cutaneous leishmaniasis caused by Leishmania major

    doi: 10.1371/journal.pntd.0006039

    Figure Lengend Snippet: Anti-NGP5B antibody response is specific against terminal α-Gal residues. (A-B) Chemiluminescent ELISA reactivity of mouse serum pools obtained at Boost 3 (n = 6) and endpoint (n = 3) from α1,3GalT-KO mice vaccinated with NGP5B, NGP5B+CpG, CpG, or PBS. Immunized groups are indicated in the legend and antigens on the microplate are shown in the Y-axis. (C) Chemiluminescent ELISA reactivity of mouse serum obtained at Boost 2. NGP5B (125 ng/well) was treated or not with green-coffee bean α-galactosidase. One-way ANOVA (compared with untreated sample): (**), P

    Article Snippet: α-Galactosidase treatment To test the specificity of the IgG antibodies elicited in mice immunized with NGP5B or NGP5B+CpG, immobilized NGP5B used as antigen was pretreated with green coffee bean α-galactosidase (G8507, Sigma-Aldrich), as previously described [ ].

    Techniques: Chemiluminescent ELISA, Mouse Assay

    Effects of EtDO-P4 and α-galactosidase A (α-Gal A) treatments on CAMs expression

    Journal:

    Article Title: Globotriaosylceramide induces oxidative stress and up-regulates cell adhesion molecule expression in Fabry disease endothelial cells

    doi: 10.1016/j.ymgme.2008.06.016

    Figure Lengend Snippet: Effects of EtDO-P4 and α-galactosidase A (α-Gal A) treatments on CAMs expression

    Article Snippet: For enzyme treatment, cells were incubated with recombinant α-galactosidase A (Shire Human Genetic Therapies, Cambridge, MA) at 0.08 IU/ml (1:500 dilution of original solution) for 4 days.

    Techniques: Expressing

    Presence of Galα1-3Gal in immune complexes in the blood of baboons, after pulmonary xenotransplantation. Immune complexes in the serum taken from baboon blood after a pulmonary xenotransplantation were diluted 5:1 with 12% PEG 8000 and 60 mmol/L EDTA in VBS for 16 hours at 4°C to precipitate immune complexes. The solution was centrifuged at 2,000 × g for 20 minutes at 4°C to pellet immune complexes. Precipitates were resuspended in digestion buffer (100 mmol/L NaCl and 50 mmol/L sodium acetate). Samples were incubated with (+) or without (−) α-galactosidase (1 U/ml) for 5 hours at 37°C. Proteins were precipitated with ethanol, reduced, separated by SDS-PAGE (7.5% PA), and transferred to PVDF. The blots were reacted with human serum as a source of xenoreactive IgM and then with anti-human IgM. Lane 1 , α-galactosidase digested proteins targeted by human IgM, Lane 2 , control proteins targeted by human IgM. Digestion of samples with α-galactosidase abrogates the binding of human xenoreactive IgM.

    Journal: The American Journal of Pathology

    Article Title: Immune Complex Formation after Xenotransplantation

    doi:

    Figure Lengend Snippet: Presence of Galα1-3Gal in immune complexes in the blood of baboons, after pulmonary xenotransplantation. Immune complexes in the serum taken from baboon blood after a pulmonary xenotransplantation were diluted 5:1 with 12% PEG 8000 and 60 mmol/L EDTA in VBS for 16 hours at 4°C to precipitate immune complexes. The solution was centrifuged at 2,000 × g for 20 minutes at 4°C to pellet immune complexes. Precipitates were resuspended in digestion buffer (100 mmol/L NaCl and 50 mmol/L sodium acetate). Samples were incubated with (+) or without (−) α-galactosidase (1 U/ml) for 5 hours at 37°C. Proteins were precipitated with ethanol, reduced, separated by SDS-PAGE (7.5% PA), and transferred to PVDF. The blots were reacted with human serum as a source of xenoreactive IgM and then with anti-human IgM. Lane 1 , α-galactosidase digested proteins targeted by human IgM, Lane 2 , control proteins targeted by human IgM. Digestion of samples with α-galactosidase abrogates the binding of human xenoreactive IgM.

    Article Snippet: Immune complexes were precipitated with PEG (described above) from the serum of baboons with pulmonary xenografts and resuspended in digestion buffer (100 mmol/L NaCl and 50 mmol/L sodium acetate, pH 5.0) containing 1 U/ml α-galactosidase (green coffee bean; Boehringer Mannheim Corp, Indianapolis, IN), or in digestion buffer alone, as a control.

    Techniques: Incubation, SDS Page, Binding Assay

    A, HPLC analysis of A. thaliana and C. reinhardtii peptidyl galactosyltransferase activity. P100 fractions were prepared from A. thaliana cells (Col-0), C. reinhardtii CC-125 cells, and C. reinhardtii cell wall-less mutant CC-503 cells. The P100 fraction (100 μg for A. thaliana or 10 μg for C. reinhardtii ) was incubated with the assay mixture described under “Experimental Procedures” containing AtEXT peptide as an acceptor at 30 °C for 10 h ( A. thaliana ) or 3 h ( C. reinhardtii ) and then analyzed by HPLC. The acceptor peptide (AtEXT) was eluted at 18.8 min, and three enzymatic reaction products were detected, indicated as products 1, 2, and 3, at 15.2, 16.1, and 13.7 min, respectively (these are indicated with vertical broken lines in A–D ). B–D, HPLC analysis of products after α- and β-galactosidase ( GSD ) treatment. B shows product 1 before and after galactosidase treatment, and C shows product 2 before and after treatment. D shows product 3 before and after treatment. α→β- GSD indicates product 3 treated with β- GSD after α-galactosidase treatment. β→α- GSD indicates product 3 treated with α-galactosidase after β-galactosidase treatment. The sequence of AtEXT peptide was FITC-Ahx-VYKSOOOOV-NH 2 . O indicates hydroxyproline. E, P100 fraction (10 μg) of C. reinhardtii CC-503 cells was incubated in an assay mixture containing VYKAOOOOV peptide as an acceptor at 30 °C for 3 h and then analyzed by HPLC. The product was collected and treated with β-galactosidase. F, ), and analyzed by HPLC using a TSKgel sugar AXI column. PA-galactose was detected at 97 min, whereas PA-glucose and PA-mannose were detected at 55 and 62 min, respectively (data not shown).

    Journal: The Journal of Biological Chemistry

    Article Title: Identification of Novel Peptidyl Serine α-Galactosyltransferase Gene Family in Plants *

    doi: 10.1074/jbc.M114.553933

    Figure Lengend Snippet: A, HPLC analysis of A. thaliana and C. reinhardtii peptidyl galactosyltransferase activity. P100 fractions were prepared from A. thaliana cells (Col-0), C. reinhardtii CC-125 cells, and C. reinhardtii cell wall-less mutant CC-503 cells. The P100 fraction (100 μg for A. thaliana or 10 μg for C. reinhardtii ) was incubated with the assay mixture described under “Experimental Procedures” containing AtEXT peptide as an acceptor at 30 °C for 10 h ( A. thaliana ) or 3 h ( C. reinhardtii ) and then analyzed by HPLC. The acceptor peptide (AtEXT) was eluted at 18.8 min, and three enzymatic reaction products were detected, indicated as products 1, 2, and 3, at 15.2, 16.1, and 13.7 min, respectively (these are indicated with vertical broken lines in A–D ). B–D, HPLC analysis of products after α- and β-galactosidase ( GSD ) treatment. B shows product 1 before and after galactosidase treatment, and C shows product 2 before and after treatment. D shows product 3 before and after treatment. α→β- GSD indicates product 3 treated with β- GSD after α-galactosidase treatment. β→α- GSD indicates product 3 treated with α-galactosidase after β-galactosidase treatment. The sequence of AtEXT peptide was FITC-Ahx-VYKSOOOOV-NH 2 . O indicates hydroxyproline. E, P100 fraction (10 μg) of C. reinhardtii CC-503 cells was incubated in an assay mixture containing VYKAOOOOV peptide as an acceptor at 30 °C for 3 h and then analyzed by HPLC. The product was collected and treated with β-galactosidase. F, ), and analyzed by HPLC using a TSKgel sugar AXI column. PA-galactose was detected at 97 min, whereas PA-glucose and PA-mannose were detected at 55 and 62 min, respectively (data not shown).

    Article Snippet: We used β-galactosidase from Aspergillus oryzae (Sigma) and α-galactosidase from guar seed (Megazyme, Wicklow, Ireland).

    Techniques: High Performance Liquid Chromatography, Activity Assay, Mutagenesis, Incubation, Sequencing

    Targeted integration of the human coagulation factor IX and α-galactosidase genes by enhanced ZFRs. (a) Bulk PCR analysis of HEK293 cells transfected with eZFRs targeting human chromosome 4 and donor plasmids harboring either the human coagulation factor IX (FIX) or α-galactosidase genes (GLA). Integration was evaluated in the forward and reverse orientations. GAPDH indicates PCR control. DO indicates donor only (no eZFRs). Genome-wide integration rates indicated beneath each lane. (b) Clonal analysis of puromycin-resistant cells transfected with eZFRs and donor plasmids containing the FIX or GLA genes.

    Journal: Journal of the American Chemical Society

    Article Title: Enhancing the Specificity of Recombinase-Mediated Genome Engineering through Dimer Interface Redesign

    doi: 10.1021/ja4130059

    Figure Lengend Snippet: Targeted integration of the human coagulation factor IX and α-galactosidase genes by enhanced ZFRs. (a) Bulk PCR analysis of HEK293 cells transfected with eZFRs targeting human chromosome 4 and donor plasmids harboring either the human coagulation factor IX (FIX) or α-galactosidase genes (GLA). Integration was evaluated in the forward and reverse orientations. GAPDH indicates PCR control. DO indicates donor only (no eZFRs). Genome-wide integration rates indicated beneath each lane. (b) Clonal analysis of puromycin-resistant cells transfected with eZFRs and donor plasmids containing the FIX or GLA genes.

    Article Snippet: ZFR donor plasmids (pDonor; previously pBABE-Puromycin) were constructed as previously described , with the following exceptions: cDNA for the human coagulation factor IX (FIX) and α-galactosidase (GLA) genes (Genecopoeia) were PCR amplified with the primers Pst I-CMV-Donor-Fwd and Bam H1-ZFR-Donor-Rev.

    Techniques: Coagulation, Polymerase Chain Reaction, Transfection, Genome Wide

    (A) Schematic representation of processing of human α‐galactosidase GLA by GH99 endomannosidase. GLA is pre‐labeled by fluorescent TB340, and contains high‐mannose N‐glycans which can be truncated by endomannosidase, resulting in a decrease in GLA molecular weight. Activity‐based labeling of endomannosidase by spiro‐epoxyglycosides 4 or 5 (prior to incubation with GLA) blocks its activity, and is therefore unable to process GLA. (B) Bt GH99 wild‐type demannosylates GLA, causing a shift in molecular weight for the protein bands. Pre‐labeling Bt GH99 wild‐type with 4 or 5 abrogates GLA demannosylation. Endo‐H cleaves high‐mannose structures, PNGase‐F cleaves full N‐linked glycan (leaving Asp‐GlcNAc). (C) Bx GH99 wild‐type demannosylates GLA, while Bx GH99 pre‐labeled with 4 or 5 is unable to do so. Bx GH99 active‐site mutants E333Q and E3336Q are unable to process GLA. (D) Fluorescent labeling of Bt GH99 (top) and Bx GH99 (bottom) by 4 or 5 competed by different concentrations of 17 , 18 , ManIFG ( 2 ) and yeast mannan. The marker is annotated with an asterisk (*).

    Journal: Chemistry (Weinheim an Der Bergstrasse, Germany)

    Article Title: Spiro‐epoxyglycosides as Activity‐Based Probes for Glycoside Hydrolase Family 99 Endomannosidase/Endomannanase

    doi: 10.1002/chem.201801902

    Figure Lengend Snippet: (A) Schematic representation of processing of human α‐galactosidase GLA by GH99 endomannosidase. GLA is pre‐labeled by fluorescent TB340, and contains high‐mannose N‐glycans which can be truncated by endomannosidase, resulting in a decrease in GLA molecular weight. Activity‐based labeling of endomannosidase by spiro‐epoxyglycosides 4 or 5 (prior to incubation with GLA) blocks its activity, and is therefore unable to process GLA. (B) Bt GH99 wild‐type demannosylates GLA, causing a shift in molecular weight for the protein bands. Pre‐labeling Bt GH99 wild‐type with 4 or 5 abrogates GLA demannosylation. Endo‐H cleaves high‐mannose structures, PNGase‐F cleaves full N‐linked glycan (leaving Asp‐GlcNAc). (C) Bx GH99 wild‐type demannosylates GLA, while Bx GH99 pre‐labeled with 4 or 5 is unable to do so. Bx GH99 active‐site mutants E333Q and E3336Q are unable to process GLA. (D) Fluorescent labeling of Bt GH99 (top) and Bx GH99 (bottom) by 4 or 5 competed by different concentrations of 17 , 18 , ManIFG ( 2 ) and yeast mannan. The marker is annotated with an asterisk (*).

    Article Snippet: Recombinant α‐galactosidase (GLA) was purchased from Genzyme (Cambridge, MA, USA).

    Techniques: Labeling, Molecular Weight, Activity Assay, Incubation, Marker