Journal: PLoS ONE

Article Title: Diabetes Alters the Expression and Translocation of the Insulin-Sensitive Glucose Transporters 4 and 8 in the Atria

doi: 10.1371/journal.pone.0146033

Figure Lengend Snippet: Higher total protein expression of A) GLUT4 and lower B) GLUT8 content in the healthy atrial compared to ventricular myocytes. Top panels: representative Western blot from total lysate of isolated rat myocytes; calsequestrin (CLSQ) was used as a loading control; representative bands were obtained from the same membrane. Bottom panels: Mean ± SE of total GLUT protein content (values expressed relative to atria; n = 6/group); * P<0.05 vs. atria. GLUT: glucose transporters. Insulin stimulation increases C) GLUT4 and D) GLUT8 protein content in the healthy atrial myocytes. Top panels: representative Western blot from total lysate of isolated rat myocytes, calsequestrin (CLSQ) was used as a loading control. Bottom panels: Mean ± SE of total GLUT protein content (values expressed relative to basal atria; n = 8-11/group); # P<0.05 vs. basal. Insulin stimulates E) GLUT4 and F) GLUT8 trafficking to the atrial and ventricular cell surface. Top panels: representative Western blot. Bottom panels: Mean ± SE of cell surface GLUT protein content (values expressed relative to labeled basal atria; n = 3-4/group); # P<0.05 vs. basal; *P<0.05 vs. atria. Methods: Cell surface GLUT measured using biotinylated photolabeling technique in the intact perfused mouse heart. L: Labeled (cell surface fraction); UL: Unlabeled (intracellular fraction).

Article Snippet: Equal protein loading was confirmed by reprobing each membrane with Calsequestrin monoclonal IgG (Thermo-Scientific PA1-903, 1:2500, polyclonal rabbit anti-dog).

Techniques: Expressing, Western Blot, Isolation, Control, Membrane, Labeling

Journal: PLoS ONE

Article Title: Diabetes Alters the Expression and Translocation of the Insulin-Sensitive Glucose Transporters 4 and 8 in the Atria

doi: 10.1371/journal.pone.0146033

Figure Lengend Snippet: A) Insulin stimulates phosphorylation of Akt at s473 and Th308 site in atrial myocytes. Top panel: representative Western blot from total lysate of isolated rat atrial myocytes incubated with (0.01μM) and without (basal) insulin; calsequestrin (CLSQ) was used as a loading control. Bottom panel: Mean ± SE of protein expression (values expressed relative to basal; n = 5/group); # P<0.05 vs. basal. B) Significant linear positive linear correlation between Akt phosphorylation (at s473 and Th308 site ) and GLUT4 expression in the healthy atria. C) Insulin stimulates phosphorylation of AS160 in atrial myocytes. Top panel: representative Western blot from total lysate of isolated rat atrial myocytes; calsequestrin (CLSQ) was used as a loading control. Bottom panel: Mean ± SE of protein expression (values expressed relative to basal; n = 6-8/group); # P<0.05 vs. basal. D) Significant linear correlation between AS160 phosphorylation and GLUT-4 and -8 expression in the healthy atria.

Article Snippet: Equal protein loading was confirmed by reprobing each membrane with Calsequestrin monoclonal IgG (Thermo-Scientific PA1-903, 1:2500, polyclonal rabbit anti-dog).

Techniques: Phospho-proteomics, Western Blot, Isolation, Incubation, Control, Expressing

Journal: PLoS ONE

Article Title: Diabetes Alters the Expression and Translocation of the Insulin-Sensitive Glucose Transporters 4 and 8 in the Atria

doi: 10.1371/journal.pone.0146033

Figure Lengend Snippet: Decreased atrial A) GLUT4 and B) GLUT8 content during type 1 diabetes (T1Dx). Top panels: representative Western blot from crude membrane extract of perfused mouse atria, calsequestrin (CLSQ) was used as a loading control. Bottom panels: Mean ± SE of GLUT protein content (values expressed relative to control; n = 7-8/group). Type 1 diabetes decreased C) GLUT4 and D) GLUT8 trafficking to the atrial cell surface. Top panels: representative Western blot. Bottom panels: Mean ± SE of cell surface GLUT protein content (values expressed relative to control; n = 4-5/group). Majority of E) GLUT4 and F) GLUT8 is intracellular under basal conditions (mean ± SE, values expressed relative to control labeled, n = 5/group). Methods: Intact perfused mouse hearts were photolabeled with bio-LC-ATB-BGPA to determine the amount of cell surface (L: labeled fraction) and intracellular (UL: unlabeled fraction) content. T1Dx: type 1 diabetic; Con: control; * P<0.05 vs. control.

Article Snippet: Equal protein loading was confirmed by reprobing each membrane with Calsequestrin monoclonal IgG (Thermo-Scientific PA1-903, 1:2500, polyclonal rabbit anti-dog).

Techniques: Western Blot, Membrane, Control, Labeling

Journal: PLoS ONE

Article Title: Diabetes Alters the Expression and Translocation of the Insulin-Sensitive Glucose Transporters 4 and 8 in the Atria

doi: 10.1371/journal.pone.0146033

Figure Lengend Snippet: Type 1 diabetes (T1 Dx) did not alter A) Akt or B) AS160 phosphorylation in the atria . Top panels: representative Western blot from total lysate of mouse atria; calsequestrin (CLSQ) was used as a loading control. Bottom panels: Mean ± SE of protein expression (values expressed relative to control; n = 4-5/group). Insulin stimulates C) GLUT4 and D) GLUT8 trafficking to the atrial cell surface in type 1 diabetic (T1 Dx) subjects . Top panels: representative Western blot. Bottom panels: Mean ± SE of cell surface GLUT protein content (values expressed relative to control basal labeled; n = 4-6/group). Methods: Intact mouse hearts were perfused with and without insulin, and photolabeled with bio-LC-ATB-BGPA to determine the amount of cell surface (L: labeled fraction) and intracellular (UL: unlabeled fraction) content. T1Dx: type 1 diabetic; *P<0.05 vs. control; # P<0.05 vs. basal.

Article Snippet: Equal protein loading was confirmed by reprobing each membrane with Calsequestrin monoclonal IgG (Thermo-Scientific PA1-903, 1:2500, polyclonal rabbit anti-dog).

Techniques: Phospho-proteomics, Western Blot, Control, Expressing, Labeling

Journal: BMC Biochemistry

Article Title: Quaternary structures of recombinant, cellular, and serum forms of Thymidine Kinase 1 from dogs and humans

doi: 10.1186/1471-2091-13-12

Figure Lengend Snippet: Amino acid sequence alignment of canine and human TK1. Residues that differ between the two sequences are shaded. Cysteines that are present in the human but not in the canine sequence are marked with squares. The Zn-binding cysteines are shaded in yellow. GenBank accession numbers are XM_540461 (dog) and KO_2582 (human).

Article Snippet: Two rabbit polyclonal anti-dog TK1 antibodies were produced, one using a 28-amino acid (28-mer) synthetic peptide (amino acids 195–223) as the antigen by Agrisera AB (Umeå, Sweden), and the other one using a 16-amino acid (16-mer) synthetic peptide (211–225) as the antigen (Figure ) by GenScript (Piscataway, NJ, USA).

Techniques: Sequencing, Binding Assay

Journal: BMC Biochemistry

Article Title: Quaternary structures of recombinant, cellular, and serum forms of Thymidine Kinase 1 from dogs and humans

doi: 10.1186/1471-2091-13-12

Figure Lengend Snippet: Steady state kinetics of recombinant canine and human TK1. Substrate saturation curve of canine (A) and human (B) TK1 with dThd (■) and AZT (●) as the variable substrate. Activity data were fitted to the Michaelis-Menten equation. The assays were repeated three times and error bars are shown.

Article Snippet: Two rabbit polyclonal anti-dog TK1 antibodies were produced, one using a 28-amino acid (28-mer) synthetic peptide (amino acids 195–223) as the antigen by Agrisera AB (Umeå, Sweden), and the other one using a 16-amino acid (16-mer) synthetic peptide (211–225) as the antigen (Figure ) by GenScript (Piscataway, NJ, USA).

Techniques: Recombinant, Activity Assay

Journal: BMC Biochemistry

Article Title: Quaternary structures of recombinant, cellular, and serum forms of Thymidine Kinase 1 from dogs and humans

doi: 10.1186/1471-2091-13-12

Figure Lengend Snippet: Temperature and pH effects on the activities of recombinant canine and human TK1. The activities of dog (A) and human (B) TK1 were measured using [ 3 H]-AZT as a substrate at different temperatures. The activities of canine (C) and human (D) TK1 were measured with AZT as substrate at different pH (indicated on the x-axis) and different temperatures: 37 °C (filled bars) and 45 °C (open bars).

Article Snippet: Two rabbit polyclonal anti-dog TK1 antibodies were produced, one using a 28-amino acid (28-mer) synthetic peptide (amino acids 195–223) as the antigen by Agrisera AB (Umeå, Sweden), and the other one using a 16-amino acid (16-mer) synthetic peptide (211–225) as the antigen (Figure ) by GenScript (Piscataway, NJ, USA).

Techniques: Recombinant

Journal: BMC Biochemistry

Article Title: Quaternary structures of recombinant, cellular, and serum forms of Thymidine Kinase 1 from dogs and humans

doi: 10.1186/1471-2091-13-12

Figure Lengend Snippet: Quaternary structure of recombinant TK1 and the effect of reducing agent. Untreated (●) or DTE-treated (○) recombinant canine (A) and human (B) TK1 was analyzed by size exclusion chromatography. Four micrograms of freshly isolated TK1 in 200 μl buffer were injected into the column and eluted. A total of 24 fractions were collected. TK1 activity in the fractions was determined by radiochemical assay and TK1 protein was detected by immunoaffinity detection method as described in Materials and Methods. Inset: activity profile of untreated recombinant canine TK1. Arrows indicate the elution position of molecular weight markers. Western blot analysis of FPLC fractions of canine (C) and human (D) TK1; untreated (−DTE), and DTE-treated (+DTE). The numbers represent FPLC fractions.

Article Snippet: Two rabbit polyclonal anti-dog TK1 antibodies were produced, one using a 28-amino acid (28-mer) synthetic peptide (amino acids 195–223) as the antigen by Agrisera AB (Umeå, Sweden), and the other one using a 16-amino acid (16-mer) synthetic peptide (211–225) as the antigen (Figure ) by GenScript (Piscataway, NJ, USA).

Techniques: Recombinant, Size-exclusion Chromatography, Isolation, Injection, Activity Assay, Radiochemical Assay, Molecular Weight, Western Blot

Journal: BMC Biochemistry

Article Title: Quaternary structures of recombinant, cellular, and serum forms of Thymidine Kinase 1 from dogs and humans

doi: 10.1186/1471-2091-13-12

Figure Lengend Snippet: Size distribution of cytosolic TK1. TK1 activity in each fraction was measured using [ 3 H]-dThd as substrate. MDCK extracts (A) and CEM extracts (B) analyzed directly (○) or treated with DTE (●) prior to chromatography. Arrows indicate the elution position of the molecular weight markers. Western blot analyses of cytosolic canine TK1 (C) and human TK1 (D) in the FPLC fractions using polyclonal anti-dog TK1 antibody or mouse monoclonal anti-TK1 antibody for detection. Pre-treatment with DTE is indicated as + DTE. The numbers represent FPLC fractions.

Article Snippet: Two rabbit polyclonal anti-dog TK1 antibodies were produced, one using a 28-amino acid (28-mer) synthetic peptide (amino acids 195–223) as the antigen by Agrisera AB (Umeå, Sweden), and the other one using a 16-amino acid (16-mer) synthetic peptide (211–225) as the antigen (Figure ) by GenScript (Piscataway, NJ, USA).

Techniques: Activity Assay, Chromatography, Molecular Weight, Western Blot

Journal: BMC Biochemistry

Article Title: Quaternary structures of recombinant, cellular, and serum forms of Thymidine Kinase 1 from dogs and humans

doi: 10.1186/1471-2091-13-12

Figure Lengend Snippet: Quaternary structures of serum TK1. (A) Thymidine kinase activity in serum fractions from dogs with acute lymphocytic leukemia (●) injected directly into the Superose 12 column, or pre-treated with 20 mM DTE (○). (B) Thymidine kinase activity in serum fractions from human patients with acute lymphocytic leukemia (♦) injected directly into Superose 12 or pre-treated with DTE (◊). Arrows indicate the elution position of molecular weight markers. (C) Western blot analyses of untreated dog serum samples (−DTE) or those pre-treated with 20 mM DTE (+DTE), using polyclonal anti-dog TK1 antibody. (D) Western blot analyses of untreated human serum samples (−DTE) or those pre-treated with DTE (+DTE), using monoclonal anti-human TK1 antibody. The numbers represent FPLC fractions.

Article Snippet: Two rabbit polyclonal anti-dog TK1 antibodies were produced, one using a 28-amino acid (28-mer) synthetic peptide (amino acids 195–223) as the antigen by Agrisera AB (Umeå, Sweden), and the other one using a 16-amino acid (16-mer) synthetic peptide (211–225) as the antigen (Figure ) by GenScript (Piscataway, NJ, USA).

Techniques: Activity Assay, Injection, Molecular Weight, Western Blot