g6pase-α #sc-25840 antibody  (Santa Cruz Biotechnology)

Journal: Nature Communications

Article Title: mRNA therapy restores euglycemia and prevents liver tumors in murine model of glycogen storage disease

doi: 10.1038/s41467-021-23318-2

Figure Lengend Snippet: a Hypothetical model of h G6PC mRNA therapy. hG6PC mRNAs are delivered to liver via lipid nanoparticles. Once the mRNA is in the cell (hepatocytes) it is translated by the cellular machinery into a functional protein that is localized to the ER membrane (likely following a co-translational translocation model), resulting in an active G6Pase-α enzyme. b Protein consensus screening by ortholog residue analysis. Top: WebLogo representation of the abundance of each alternative amino acid used at indicated residue positions. Bottom: The degree of conservation of amino acids at each position was quantified as relative entropy (Kullback–Leibler divergence). c Relative hG6Pase-α protein expression (solid circle) and hG6Pase-α enzymatic activity (solid square) in HeLa cells treated with the top ten h G6PC mRNA variants generated using protein consensus analysis. Data were shown as percentage of wild-type (WT) group and presented as mean ± SD of n = 2 (for protein expression), 3 (for enzymatic activity, Q247R), or 4 (for enzymatic activity, all other groups) biologically independent samples. d Subcellular localization of WT hG6Pase-α and S298C variant in HeLa cells. Green: hG6Pase-α, Red: Calnexin, an ER marker (top); TOM20, mitochondrial marker (bottom). Scale bars are 10 µm. The ratio of colocalized signal over total signal was calculated by Mander’s colocalization coefficient analysis (bottom panel). Data were presented as mean ± SD of n = 2 biologically independent samples. Source data are provided as a Source Data File.

Article Snippet: Antibodies against G6Pase-α were quantified on Nunc Immuno Maxisorp plates (ThermoFisher, #442404) coated with 0.5 μg/mL recombinant G6Pase-α protein (Viva Biotech) in 50 mM Na 2 CO 3 for 1 h at room temperature and blocked with SuperBlock (PBS) Blocking Buffer (ThermoFisher, #37515).

Techniques: Functional Assay, Membrane, Translocation Assay, Residue, Expressing, Activity Assay, Generated, Variant Assay, Marker

Journal: Nature Communications

Article Title: mRNA therapy restores euglycemia and prevents liver tumors in murine model of glycogen storage disease

doi: 10.1038/s41467-021-23318-2

Figure Lengend Snippet: a hG6Pase-α protein expression (left panel) and enzymatic activity (right panel) of wild-type hG6PC (WT), codon optimized wild-type h G6PC (WT_CO), hG6PC_S298C (S298C), and codon optimized hG6PC_S298C (S298C_CO) mRNAs evaluated in Hep3B cells. Control cells were treated with eGFP mRNA. Data were presented as mean ± SD of n = 3 biologically independent samples. b hG6Pase-α protein expression (left panel) and enzymatic activity (right panel) of WT and codon optimized h G6PC mRNAs as evaluated in male CD-1 mice. Control animals were treated with eGFP mRNA. Data were presented as mean ± SD of n = 4 mice. For statistical analysis, raw values were Log2 transformed and subjected to one-way ANOVA, followed by the Dunnett’s multiple comparisons test, compared to the non-codon optimized WT hG6PC mRNA. Statistically significant P values ( p ≤ 0.05) are shown in the graphs. Source data are provided as a Source Data File.

Article Snippet: Antibodies against G6Pase-α were quantified on Nunc Immuno Maxisorp plates (ThermoFisher, #442404) coated with 0.5 μg/mL recombinant G6Pase-α protein (Viva Biotech) in 50 mM Na 2 CO 3 for 1 h at room temperature and blocked with SuperBlock (PBS) Blocking Buffer (ThermoFisher, #37515).

Techniques: Expressing, Activity Assay, Control, Transformation Assay

Journal: Nature Communications

Article Title: mRNA therapy restores euglycemia and prevents liver tumors in murine model of glycogen storage disease

doi: 10.1038/s41467-021-23318-2

Figure Lengend Snippet: Wild-type (CD-1) male mice were i.v. administered with 1.0 mg/kg of eGFP, h G6PC -wild type (WT), or codon-optimized hG6PC -S298C (hG6PC_S298C_CO) mRNA-LNP and sacrificed at 6, 24, 72, 168, 336 h ( n = 4/group/sacrifice time point). a h G6PC mRNA levels (h G6PC -WT and S298C mRNAs). b Hepatic protein levels in mice treated with eGFP mRNA, mRNA encoding hG6Pase-α WT, or codon-optimized mRNA encoding hG6PC-S298C protein variant. c Hepatic enzymatic activity levels in mice treated with eGFP mRNA, mRNA encoding hG6Pase-α WT, or codon-optimized mRNA encoding hG6PC-S298C protein variant. Data were presented as mean ± SD ( n = 3–4). Source data are provided as a Source Data File.

Article Snippet: Antibodies against G6Pase-α were quantified on Nunc Immuno Maxisorp plates (ThermoFisher, #442404) coated with 0.5 μg/mL recombinant G6Pase-α protein (Viva Biotech) in 50 mM Na 2 CO 3 for 1 h at room temperature and blocked with SuperBlock (PBS) Blocking Buffer (ThermoFisher, #37515).

Techniques: Variant Assay, Activity Assay

Journal: Nature Communications

Article Title: mRNA therapy restores euglycemia and prevents liver tumors in murine model of glycogen storage disease

doi: 10.1038/s41467-021-23318-2

Figure Lengend Snippet: a Blood glucose levels following administration of h G6PC S298C mRNA-LNP in L. G6pc −/− mice. WT, wild-type mice. (WT treated with PBS, n = 8 per group; L.G6 pc −/− treated with eGFP, n = 6, 5, 5, and 5 per group for fasting duration of 0, 2.5, 6, and 24 h, respectively; L.G6 pc −/− treated with h G6PC S298C at 0.2 mg/kg, n = 7 per group for all time points; L. G6pc −/− treated with hG6PC S298C at 0.5 mg/kg, n = 7, 6, 6, and 6 per group for fasting duration of 0, 2.5, 6, and 24 h, respectively; L.G6 pc −/− treated with h G6PC S298C at 1.0 mg/kg, n = 7 per group for all time points). Data were presented as mean ± SD. b Liver morphology (left panel) and liver weight (right panel) following administration of h G6PC S298C mRNA in L. G6pc −/− mice. Representative liver images are shown from n = 8, 5, 7, 6, and 6 mice per group from WT treated with PBS, L. G6pc −/− treated with eGFP, and L. G6pc −/− treated with hG6PC S298C mRNA at 0.2, 0.5, or 1.0 mg/kg, respectively. c hG6Pase-α S298C protein expression and enzymatic activity in livers of L. G6pc −/− mice. d Hepatic biomarker analysis following administration of h G6PC S298C mRNA-LNP in L. G6pc −/− mice. Liver G6P (left panel), liver glycogen (middle panel), liver triglycerides (right panel). e Serum triglycerides following administration of h G6PC S298C mRNA-LNP in L. G6pc −/− mice. h G6PC S298C mRNA-LNP dose range: 0.2, 0.5, and 1.0 mg/kg. For b–e , quantitative data were presented as mean ± SD ( n = 8, 5, 7, 6, and 6 mice per group for WT treated with PBS, L. G6pc −/− treated with eGFP, and L. G6pc −/− treated hG6PC S298C mRNA at 0.2, 0.5, or 1.0 mg/kg, respectively). For statistical analysis, raw values were Log2 transformed and subjected to one-way ANOVA, followed by the Dunnett’s multiple comparisons test, compared to the eGFP mRNA treated group. Statistically significant P values ( p ≤ 0.05) are shown in the graphs. Source data are provided as a Source Data File.

Article Snippet: Antibodies against G6Pase-α were quantified on Nunc Immuno Maxisorp plates (ThermoFisher, #442404) coated with 0.5 μg/mL recombinant G6Pase-α protein (Viva Biotech) in 50 mM Na 2 CO 3 for 1 h at room temperature and blocked with SuperBlock (PBS) Blocking Buffer (ThermoFisher, #37515).

Techniques: Expressing, Activity Assay, Biomarker Discovery, Transformation Assay

Journal: Nature Communications

Article Title: mRNA therapy restores euglycemia and prevents liver tumors in murine model of glycogen storage disease

doi: 10.1038/s41467-021-23318-2

Figure Lengend Snippet: a Single-dose duration of action of h G6PC S298C mRNA-LNP (0.5 or 1.0 mg/kg) administered i.v. in L. G6pc −/− mice. Blood glucose levels were measured at fed (0 h) or fasting conditions (2.5- or 6-h post-fasting). Data were presented as mean ± SD ( n = 8, 9, 10, 10, and 10 mice per group for wild-type (WT) treated with PBS, L. G6pc −/− treated with eGFP, hG6PC S298C mRNA at 0.5 or 1.0 mg/kg, respectively). For statistical analysis, two-sample t -test (two-sided) was performed and corrected for multiple testing by using a Bonferroni adjusted level of 0.005. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001, comparing h G6PC S298C mRNA 1.0 mg/kg with eGFP ( p values are 0.0017 [day 0, 2.5 h], 0.0016 [day 0, 6 h], 0.0006 [day 2, 2.5 h], 0.0005 [day 2, 6 h], 0.001 [day 4, 2.5 h], 0.001 [day 4, 6 h], and 0.048 [day 7, 6 h], respectively). † P ≤ 0.05, †† P ≤ 0.01, ††† P ≤ 0.001, †††† P ≤ 0.0001, comparing h G6PC S298C mRNA 0.5 mg/kg with eGFP ( p values are 0.033 [day 0, 2.5 h], 0.001 [day 0, 6 h], 0.0004 [day 2, 2.5 h], 0.00003 [day 2, 6 h], 0.002 [day 4, 2.5 h], 0.001 [day 4, 6 h], and 0.009 [day 7, 6 h], respectively) b Blood glucose levels following repeat (five doses) i.v. administrations of h G6PC S298C mRNA-LNP (0.25 mg/kg) in L. G6pc −/− mice. Arrows indicate dose administration. Blood glucose levels were measured at 2.5-h post-fasting. Data were presented as mean ± SD ( n = 8, 7, and 9 mice per group for WT treated with PBS, L. G6pc −/− treated with eGFP, and L. G6pc −/− treated with h G6PC S298C mRNA, respectively). For statistical analysis, two-sample t -test (two-sided) was performed and corrected for multiple testing by using a Bonferroni adjusted level of 0.005. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, *** P ≤ 0.0001 comparing hG6PC S298C mRNA with eGFP ( p values are 0.005 [day 11], 4 × 10 −6 [day 25], 0.0007 [day 28], 0.0192 [day38], 1.3 × 10 −5 [day 39], 0.001 [day 42], 2 × 10 −6 [day 52], and 5 × 10 −5 [day 53], respectively). c Serum proinflammatory cytokines (from left to right): IFNɣ, IL-1β, TNFα, and IL6 from the dose-ranging study. d serum ALT (mU/mL) levels from the dose-ranging study. For c and d , data were presented as mean ± SD ( n = 6, 5, 8, 7, and 6 mice per group for WT treated with PBS, L. G6pc −/− treated with eGFP, and L. G6pc −/− treated with hG6PC S298C mRNA at 0.2, 0.5, or 1.0 mg/kg, respectively). e Serum proinflammatory cytokines (from left to right): IFNɣ, IL-1β, TNFα, and IL6 from repeat-dose study. Data were presented as mean ± SD ( n = 10, 10, and 7 mice per group for WT treated with PBS, L. G6pc −/− treated with eGFP, or hG6PC S298C mRNA). f Antidrug antibody assay measuring anti-G6Pase-α antibodies in sera of mice treated with five doses of h G6PC S298C mRNA-LNP (0.5 mg/kg). Data were presented as mean ± SD ( n = 9, 7, 7, 6 mice per group for WT treated with PBS, L.G6 pc −/− treated with eGFP, L. G6pc −/− treated with hG6PC S298C mRNA, and positive sera, respectively). g Body weight of L. G6pc −/− mice prior to each repeat i.v. dose treatment of h G6PC mRNA -LNP (0.25 mg/kg) for repeat dose study. Data were presented as mean ± SD ( n = 8, 7, and 9 mice per group for WT treated with PBS, L.G6 pc −/− treated with eGFP, and L. G6pc −/− treated with hG6PC S298C mRNA, respectively). For statistical analysis of c–f , raw values were Log2 transformed and subjected to one-way ANOVA, followed by the Dunnett’s multiple comparisons test, compared to the eGFP mRNA treated group. P values are shown in the graphs ( c–f ). Source data are provided as a Source Data File.

Article Snippet: Antibodies against G6Pase-α were quantified on Nunc Immuno Maxisorp plates (ThermoFisher, #442404) coated with 0.5 μg/mL recombinant G6Pase-α protein (Viva Biotech) in 50 mM Na 2 CO 3 for 1 h at room temperature and blocked with SuperBlock (PBS) Blocking Buffer (ThermoFisher, #37515).

Techniques: Transformation Assay

Journal: Nature Communications

Article Title: mRNA therapy restores euglycemia and prevents liver tumors in murine model of glycogen storage disease

doi: 10.1038/s41467-021-23318-2

Figure Lengend Snippet: a Number of mice with tumors (left), number of tumors per mouse (middle), and tumor burden/area (right). Data were presented as mean ± s.e.m ( n = 21, 26, and 34 mice per group for wild-type (WT) treated with PBS, L. G6pc −/− treated with eGFP, and hG6PC S298C mRNA, respectively). b Liver morphology (tumor-circled in yellow) (top panels) and liver histology (bottom panels) of WT and L. G6pc −/− mice treated with either eGFP mRNA or h G6PC S298C mRNAs. c HCA/HCC biomarkers (protein expression). Results are expressed as mean ± SD ( n = 15, 27, and 35 mice per group for WT treated with PBS, L. G6pc −/− treated with eGFP, and L.G6 pc −/− treated with hG6PC S298C mRNA, respectively). For statistical analysis, raw values were subjected to one-way ANOVA, followed by the Dunnett’s multiple comparisons test, compared to the eGFP mRNA treated group. Statistically significant P values ( p ≤ 0.05) are shown in the graphs. Source data are provided as a Source Data File.

Article Snippet: Antibodies against G6Pase-α were quantified on Nunc Immuno Maxisorp plates (ThermoFisher, #442404) coated with 0.5 μg/mL recombinant G6Pase-α protein (Viva Biotech) in 50 mM Na 2 CO 3 for 1 h at room temperature and blocked with SuperBlock (PBS) Blocking Buffer (ThermoFisher, #37515).

Techniques: Expressing