Results: 5

1.
Figure 4

Figure 4. Apoptotic MRC5-SV2 cells overexpressing 462S demonstrate increased caspase-mediated cleavage as compared to 462P wild-type. From: An Ethnic-Specific Polymorphism in the Catalytic Subunit of Glutamate-Cysteine Ligase Impairs the Production of Glutathione Intermediates In Vitro.

A) Transfected MRC5-SV2 cells were incubated with staurosporine to induce apoptosis. α-GCLC western blot analysis was performed on lysates. Molecular weight in kD is indicated on left. B) Band signals were quantitated and cleaved:total protein ratios were calculated. Graph values indicate mean cleaved:total protein ratios ± SEM over several experiments. P-value was calculated using an unpaired t-test.

Truc M. Le, et al. Mol Genet Metab. ;101(1):55-61.
2.
Figure 5

Figure 5. Apoptotic MRC5-SV2 cells overexpressing 462S undergo increased DNA fragmentation as compared to wild-type. From: An Ethnic-Specific Polymorphism in the Catalytic Subunit of Glutamate-Cysteine Ligase Impairs the Production of Glutathione Intermediates In Vitro.

Transfected MRC5-SV2 cells were incubated with staurosporine to induce apoptosis. Cell lysates were collected, and DNA was separated on a 1% agarose gel and visualized by UV light following ethidium bromide staining. Similar results were obtained using Hœchst nuclear staining (data not shown).

Truc M. Le, et al. Mol Genet Metab. ;101(1):55-61.
3.
Figure 3

Figure 3. Mammalian cells overexpressing the 462S GCLC enzyme have significantly lower intracellular glutathione levels compared to cells overexpressing the wild-type enzyme. From: An Ethnic-Specific Polymorphism in the Catalytic Subunit of Glutamate-Cysteine Ligase Impairs the Production of Glutathione Intermediates In Vitro.

Plasmids encoding either the wild-type or 462S GCLC polymorphism were transiently transfected into MRC5-SV2 cells and incubated for 48 hours. Total glutathione levels were measured in the cell lysate. Bar graphs indicate percent increase in glutathione levels relative to mock-transfected MRC5 lysate. Graph values represent the mean ± SEM over several experiments. P-values were calculated using a paired t-test.

Truc M. Le, et al. Mol Genet Metab. ;101(1):55-61.
4.
Figure 2

Figure 2. Mammalian cell-expressed GCLC 462S enzyme has significantly decreased enzymatic activity in vitro. From: An Ethnic-Specific Polymorphism in the Catalytic Subunit of Glutamate-Cysteine Ligase Impairs the Production of Glutathione Intermediates In Vitro.

Plasmids encoding either the 462P wild-type or 462S GCLC polymorphism were transiently transfected into MRC5-SV2 cells and incubated for 48 hours. γ-Glutamylcysteine levels in the cell media were measured using an amino acid analyzer. Bar graphs indicate proportion of γ-GC production relative to control MRC5 lysate. Graph values represent the mean ± SEM over several experiments. P-values are based on the difference between the two polymorphisms and were calculated using a paired t-test.

Truc M. Le, et al. Mol Genet Metab. ;101(1):55-61.
5.
Figure 1

Figure 1. Bacterially-expressed GCLC 462S enzyme has significantly decreased enzymatic activity in vitro. From: An Ethnic-Specific Polymorphism in the Catalytic Subunit of Glutamate-Cysteine Ligase Impairs the Production of Glutathione Intermediates In Vitro.

Plasmids encoding either the 462P wild-type or 462S GCLC polymorphism were used in an E. coli expression system. Bacterial lysates were used to produce γ-glutamylcysteine, which was measured using a fluorescent-based assay. Bar graphs indicate proportion of activity relative to an equivalent amount of endogenous E. coli lysate. Graph values represent the mean ± SEM over several experiments. P-values are based on the difference between the two polymorphisms and were calculated using a paired t-test.

Truc M. Le, et al. Mol Genet Metab. ;101(1):55-61.

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