Journal:

Article Title: The Pharmacological Chaperone 1-Deoxygalactonojirimycin Reduces Tissue Globotriaosylceramide Levels in a Mouse Model of Fabry Disease

doi: 10.1038/mt.2009.220

Figure Lengend Snippet: hR301Q α-Gal A Tg/KO mice have reduced α-Gal A and elevated GL-3 levels. (a) α-Gal A and GL-3 levels were assessed in tissue lysates prepared from skin, heart, and kidney of 12-week-old male GLA KO (open bars), hR301Q α-Gal A Tg/KO (gray bars), and wild-type C57BL/6 (black bars) mice. α-Gal A activity in hR301Q α-Gal A Tg/KO and GLA KO mice was significantly lower than that of wild-type mice (*P < 0.05, t-test); α-Gal A activity in hR301Q α-Gal A Tg/KO mice was significantly higher than that of GLA KO mice (#P < 0.05, t-test). GL-3 levels in hR301Q α-Gal A Tg/KO and GLA KO mice were significantly higher than those of wild-type mice (*P < 0.05, t-test). Each bar represents the mean ± SEM from four mice per group analyzed in triplicate. (b) Cell-type specific GL-3 staining was assessed by immunohistochemistry in GLA KO, hR301Q α-Gal A Tg/KO, and wild-type C57BL/6 mouse tissues. GL-3 staining is represented as brown spots (black arrows); nuclei are represented as green spots (stained with methyl green). The data shown are representative photomicrographs from four 12-week-old male mice using ×20 magnification, except for the glomeruli, which are representative of four 7-month-old male mice using ×40 magnification. α-Gal A, α-galactosidase A; GL-3, globotriaosylceramide; KO, knockout; Tg, transgenic.

Article Snippet: 49 Western blotting of tissue α-Gal A. Tissue lysates (50 µg total protein) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis on 12% polyacrylamide gels (Bio-Rad, Hercules, CA), transferred to polyvinylidene fluoride membranes (Bio-Rad), and immunoblotted with rabbit antihuman α-Gal A primary antibody (1:1,000 dilution).

Techniques: Activity Assay, Staining, Immunohistochemistry, Knock-Out, Transgenic Assay

Journal:

Article Title: The Pharmacological Chaperone 1-Deoxygalactonojirimycin Reduces Tissue Globotriaosylceramide Levels in a Mouse Model of Fabry Disease

doi: 10.1038/mt.2009.220

Figure Lengend Snippet: DGJ administration increases α-Gal A activity and reduces GL-3 in hR301Q α-Gal A Tg/KO mice. Eight-week-old male hR301Q α-Gal A Tg/KO mice were administered DGJ ad libitum in drinking water for 4 weeks at the indicated doses. Lysates from skin, heart, and kidney were subsequently tested for (a) α-Gal A activity and (b,c) GL-3 levels. (a) Significant increases in α-Gal A activity were seen in all three tissues (*P < 0.05 versus untreated, t-test). The effect was also significant for a linear trend, indicating a dose-dependent increase in α-Gal A activity (post hoc analysis, P < 0.05). Insets: Tissue α-Gal A protein levels were determined by western blotting. A dose-dependent increase in the 46 kd mature form of α-Gal A was seen in all tissues. Each lane on the blot contains tissue lysate from a single animal. The western blot shown is representative of two experiments. (b) Significant reductions in GL-3 levels were seen in all three tissues as measured by LC-MS/MS (*P < 0.05 versus untreated, t-test). Blue bars (a,b) represent α-Gal A and GL-3 levels from age-matched untreated wild-type C57BL/6 mice. Each bar represents pooled data from three independent studies with the mean ± SEM of 21 hR301Q α-Gal A Tg/KO mice/group or four untreated wild-type mice, all analyzed in triplicate. (c) Cell type–specific reduction of GL-3 in hR301Q α-Gal A Tg/KO mice administered DGJ (100 mg/kg per day) ad libitum in drinking water was assessed by immunohistochemistry. GL-3 staining is represented as brown spots denoted with black arrows. The data shown are representative photomicrographs from 7–8 mice/group (magnification: ×20, insets: ×40). α-Gal A, α-galactosidase A; DGJ, 1-deoxygalactonojirimycin; GL-3, globotriaosylceramide; KO, knockout; Tg, transgenic.

Article Snippet: 49 Western blotting of tissue α-Gal A. Tissue lysates (50 µg total protein) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis on 12% polyacrylamide gels (Bio-Rad, Hercules, CA), transferred to polyvinylidene fluoride membranes (Bio-Rad), and immunoblotted with rabbit antihuman α-Gal A primary antibody (1:1,000 dilution).

Techniques: Activity Assay, Western Blot, Liquid Chromatography with Mass Spectroscopy, Immunohistochemistry, Staining, Knock-Out, Transgenic Assay

Journal:

Article Title: The Pharmacological Chaperone 1-Deoxygalactonojirimycin Reduces Tissue Globotriaosylceramide Levels in a Mouse Model of Fabry Disease

doi: 10.1038/mt.2009.220

Figure Lengend Snippet: Long-term (24-week) administration of DGJ results in greater GL-3 reduction compared to 4-week administration. (a) Four-week-old male hR301Q α-Gal A Tg/KO mice were administered DGJ ad libitum in drinking water for 24 weeks at the indicated doses. GL-3 levels were subsequently measured in skin, heart, and kidney lysates by LC-MS/MS. Significant reductions in GL-3 were seen in all three tissues (*P < 0.05 versus untreated, t-test). The effect was also significant for a linear trend, indicating a dose-dependent decrease in GL-3 levels (post hoc analysis, P < 0.05). Each bar represents the mean ± SEM of 7–8 mice/dose group analyzed in triplicate. (b) Cell type–specific GL-3 reductions were assessed by immunohistochemistry. GL-3 staining is represented as brown spots. Data shown are representative photomicrographs from 7–8 mice/group (magnification: ×20, except glomeruli: ×40). DGJ, 1-deoxygalactonojirimycin; GL-3, globotriaosylceramide; KO, knockout; Tg, transgenic.

Article Snippet: 49 Western blotting of tissue α-Gal A. Tissue lysates (50 µg total protein) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis on 12% polyacrylamide gels (Bio-Rad, Hercules, CA), transferred to polyvinylidene fluoride membranes (Bio-Rad), and immunoblotted with rabbit antihuman α-Gal A primary antibody (1:1,000 dilution).

Techniques: Liquid Chromatography with Mass Spectroscopy, Immunohistochemistry, Staining, Knock-Out, Transgenic Assay

Journal:

Article Title: The Pharmacological Chaperone 1-Deoxygalactonojirimycin Reduces Tissue Globotriaosylceramide Levels in a Mouse Model of Fabry Disease

doi: 10.1038/mt.2009.220

Figure Lengend Snippet: Effect of 4- and 24-week DGJ administration on tissue GL-3 levels in hR301Q α-Gal A Tg/KO mice

Article Snippet: 49 Western blotting of tissue α-Gal A. Tissue lysates (50 µg total protein) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis on 12% polyacrylamide gels (Bio-Rad, Hercules, CA), transferred to polyvinylidene fluoride membranes (Bio-Rad), and immunoblotted with rabbit antihuman α-Gal A primary antibody (1:1,000 dilution).

Techniques:

Journal:

Article Title: The Pharmacological Chaperone 1-Deoxygalactonojirimycin Reduces Tissue Globotriaosylceramide Levels in a Mouse Model of Fabry Disease

doi: 10.1038/mt.2009.220

Figure Lengend Snippet: Less frequent DGJ administration results in greater GL-3 reduction. (a) Eight-week-old male hR301Q α-Gal A Tg/KO mice were administered drinking water (red dotted lines) or DGJ [100 mg/kg per day (red solid lines) ad libitum in drinking water] for 4 weeks, followed by a washout period (drinking water only) of up to 7 days. Groups of mice were then euthanized on days 0, 1, 3, 5, or 7 after DGJ withdrawal. Skin, heart, and kidney were collected at each time point, and α-Gal A activity and DGJ levels (blue lines) were measured in tissue lysates. Significantly increased α-Gal A activity was sustained in all three tissues for up to 7 days (*P < 0.05 versus untreated, t-test). For the α-Gal A activity, each data point represents the mean ± SEM of 7–8 mice/time point analyzed in triplicate; data were normalized to untreated levels. For tissue DGJ levels, each data point represents the mean ± SEM for five mice/time point. The limit of quantitation for DGJ in skin, heart, and kidney was 6, 4, and 10 ng/g, respectively. (b) Eight-week-old male hR301Q α-Gal A Tg/KO mice were administered DGJ (300 mg/kg per day) ad libitum in drinking water for 4 weeks either daily or less frequently using four cycles of a “4 on/3 off” regimen. GL-3 levels were subsequently measured in lysates from skin, heart, and kidney by LC-MS/MS. Significant reductions in GL-3 levels were seen in all tissues (*P < 0.05 versus untreated, t-test). GL-3 levels in skin, heart, and kidney also showed significantly greater reductions with less frequent compared to daily administration (#P < 0.05 daily versus “4 on/3 off”, t-test). Each bar represents pooled data from three independent studies with the mean ± SEM of 21 mice/group analyzed in triplicate. DGJ, 1-deoxygalactonojirimycin; GL-3, globotriaosylceramide; KO, knockout; Tg, transgenic.

Article Snippet: 49 Western blotting of tissue α-Gal A. Tissue lysates (50 µg total protein) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis on 12% polyacrylamide gels (Bio-Rad, Hercules, CA), transferred to polyvinylidene fluoride membranes (Bio-Rad), and immunoblotted with rabbit antihuman α-Gal A primary antibody (1:1,000 dilution).

Techniques: Activity Assay, Quantitation Assay, Liquid Chromatography with Mass Spectroscopy, Knock-Out, Transgenic Assay

Journal:

Article Title: The Pharmacological Chaperone 1-Deoxygalactonojirimycin Reduces Tissue Globotriaosylceramide Levels in a Mouse Model of Fabry Disease

doi: 10.1038/mt.2009.220

Figure Lengend Snippet: Long-term DGJ administration reduces accumulated tissue GL-3 in aged hR301Q α-Gal A Tg/KO mice. Twenty-four-week-old male hR301Q α-Gal A Tg/KO mice were administered DGJ ad libitum in drinking water for 24 weeks either daily or less frequently using a “4 on/3 off” regimen at the indicated doses. GL-3 levels were measured in lysates of skin, heart, and kidney by LC-MS/MS. Dose-dependent and significant reductions in GL-3 were seen in all three tissues (*P < 0.05 versus untreated, t-test). Reduction of GL-3 in skin and heart was significantly greater after less frequent compared to daily administration of 300 mg/kg DGJ (#P < 0.05 daily versus “4 on/3 off”, t-test). Each bar represents the mean ± SEM of 7–8 mice/group analyzed in triplicate. DGJ, 1-deoxygalactonojirimycin; GL-3, globotriaosylceramide; KO, knockout; Tg, transgenic.

Article Snippet: 49 Western blotting of tissue α-Gal A. Tissue lysates (50 µg total protein) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis on 12% polyacrylamide gels (Bio-Rad, Hercules, CA), transferred to polyvinylidene fluoride membranes (Bio-Rad), and immunoblotted with rabbit antihuman α-Gal A primary antibody (1:1,000 dilution).

Techniques: Liquid Chromatography with Mass Spectroscopy, Knock-Out, Transgenic Assay

Journal:

Article Title: The Pharmacological Chaperone 1-Deoxygalactonojirimycin Reduces Tissue Globotriaosylceramide Levels in a Mouse Model of Fabry Disease

doi: 10.1038/mt.2009.220

Figure Lengend Snippet: Effect of DGJ on plasma GL-3 levels in hR301Q α-Gal A Tg/KO mice

Article Snippet: 49 Western blotting of tissue α-Gal A. Tissue lysates (50 µg total protein) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis on 12% polyacrylamide gels (Bio-Rad, Hercules, CA), transferred to polyvinylidene fluoride membranes (Bio-Rad), and immunoblotted with rabbit antihuman α-Gal A primary antibody (1:1,000 dilution).

Techniques:

Journal:

Article Title: The Pharmacological Chaperone 1-Deoxygalactonojirimycin Reduces Tissue Globotriaosylceramide Levels in a Mouse Model of Fabry Disease

doi: 10.1038/mt.2009.220

Figure Lengend Snippet: DGJ increases α-Gal A and decreases GL-3 levels in brain of aged hR301Q α-Gal A Tg/KO mice. Twenty-four-week-old male hR301Q α-Gal A Tg/KO mice were administered DGJ ad libitum in drinking water for 12 weeks either daily or less frequently using a “4 on/3 off” regimen at the indicated doses. Brain α-Gal A and GL-3 levels were subsequently measured. Significant increases in α-Gal A and concomitant reductions in GL-3 were seen with DGJ administration (*P < 0.05 versus untreated, t-test). Each bar represents the mean ± SEM of 7–8 mice/group analyzed in triplicate. DGJ, 1-deoxygalactonojirimycin; GL-3, globotriaosylceramide; KO, knockout; Tg, transgenic.

Article Snippet: 49 Western blotting of tissue α-Gal A. Tissue lysates (50 µg total protein) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis on 12% polyacrylamide gels (Bio-Rad, Hercules, CA), transferred to polyvinylidene fluoride membranes (Bio-Rad), and immunoblotted with rabbit antihuman α-Gal A primary antibody (1:1,000 dilution).

Techniques: Knock-Out, Transgenic Assay

Journal: Frontiers in neuroscience

Article Title: Fluorescence microscopy-based sensitive method to quantify dopaminergic neurodegeneration in a Drosophila model of Parkinson's disease.

doi: 10.3389/fnins.2023.1158858

Figure Lengend Snippet: FIGURE 3 Characterization of DAergic neurodegeneration in the whole fly brain through anti-TH antibody immunostaining and quantification of brain TH protein using western blotting: the image depicts the whole brain mount of the adult Drosophila under control and PQ-treated conditions (A) (CTR, Control; TD, PQ-treated). Quantification of DAergic neurons reveals that the neuronal number remains unaffected (B,C), whereas the fluorescence intensity (of fluorescently labeled secondary antibodies that target primary antibody anti-TH) is significantly decreased in all the clusters (D), and in toto (E) between the control and treated group. The scale bar of the brain images in the panel is 20 μm. (CTR, Control; TD, Treated with 10 mM PQ; Represented images are “merged” Z-stacking images; however, the quantification of DA neuronal number and fluorescence intensity is performed in 3D Z-stack images; PAL, Protocerebral anterior lateral; PPL, Protocerebral posterior lateral; PPM, Protocerebral posterior medial). (F) Stain Free Western Blot analysis shows a reduction of brain TH protein (15%) upon PQ treatment in the fly model of PD [Bio-Rad Stain-Free Western Blotting using total protein normalization method (TPN) (M-protein ladder; CTR-control; PQ- paraquat treated)]. Statistical analysis was performed using a t-test (compared to control). *p < 0.05, **p < 0.01, ***p < 0.001; NS, not-significant.

Article Snippet: The scale bar of the brain images in the panel is 20 μm. (CTR, Control; TD, Treated with 10 mM PQ; Represented images are “merged” Z-stacking images; however, the quantification of DA neuronal number and fluorescence intensity is performed in 3D Z-stack images; PAL, Protocerebral anterior lateral; PPL, Protocerebral posterior lateral; PPM, Protocerebral posterior medial). (F) Stain Free Western Blot analysis shows a reduction of brain TH protein (15%) upon PQ treatment in the fly model of PD [Bio-Rad Stain-Free Western Blotting using total protein normalization method (TPN) (M-protein ladder; CTR-control; PQ- paraquat treated)].

Techniques: Immunostaining, Western Blot, Control, Fluorescence, Labeling, Staining