Journal: bioRxiv

Article Title: Early endosome disturbance and endolysosomal pathway dysfunction in Duchenne muscular dystrophy

doi: 10.1101/2024.12.16.628552

Figure Lengend Snippet: The expression of Rab5 mRNA analyzed by RT-PCR (top) and Rab5 protein analyzed by western-blot (bottom) in (A) DMD myoblasts (n = 3 independent experiments) (B) dog muscles (n = 5 biopsies from 5 WT dogs and n=5 biopsies from 4 GRMD dogs) and (C) mdx mouse muscles (n = 4 mice per group). RT-qPCR were performed in duplicate. The data are represented as the mean ± SEM. Statistics: one-way ANOVA with a post hoc Bonferroni test for (A) DMD myoblasts comparison and unpaired Student t-test (two tailed) for (B) dog and (C) mice comparisons. (D) Western-blot analysis and quantification (n=3) of Rab5 protein showing the efficacy of Rab5 silencing in human DMDΔ45-52 myoblasts transfected with siRNA directed against Rab5 (siRab5) compared to DMD and control myoblasts (Ctrl) treated with a scrambled siRNA control (siScr). (E) The number of EEA1 positive endosomes is reduced in human DMD cells treated with siRab5, to a level equivalent to that of control cells (Ctrl). The data are represented as the mean ± SEM of 3 independent experiments for western-blot and at least 300 cells of three independent experiments analyzed on confocal images for early endosome counting. Statistics: one-way ANOVA with a post hoc Bonferroni test. *p<0.05, **p<0.01, ***p<0.01, ****p<0.0001. ns: non-significant.

Article Snippet: Membranes were blocked in Tris-buffered saline (TBS) 0.1% Tween-20 with 5% non-fat dry milk 1h at RT and incubated overnight at 4°C with mouse monoclonal antibody against Rab5 (1/500; Santa Cruz Biotechnology, Heidelberg, Germany), with mouse antibody against LAMP1 (1/1000; Santa Cruz Biotechnology, Heidelberg, Germany) or with rabbit monoclonal Actin antibody (1/1000; Sigma-Aldrich Chimie, Saint Quentin Fallavier, France).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Muscles, Quantitative RT-PCR, Comparison, Two Tailed Test, Transfection, Control

Journal: bioRxiv

Article Title: Early endosome disturbance and endolysosomal pathway dysfunction in Duchenne muscular dystrophy

doi: 10.1101/2024.12.16.628552

Figure Lengend Snippet: (A) The expression of Rab5 mRNA analyzed by RT-PCR (left) and Rab5 protein analyzed by western blot (right) in muscles of WT dogs (n=5 for mRNA or n=1 for protein), GRMD (n= 5 dogs for mRNA or n=2 dogs for protein), AAV-U7snRNA treated GRMD to restore dystrophin (n = 7 biopsies from 3 treated GRMD for mRNA, NaCl-treated controls (NaCl-GRMD, n=1), or n=2 biopsies per dog from 2 treated GRMD (for protein) showing that expression of Rab5 is decreased by dystrophin restoration in treated GRMD. Actin was used as a loading buffer in western blot. (B) Representative sections of muscle biopsies isolated from moderately affected GRMD and AAV-U7snRNA treated GRMD labeled with DAPI to mark nuclei (blue), anti-caveolin antibody to mark the plasma membrane (white) and anti-EEA1 antibody to mark early endosomes (green). Quantification of EEA1 positive puncta showed a significant decrease in early endosome staining in GRMD treated with an AAV-U7snRNA (n=2 biopsies from 1 treated GRMD) compared to NaCl-treated controls (NaCl-GRMD, n=1) showing that the therapeutically restored dystrophin allowed a partial restoration of early endosomes number compared to controls. The data are represented as the mean ± SEM of at least 500 fibers per dog analyzed on confocal images. Sale Bar = 50 µm. Statistics: one-way ANOVA with a post hoc Bonferroni test for panel A (Rab5 mRNA) and unpaired Student’s t-test (two tailed) for panel B (EEs quantification), *p<0.05; ****p<0.0001, ns: non-significant.

Article Snippet: Membranes were blocked in Tris-buffered saline (TBS) 0.1% Tween-20 with 5% non-fat dry milk 1h at RT and incubated overnight at 4°C with mouse monoclonal antibody against Rab5 (1/500; Santa Cruz Biotechnology, Heidelberg, Germany), with mouse antibody against LAMP1 (1/1000; Santa Cruz Biotechnology, Heidelberg, Germany) or with rabbit monoclonal Actin antibody (1/1000; Sigma-Aldrich Chimie, Saint Quentin Fallavier, France).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Muscles, Isolation, Labeling, Clinical Proteomics, Membrane, Staining, Two Tailed Test

Journal: The Journal of biological chemistry

Article Title: Purification and characterization of a nitric-oxide synthase from rat liver mitochondria.

doi: 10.1074/jbc.273.18.11044

Figure Lengend Snippet: FIG. 1. SDS-PAGE, native PAGE, and Western blot analysis of mtNOS. Panel A, SDS-PAGE was performed using a 10% polyacryl- amide precast gel (Novex, San Diego, CA) under reducing conditions. The proteins were stained with Coomassie Blue. The mitochondrial fractions were, from left to right: I, 8,000 3 g pellet; II, 150,000 3 g supernatant; III, NADPH eluate from the 29,59-ADP Sepharose 4B column. Mac-Lysate, a lysate of the mouse macrophage RAW 264.7 cell line. The molecular mass of protein markers is indicated in kDa. Panel B, native PAGE was performed using 4–15% gradient polyacrylamide gel stained with Coomassie Blue using PhastSystem from Amersham Pharmacia Biotech. In the same gel, catalase (232 kDa) was run under identical conditions. Panel C, for Western blot analysis, proteins were separated with SDS-PAGE gel under the conditions described under “Materials and Methods.” The proteins were transferred to nitrocellu- lose membranes, and later were incubated with mouse monoclonal antibodies against macNOS. The immunocomplexes were developed using enhanced horseradish peroxidase/luminol chemiluminescence re- action, detected with photographic film.

Article Snippet: The membranes were thoroughly washed with 0.05% Tween 20 in TBS, and incubated with mouse monoclonal antibody against macNOS (1/2, 500 in 0.05% Tween 20 in TBS) for 2 h. The membranes were extensively washed with 0.05% Tween 20 in TBS, and subsequently incubated with goat antibodies against mouse IgG conjugated with horseradish peroxidase (1/30,000; Bio-Rad) for 1 h. After washing the membranes with 0.05% Tween 20 in TBS, the immunocomplexes were developed using enhanced horseradish peroxidase/luminol chemiluminescence reaction, detected with photographic film (Hyperfilm ECL; Amersham Pharmacia Biotech) recorded after 30 s to 7 min of exposure.

Techniques: SDS Page, Clear Native PAGE, Western Blot, Staining, Incubation, Bioprocessing