Journal: Molecular Medicine

Article Title: d -Amino Acid Substitution of Peptide-Mediated NF-κB Suppression in mdx Mice Preserves Therapeutic Benefit in Skeletal Muscle, but Causes Kidney Toxicity

doi: 10.2119/molmed.2013.00141

Figure Lengend Snippet: Urinalysis from untreated and 8K-NBD peptide-treated mdx mice at multiple time points. Urine samples (7.5 μL) were electrophoresed on a 10% resolving SDS-PAGE gel followed by visualization of separated proteins by Coomassie Blue R-250 staining. Representative urine samples collected after 1 wk of treatment (early urinalysis) and after 4 wks of treatment (final urinalysis) are shown in (A). Immunoblotting for albumin in mouse urine using an anti-mouse albumin antibody is shown in the far right lane of (A), confirming presence of the 68-kDa albumin protein in urine. Densitometric measurements of the 68-kDa albumin band present in all urine samples (see arrow in [A]) is shown in (B) as average band density ± standard error. In (B), an asterisk (*) indicates a significant difference between early urinalysis groups compared with untreated mdx (white bars); two asterisks (**) indicate a significant difference between late urinalysis groups compared with untreated mdx mice (black bars) and § indicates a significant difference between early and late treatment samples in specified treatment groups (p < 0.05). To represent relative amounts and timing of proteinuria in 8K-NBD peptide-treated mdx mice, the total number of protein bands after SDS-PAGE electrophoresis of urine samples from each mouse in the study was quantified and the data is presented as the average number of protein bands present in the gel ± standard error (C). In (C), an asterisk (*) indicates the 8K-NBD peptide treatment group is significantly different than untreated mdx (p < 0.05). In (D), 10 μL urine samples, collected from mdx mice prior to, and 30 min and 24 h after 1, 2 or 3 high-dose, d-8K-wild-type-NBD peptide injections were electrophoresed and stained as in (A) to show early onset of severe proteinuria in this treatment group. Age-matched mdx mice served as the untreated mdx group. n = number of animals per group.

Article Snippet: The albumin band was confirmed by immunoblotting with a 1:1000 diluted goat anti-mouse albumin antibody (Bethyl Laboratories Inc.), followed by a 1:30,000 diluted IRDye 800CW goat anti-mouse IgG (H + L) conjugated secondary antibody (LI-COR Biosciences).

Techniques: SDS Page, Staining, Western Blot, Electrophoresis