Journal: Science translational medicine
Article Title: Linker proteins restore basement membrane and correct LAMA2-related muscular dystrophy in mice
Figure Lengend Snippet: Muscles of LAMA2 MD patients and dy W /dy W mice contain high amounts of laminin-α4 and show deficits in BM ( A ) Representative immunofluorescence images (magnified images in insets) of human muscle biopsy cross sections stained for laminin-α4 and laminin-β1γ1. n = 3 LAMA2 MD patients or healthy controls. ( B ) Western blot analysis and quantification of laminin chains in human muscle biopsies. α-Actinin was used as loading control. n = 4 controls, n = 3 LAMA2 MD patients. ( C ) Representative immunofluorescence images (magnified images in insets) of triceps muscle from 8-week-old mice stained for laminin-α4 and laminin-β1γ1. n = 4 mice per genotype. ( D ) Western blot analysis and quantification of laminin-α4 (left graph), laminin-β1γ1 (middle graph), and Lama4 transcripts (right graph) from triceps muscle of 8-week-old mice. n = 3 mice per genotype (Western blot) and n = 4 mice per genotype (quantitative reverse transcription polymerase chain reaction). ( E ) Schematic of subcellular fractionation. ( F ) Western blot analysis of S1 to S4 of a muscle biopsy from a control patient. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Na + /K + -ATPase (sodium-potassium adenosine triphosphatase) were used as markers for cytosolic and membrane proteins, respectively. ( G ) Western blot analysis of S3 and S4 fractions from control and LAMA2 MD patient muscle biopsies probed for the indicated proteins. Right: Ratio of the laminin-β1γ1 intensity in S3 and S4 fraction. n = 4 healthy controls, n = 3 LAMA2 MD patients. Data are means ± SEM. * P
Article Snippet: For immunostaining and Western blot analysis, the following antibodies were used: α-actinin (1:5000; Sigma, catalog no. A7732), agrin [chicken, for detection of mag, produced in-house , 1:1000 for Western blots, 1:200 for immunostainings], eMHC (1:100 for Western blot, 1:1200 for immunostainings; developed by H. Blau, obtained from Developmental Studies Hybridoma Bank, catalog no. F1.652), F4/80 (1:100; Abcam, catalog no. ab6640), laminin-α1 (for detection of αLNNd by Western blot, 1:2000; R & D Systems, catalog no. AF4187), αLNNd [for detection of αLNNd by immunostainings, 1:100 ( )], laminin-α2 [for Western blot analysis , 1:500], laminin-α2 (N-terminal, for immunostainings of human samples, clone 4H8-2, 1:400; Sigma, catalog no. L0663), laminin-α2 (C-terminal, for immunostainings of human samples, clone 5H2, 1:5000; Merck Millipore, catalog no. MAB1922), laminin-α4 ( ) (1:1000 for Western blots, 1:200 for immunostainings), laminin-α5 [clone 504, gift from L. Sorokin , 1:1000], laminin-β1γ1 (1:1000 for Western blots, 1:100 for immunostainings; Sigma, catalog no. L9393), laminin-γ1 (1:200; Millipore, catalog no. MAB1914), Na+ /K+ -ATPase (1:1000; Cell Signaling, catalog no. 3010), GAPDH (1:1000; Cell Signaling, catalog no. 2118), and CD31 (1:100; Abcam, catalog no. ab9498).
Techniques: Mouse Assay, Immunofluorescence, Staining, Western Blot, Reverse Transcription Polymerase Chain Reaction, Fractionation