β dystroglycan  (Developmental Studies Hybridoma Bank)

Journal: Skeletal Muscle

Article Title: Functional and structural pathologies in skeletal muscle of a rat model of Duchenne muscular dystrophy

doi: 10.1186/s13395-026-00419-4

Figure Lengend Snippet: CRISPR/Cas9-mediated deletion of full-length rat dystrophin. A Organization of the rat Dmd that spans ~ 2.3 mbp of the X chromosome. The 5 exons targeted for removal – 22–26 (in gray) and the two flanking exons (21 & 27 – in green) are shown in more detail (WT). The two guide RNAs (5’ and 3’ gRNAs) aided in the Cas9-mediated deletion of 18,307 bps that include exons 22–26 (MDR). B The 5’ and 3’ untranslated regions (unfilled magenta boxes) flank the 79 exons (filled magenta boxes) that contribute coding sequence of full-length dystrophin protein. Indicated with a closer view of exons 21–27 is the number of nucleotides each exon contributes (in parentheses). The 5 deleted exons (in gray) together contribute 800 bp of protein coding sequence, whose removal leads to a frame shift and a premature termination codon within the region of the transcript coded by exon 27 (*, MDR). C Immunoblotting confirms loss of full-length dystrophin (427 kDa; black arrow) and presence of predicted 106 kDa N-terminal fragment (gray double arrow) in 4 mo MDR gastrocnemius and heart lysates, and D immunofluorescence confirms loss of dystrophin staining at the sarcolemma of muscle fibers and cardiomyocytes (scale bars = 25 μm). E-F Immunoblotting was also performed for dystrophin glycoprotein complex (DGC) members β-dystroglycan (β-DG), α-dystrobrevin (α-DB), and syntrophins ( n = 3). Data are displayed as mean ± SEM with individual values and were analyzed using Student’s T-tests (α = 0.05; * p < 0.05, ** p < 0.01, *** p < 0.001)

Article Snippet: The following primary antibodies were used for immunofluorescence in the present study: dystrophin (1:100; MANEX1011B, Clone 1C7; DSHB), β-dystroglycan (1:100; MANDAG2, Clone 7A11; DSHB), utrophin (1:50; VP-U579; Vector Laboratories), α-sarcoglycan (1:50; VP-A105; Vector Laboratories); syntrophins (1:2000; #11425; Abcam), perilipin 1 (1:500; #9349; Cell Signaling Technology), anti-Laminin β2 antibody (1:10,000; [ ]), embryonic MHC (1:100; F1.652; DSHB), type I myofibers (1:50; BA-D5; DSHB), Type IIa myofibers (1:50; SC-71; DSHB), type IIx myofibers (1:20; 6H1; DSHB), pan-muscle MHC(1:100; MF 20; DSHB), COMP (1:800; #NBP2-92658; Noveus Biologicals), α-SMA (1:1000; #7817; Abcam), SMOC2 (1:500; #AF5140; R&D Systems), and PDGFRα (1:500; #AF1062; R&D Systems).

Techniques: CRISPR, Sequencing, Western Blot, Immunofluorescence, Staining

Journal: Skeletal Muscle

Article Title: Functional and structural pathologies in skeletal muscle of a rat model of Duchenne muscular dystrophy

doi: 10.1186/s13395-026-00419-4

Figure Lengend Snippet: CRISPR/Cas9-mediated deletion of full-length rat dystrophin. A Organization of the rat Dmd that spans ~ 2.3 mbp of the X chromosome. The 5 exons targeted for removal – 22–26 (in gray) and the two flanking exons (21 & 27 – in green) are shown in more detail (WT). The two guide RNAs (5’ and 3’ gRNAs) aided in the Cas9-mediated deletion of 18,307 bps that include exons 22–26 (MDR). B The 5’ and 3’ untranslated regions (unfilled magenta boxes) flank the 79 exons (filled magenta boxes) that contribute coding sequence of full-length dystrophin protein. Indicated with a closer view of exons 21–27 is the number of nucleotides each exon contributes (in parentheses). The 5 deleted exons (in gray) together contribute 800 bp of protein coding sequence, whose removal leads to a frame shift and a premature termination codon within the region of the transcript coded by exon 27 (*, MDR). C Immunoblotting confirms loss of full-length dystrophin (427 kDa; black arrow) and presence of predicted 106 kDa N-terminal fragment (gray double arrow) in 4 mo MDR gastrocnemius and heart lysates, and D immunofluorescence confirms loss of dystrophin staining at the sarcolemma of muscle fibers and cardiomyocytes (scale bars = 25 μm). E-F Immunoblotting was also performed for dystrophin glycoprotein complex (DGC) members β-dystroglycan (β-DG), α-dystrobrevin (α-DB), and syntrophins ( n = 3). Data are displayed as mean ± SEM with individual values and were analyzed using Student’s T-tests (α = 0.05; * p < 0.05, ** p < 0.01, *** p < 0.001)

Article Snippet: Protein extraction and immunoblotting were performed on snap-frozen heart and gastrocnemius muscles, as previously described [ – ], using the following primary antibodies: dystrophin [1:100; MANHINGE1B, Clone 10F9; Developmental Studies Hybridoma Bank (DSHB)], dystrophin (1:100; MANEX1011B; Clone 1C7; DSHB), dystrophin (1:100; MANEX1011C; Clone 4F9; DSHB), β-dystroglycan (1:1000; 11017-1-AP; ProteinTech), dystrobrevin (1:1000; #610766; BD Biosciences), syntrophins (1:2000; #11425; Abcam), and perilipin 1 (1:500; #9349; Cell Signaling Technology).

Techniques: CRISPR, Sequencing, Western Blot, Immunofluorescence, Staining