Ncl dysb

Isopentane-frozen diaphragm strip, heart, and quadriceps muscle specimens obtained at the conclusion of the study were sectioned at 8-μm thickness and immunostained per standard techniques for dystrophin/microdystrophin (Leica, NCL-DYSB) as previously described. (Hamm et al., 2021 (link)). Evaluation of percent dystrophin- or microdystrophin-positive fibers was performed by a neuropathologist using a standard fluorescent microscope and estimated to the nearest 5%.

For dystrophin immunoblots, denaturated protein samples from cell lysates were run in 4–10% gradient polyacrylamide gel for 70 min/170 V/400 mA, blotted at 100 V/300 mA for 2 h and labeled with antibody against N terminal (NCL-DYSB, Leica) and C terminal (ab15277, abcam) of dystrophin protein. As a loading control, tubulin or laminB were used. Human heart atrial tissue was obtained as procedural surplus material, available during bicaval orthotopic heart transplantation from donor organs. For western blot analysis, the sample was homogenized and dissolved in RIPA buffer (10 mM Tris-Cl (pH 8.0), 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl, 1 mM phenylmethanesulfonyl fluoride) and the supernatant was used.
For DNA repair protein expression, 10% polyacrylamide gel was run for 60 min/170 V/400 mA and blotted for 1 h. Proteins were labeled with antibodies against ligase1, ligase 3, ligase 4, NBS1, Rad51 and APE1 in dilution as stated in Supplementary material (Table S10).

Immunohistochemical analysis involved the use of immunoperoxidase techniques on frozen sections. MANDYS110 clone 3H10, provided by the Wolfson Centre for Inherited Neuromuscular Disease, NCL-DYSB and NCL-DYS2 (Novocastra Laboratories, Newcastle on Tyne, UK) mouse monoclonal antibodies were used for dystrophin protein detection (1∶50 for each ones). Other primary antibodies used were IVD3₁A9 (1∶50, Developmental Studies Hybridoma Bank, Iowa City, IA), NCL-g-Sarc (1∶10, Novocastra Laboratories) and NCL-DRP2 (1∶60, Novocastra Laboratories) for alpha-sarcoglycan, gamma-sarcoglycan, and utrophin detection respectively, NCL-MHCd (1∶20, Novocastra Laboratories) for developmental myosin heavy chain isoform, anti-complement 5b-9 (1∶250, Calbiochem, Strasbourg, France) and anti-CD3 (1∶100, Dako, Glostrup, Denmark) for lymphocytes. Briefly, transverse cryosections were incubated in PBS with 5% normal goat serum (Dako) for 1 hour at room temperature. They were then incubated with primary antibody in 5% rat serum overnight at 4°C and with biotinylated secondary antibodies (E433, 1∶300, Dako) in PBS with 5% rat serum for 1 hour. Bound antibodies were detected either with streptavidin (P397; Dako) and DAB Liquid Substrate (Dako) for immunoperoxidase. Fiber type was determined using histochemical myosin-ATPase reaction after preincubation at pH 4.2, 4.35, and 10.4 as previously described [22] .