Ncl dys1

Protein extracts were loaded onto a NuPAGE Tris‐Acetate Minigel 3–8% 1 mm (Invitrogen). Running and blotting were performed in an XCell SureLock Minicell (Invitrogen) according to the manufacturer's instructions, and proteins were transferred to a nitrocellulose transfer membrane (Amersham Protran). Membranes were blocked with 10% non‐fat dry milk and incubated O/N with primary antibodies. Protein detection was carried out with Clarity ECL Western Blotting Substrate (Bio‐Rad). Primary antibodies were as follows: anti‐dystrophin (NCL‐DYS1 Novocastra Laboratories, 1:80); anti‐actinin (ACTN sc‐15335 Santa Cruz, 1:1,000) Secondary antibody was as follows: ImmunoPure Goat Anti‐Mouse IgG Peroxidase Conjugated (31430 Pierce, 1:10,000).

Immunostaining of muscle biopsy samples was performed as previously described [31 (link)]. Briefly, muscle sections were thawed at room temperature and then fixed in 100% cold acetone for 10 min. In order to reveal only the proteins trapped in aggregates, the muscle sections were then incubated in 1 M potassium chloride (KCl) to remove all soluble proteins. Primary antibodies against PABPN1 (ab75855 Abcam; 1:100), HSP70 (MA1-90,504 Invitrogen; 1:100), PRMT1 (ab73246 Abcam; 1:100), dystrophin (NCL-dys1 Novocastra; 1:10), laminA/C (ab40567 Abcam; 1:100), p62/SQSTM1 (MBL PM045 1:200), RPL24 (GTX114729 1:100), GRP78/BiP (CST3177 Cell signaling 1:200) were then incubated overnight at 4 °C. When necessary a directly conjugated PABPN1-488 antibody (ab206056 Abcam) was used. The next day, the muscle sections were rinsed and incubated with the appropriated fluorescent secondary antibodies prior to counterstaining nuclei with Hoechst. For the eMyHC, spectrin and laminA/C staining, slides were thawed at RT and blocked in PBS containing 2% FBS for 30 min at RT. Primary antibodies for embryonic myosin (F1.652 DSHB; 1:4), human spectrin (NCL-SPEC Novocastra; 1:50) human laminA/C (ab40567 Abcam, 1:300) and laminin (Z0097 Dako; 1:400) were incubated for 1 h at RT, sections were rinsed and incubated with appropriate secondary antibodies prior to counterstaining with Hoechst.

Frozen sections of 7 micrometer thickness from muscle biopsy specimens were processed for histology, histochemistry, and immunohistochemistry according to standard protocols [48 ].
Regarding the detection of sarcolemmal and sarcolemma-associated proteins, immunohistochemistry was performed by using antibodies targeting different dystrophin epitopes: NCL-DYS1 (clone: DY4/6D3), NCL-DYS2 (clone: DY8/6C5) and NCL-DYS3 (clone: DY10/12B2), corresponding to central/core, C-terminal and N-terminal regions, respectively (Novocastra, Newcastle, UK, primary antibody dilution was 1:20 in all cases). The following antibodies were used targeting sarcoglycan alpha, beta, gamma, and delta: NCL-L-a-SARC, clone AD1/20A6; NCL-L-b-SARC, clone BEATASARC1/5B1; NCL-g-SARC, clone 35DAG/21B5; NCL-d-SARC, clone DELTASARC/12C1; (Novocastra, 1:50, 1:100, 1:100, and 1:50, respectively); merosin: NCL-MEROSIN, clone: MER3/22B2 (Novocastra, 1:100) and spectrin NCL-SPEC1, clone RBC2/3D5 (Novocastra, 1:100). Primary antibodies were incubated on slides for 1 h at room temperature.
As negative control, a muscle biopsy sample from an ALS (Amyotrophic Lateral Sclerosis) patient was used. We have succesfully applied the settings described above to detect dystrophinopathy recently [49 (link)].

For dystrophin protein quantification, transverse cryosections (8 μm thick) were lysed in buffer containing 75 mm/l Tris–HCl (pH 6.5), 10% sodium dodecyl sulphate, 5% 2-mercaptoethanol, centrifuged at 13 000 rpm for 10 min and supernatant was collected and heated at 100°C for 3 min. Proteins were resolved on a 3–8% Tris–Acetate gel (Invitrogen) and transferred to PVDF membranes for 100 minutes at 30 V (Millipore, Hertfordshire, UK). Subsequently, membranes were probed with monoclonal anti-dystrophin (1:200, NCL-DYS1, Novocastra, Milton Keynes, UK) and anti-vinculin (loading control, 1:100 000, hVIN-1, Sigma) antibodies and detected with secondary antibody IRDye 800CW goat anti-mouse (LiCOR, Cambridge, UK). Fluorescence was recorded using the Odyssey imaging system. To quantify dystrophin expression, the ratio between dystrophin and vinculin signals was plotted and referred to the ratio of C57BL10 protein standard dilutions on each gel, set as 100%.