Quantitative Analysis of SMN Protein

Analysis of the level of SMN protein were performed according to the protocol described by Narcís et al. ^{27 (link)}. Briefly, spinal cords and skeletal TA muscle samples from WT (n = 3), SMN∆7 (n = 3) and nusinersen-treated SMN∆7 (n = 4) mice were lysed at 4 °C in a buffer containing 50 mM Tris (pH 8), 150 mM NaCl, 2% Nonidet NP-40, 1 mM MgCl₂, 1 mM dithiothreitol, and 10% glycerol and supplemented with EDTA-free complete protease inhibitor cocktail and PhosphoSTOP (Roche). Samples were sonicated and cleared by centrifugation at 14,000 rpm for 10 min at 4 °C. The proteins were separated on 4–20% NuPage TG SDS–PAGE gels (Invitrogen) and transferred to nitrocellulose membranes using standard procedures. Mouse monoclonal anti-SMN (diluted 1:500) and rabbit polyclonal anti-Lamin A/C (diluted 1:1,000, generously donated by Prof. Gerace) were used. Protein bands were detected with an Odyssey Infrared-Imaging System (Li-Cor Biosciences) according to the Odyssey Western-Blotting Protocol. For the quantitative analysis of the blots, ImageJ software was used (U.S. National Institutes of Health, Bethesda, Maryland, USA, https://imagej.nih.gov/ij).