Ab610646

Expression of the ExSpeU1 was determined using RT–PCR and northern blotting on RNA from organs extracted from control and SMA-rescued mice. For the RT–PCR, ExSpeU1 forward and U1 cassette reverse primers were used on DNase-treated RNA samples. Northern blotting was performed as described below. For the SMN2 exon 7 splicing patterns, we used primers listed in Supplementary Table 1. SMN proteins levels were determined by western blotting in the brain, skeletal muscle and heart of P2–3 animals using an SMN-specific antibody (ab610646,BD Biosciences, USA, 1:2,000) and the levels were normalized to GAPDH (ab8245, Abcam, USA, 1:5,000). Uncropped blots are shown in Supplementary Fig. 11

Protein samples were obtained by tissue lysis with ice-cold RIPA buffer (Sigma, St Louis, MO USA) and Proteinase Inhibitor Cocktail (Roche). A total of 20 μg of protein was separated on NuPAGE 4–12% Bis-Tris precast gels (ThermoFisher, Waltham, MA USA) and transferred to nitrocellulose membranes. The membranes were blocked for 1 h in 5% non-fat milk and successively blotted overnight at 4°C in 5% milk with a primary SMN-specific antibody (ab610646,BD Biosciences, USA, 1:2000) and a primary GAPDH antibody (ab8245, Abcam, USA, 1:5000).
Membranes were washed and incubated with specific secondary antibodies for 1 h at room temperature (anti-Mouse 1:5000). Protein bands were detected with ECL Western blotting substrate (ThermoFisher, Waltham, MA, USA) followed by exposure to X-ray film (Kodak) or to UVItec CAMBRIDGE Alliance.