Irdye680dx

Immunoblotting was carried out in whole-cell SDH lysates of SDH and DRG. Equal amounts of whole-cell protein (50 µg) were resolved on a 12.5% SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane using standard procedures. The following primary antibodies were used: rabbit anti-DNMT3a (Abcam, ab 2851) 1:500, rabbit anti-DNMT3b (Abcam, sc 374015) 1:1000, and rabbit anti Lamb1 (Abcam, ab 108536) 1:1000, followed by the secondary antibody at a 1:10,000 dilution (IRDye680DX, Rockland Immunochemicals 926-68071). Immunoreactivity was detected with an OdysseyTM Infrared-Imaging System (LI-COR Biosciences, Lincoln, NE, USA), according to the OdysseyTM Western Blotting Protocol. The integrated optical density of the bands was determined using Image J software (U.S. National Institutes of Health, Bethesda, MD, USA, http://imagej.nih.gov/ij, accessed on 1 June 2020). The nuclear marker laminin-1β (Lamb1) was used as an internal loading control.

Spinal cords from WT (n = 3) and SMNΔ7 mice (n = 5) were lysed at 4 °C in a buffer containing 50 mM Tris (pH 8), 150 mM NaCl, 2% Nonidet P-40, 1 mM MgCl₂, 1 mM dithiothreitol, and 10% glycerol, and supplemented with EDTA-free complete protease inhibitor cocktail and PhosSTOP (Roche). The spinal cord samples were sonicated for 5 cycles of 30 seconds ON/OFF at 4 °C using a Bioruptor Plus (Diadode) and left on ice for 20 min. The samples were then 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-Tubulin ß-III (Sigma T8660) and rabbit polyclonal anti-PABPN1 and anti-Sam68 were used. Protein bands were detected with an Odyssey^TM Infrared-Imaging System (Li-Cor Biosciences) according to the Odyssey^TM Western-Blotting Protocol. Immunoblots were developed with anti-mouse IRDye800DX or anti-rabbit IRDye680DX (Rockland Immunochemicals, USA) secondary antibodies. For the quantitative analysis of the blots, ImageJ software was used (U. S. National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij).