Ubiquitin

IB analysis of protein expression and immunofluorescence staining on cells or tissue sections were performed as previously described (Bao et al., 2006a (link); Guryanova et al., 2011 (link); Cheng et al., 2013 (link); Zhou et al., 2015 (link)). Specific antibodies against USP13 (Abcam), c-Myc (Cell Signaling Technology or Santa Cruz Biotechnology, Inc.), SOX2 and OLIG2 (EMD Millipore or Santa Cruz Biotechnology, Inc.), Flag and α-tubulin (Sigma-Aldrich), GFAP (BioLegend or BD), MAP2 (Covance), TUJ1 (Covance), CD31 (Dako), FBXL14 (Santa Cruz Biotechnology, Inc.), CD133 (Miltenyi Biotec), ubiquitin (BioLegend), hemagglutinin (HA; Santa Cruz Biotechnology, Inc.), and Ki-67 (Abcam) were used for IB analysis or immunofluorescent staining. IHC staining on tumor and normal tissue sections was performed with an avidin–biotin complex kit and a 3,3′-diaminobenzine detection kit (Vector Laboratories) as previously described (Bao et al., 2006b (link); Guryanova et al., 2011 (link); Zhou et al., 2015 (link)).

Total protein lysate was collected by homogenization of flash frozen spinal cord, liver and hindlimb muscles in RIPA lysis buffer (Cell Signaling). Protein concentrations were determined using the Bradford assay (Bio-Rad). Protein extracts were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and examined by immunoblot, as previously described [24] (link) with modified blocking conditions where Odyssey blocking buffer (Li-Cor 927–40000) replaced 5% milk. Revert Total protein stain (Li-Cor 926–11010) was used as per the manufacturer's protocol. Primary antibodies used were as follows: Anti-Smn (BD Transduction, 610647 - 1:1000–2000), alpha-tubulin (Abcam, ab4074 - 1:5000–15,000) and ubiquitin (Biolegend P4D1- 1:1500), Secondary antibodies used were IRDye (Li-Cor) 680 or 800 (Li-Cor - 1:10,000 to 1:20,000). Signals were detected with Odyssey CLx (Li-Cor). Results were normalized to total protein or tubulin. Full western blots can be visualized in Supp. Fig. 6.