## 2 Materials and Methods
### Pulse-Chase Assay  
On day in vitro 1 (DIV1) 1 \(\times\) 10\({}^{6}\) NSC34 cells were seeded in 10 mL proliferation medium per 100 \(\times\) 20 mm cell culture dish and cultivated in a humidified atmosphere at 37 \({}^{\circ}\)C and 5% CO\({}_{2}\). On DIV2, medium was changed to differentiation medium with low serum conditions (1% FBS). Cells were co-transfected with a siRNA against endogenous murine \(Smn\) and plasmid constructs coding for full-length SMN (pSMN1-294-EGFP) and the mutants pSMNS290A-EGFP or pSMNS290D-EGFP, respectively, as described above. Differentiation medium was changed after 6 h. On DIV3, cells were starved for 30 min in 4 mL methionine and cysteine depleted differentiation medium (DMEM high glucose without glutamine, methionine, and cysteine (Gibco(tm), Thermo Fisher Scientific, Schwerte, Germany, 21013024), 1% dialyzed FBS, 100 U mL\({}^{-1}\) penicillin and 0.1 mg/mL streptomycin, 2 mM L-glutamine) followed by a radioactive pulse for 1 h: 100 \(\upmu\)Ci of [S35]-labeled methionine and cysteine mixture (Hartmann Analytic, Braunschweig, Germany, SRIS-103, 10 mCi/mL) were added to each dish. The radioactive pulse was stopped with 6 mL of ice-cold stop medium (differentiation medium supplemented with 100 \(\upmu\)g/mL cysteine and 60 \(\upmu\)g/mL methionine). Dishes were kept on ice for the subsequent steps. Medium was discarded and cells were washed twice with 5 mL ice-cold PBS. The chase was started with 10 mL pre-warmed chase medium (differentiation medium supplemented with 100 \(\upmu\)g/mL cysteine and 60 \(\upmu\)g/mL methionine) and incubated in a humidified atmosphere except for the first (0 h) time point. Cell samples were collected at different time points. The following steps were equal for all samples and time points. Chase medium was discarded, and cells were washed twice with ice-cold PBS followed by cell lysis with 300 \(\upmu\)L RIPA (see above) buffer per dish. Cells were scratched off and dishes were washed with an additional 200 \(\upmu\)L RIPA buffer. 9 \(\upmu\)L DNase was added to each sample and homogenization was performed with a 27-gauge needle by pulling up and down ten times. Samples were centrifuged (20 min, 13,000 refd, 4 \({}^{\circ}\)C) and supernatants were snap-frozen with liquid nitrogen and stored at \(-\)80 \({}^{\circ}\)C until further processing. Immunoprecipitation was subsequently performed at 4 \({}^{\circ}\)C. 400 \(\upmu\)L of each sample was mixed with 2 \(\upmu\)g of mouse \(\alpha\)-GFP antibody (Roche 11814460001, LOT 14442000) or mIgG (Roche) as a control. After incubation overnight, 270 \(\upmu\)g magnetic Dynabead(tm) Protein G (Invitrogen) were added and samples were rotated for 1 h. The supernatant was separated using magnetic racks and frozen at \(-\)80 \({}^{\circ}\)C. Beads were washed twice with RIPA buffer and three times with PBS before resuspension in 60 \(\upmu\)L Laemmli buffer and boiling at 95 \({}^{\circ}\)C for 3 min.  
Beads were pelleted (13,000 rcf, 1 min) and supernatant was stored at 4 \({}^{\circ}\)C until analysis by SDS-PAGE. After electrophoretic separation, gels were fixed in 10% (_p/v_) acetic acid, 25% (_v/v_) isopropanol for 30 min by shaking at room temperature. After fixation, gels were incubated for 30 min with scintillation solution (NAMP100, GE Healthcare) at room temperature. Gels were dried at 70 \({}^{\circ}\)C for 3 h and exposed to ECL films (Hyperfilm ECL, Thermo Fisher) backed by intensifying screens (Sigma Lite Plus, 104102 LP) at -80 \({}^{\circ}\)C. Autoradiographs were digitalized and analyzed using ImageJ ([PERSON]; Version 1.41o).