Hrp anti mouse

Whole protein extraction was performed with RIPA buffer (ThermoFisher) and Protease inhibitor (Peprotech) and proteins were quantified using the Pierce^TM BCA Protein Assay Kit (Thermo Fisher Scientific) according to manufacturer’s instructions in a 96-well format. 20 µg of protein were loaded on 10% SDS-page gel and run at 120 V for 1 h. Semi Dry transfer to PVDF membranes was performed by using the Trans-Blot Turbo Transfer System (BioRad) for 30 min at 35 V. After that, the membranes were blocked with 3% skimmed milk in TBS with 0.1% Tween20 for 1 h at room temperature. After 1 h blocking at room temperature, the membrane was incubated with the SMN antibody (1:10,000 in blocking solution) or Beta-actin antibody (1:20,000 in blocking solution) over night at 4 °C under continuous shaking. The next day, the membrane was washed 3 times with TBS with 0.1% Tween20 (washing solution) for 5 min under continuous shaking. Then, the membrane was incubated with the secondary antibody HRP anti-mouse (Sigma; 1:10,000 in blocking solution) for 2 h at room temperature under continuous shaking. The membrane was washed 3 times with washing solution for 5 min. Finally, the protein bands were detected using the ECL^TM Start Western Blotting Detection Reagent according to manufacturer’s instructions and the Chemidoc MP Imaging System (Bio-Rad). All antibodies used are listed in Table S2.