SMN is ubiquitinated and degraded by the UPS. (A) Treatment of 3813 SMA patient-derived fibroblast with the proteasome inhibitor, MG132 (10 μM); the cell-permeable calpain inhibitor, Calpeptin; the lysosome inhibitor, ammonium chloride (NH4Cl); and an autophagy inhibitor, 3-MA. Quantification is shown in lower panel. (B) 3813 cells treated with 1, 5, or 10 μM concentrations of the proteasome inhibitor lactacystin. The data represent mean ± the standard error of the mean (SEM) of five independent experiments. *, P < 0.05. (C) Cell lysates were denatured with 1% SDS and then renatured with 4.5% Triton X-100 prior to immunoprecipitation with anti-SMN antibody. In addition, cell lysis buffer was supplemented with 2.5 μM ubiquitin aldehyde to inhibit deubiquitinating enzymes. Western bots were probed with antibodies to SMN, Gemin 2, Gemin 3, and Gemin 6. (D) Western blot probed with an antibody to ubiquitin after immunoprecipitation of SMN from cells in the presence or absence of 10 μM lactacystin.