The D358G mutation in SMPD3 causes protein instability. (A) nSMase assay for wild-type and D358G SMPD3 lysate. Crude membrane fractions from F4328 cells expressing retrovirally transduced wild-type or D358G SMPD3 were incubated with ¹⁴C-labeled sphingomyelin in the presence of the Mg²⁺ for 30 minutes at 37°C. The release of ¹⁴C-phosphorylcholine in the aqueous phase was measured by scintillation counter. Error bars indicate standard error of the mean. (B,C) F4328 cells were transiently transfected with empty vector, V5-tagged wild-type, or D358G SMPD3. Cells were harvested 48 hours after transfection and were split for Northern (B) and Western blot analysis (C). (D) F4328 cells transiently transfected with either V5-tagged wild-type or D358G SMPD3 were treated with 100 μg/mL cycloheximide for the indicated times, and the total cell lysates were prepared for Western blot by anti-V5 antibody. The middle panel shows a longer exposure of the same blot. (E) Western blot results were quantitated by QuantityOne software (Bio-Rad, Hercules, CA) and protein stability was plotted by percentage of the intensity of band compared with that of the untreated sample. □ indicates wild-type SMPD3; ■, D358G SMPD3.