Anti smpd1

The reagents used were as follows: RIPA buffer for preparing cell lysates (Thermo Fisher Scientific Inc., Waltham, MA, USA), Protease Inhibitor Cocktail (Sigma-Aldrich Co. LLC, St. Louis, MO, USA), polyacrylamide gels for SDS-PAGE (Wako Pure Chemical Industries, Ltd. Osaka, Japan), PVDF membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA), PVDF Blocking Reagent for Can Get Signal (TOYOBO CO., LTD., OSAKA, JAPAN), and Luminata Forte Western HRP Substrate (Millipore Corporation, Billerica, MA, USA). The immunoblots were visualized by Fusion-FX7 (Vilber Lourmat, Marne-la-Vallée, France). Primary antibodies used were as follow anti-SMPD1 (Proteintech Group, Inc., Chicago, USA, 14609–1-AP) and anti-β-actin (Sigma-Aldrich Co. LLC, A2228). Also, β-actin was used as an internal control.

Acid sphingomyelinase staining protocol: Tissue slides were deparaffinized and rehydrated in xylene substitute and gradients of ethanol after heating for 1 h at 65 °C. Permeabilization was done with 1% Triton X-100 for 15 min on a shaker plate and then washed with PBS-Tween buffer. Antigen retrieval was done with Tris–EDTA, pH 9.0 buffer by using Thermo-Electron Corporation Shandon Tissue Wave 2, HIER1 program. Slides were cooled to room temperature, washed with PBS-Tween buffer, and then treated with 3% H₂O₂ for 1 h at 37 °C to block endogenous peroxidase. Specimens were then incubated with rabbit primary antibody, anti-Smpd1 (Proteintech, Cat# 14,609–1-AP) at 1:200 dilution at 4 °C overnight. The sections were extensively washed with PBS-Tween buffer and then incubated with biotinylated goat anti-rabbit secondary antibody at room temperature for 30 min. The specimens were washed with PBS-Tween buffer and then incubated with Streptavidin-HRP pre-diluted (SAV-HRP, # BD550946, ready to use) for 30 min at room temperature. The specimens were then treated with diaminobenzidine DAB (HRP substrate, ThermoScientific, TA-125-QHDX) for about 1 min and the reaction stopped by transfer to distilled water. The specimens were then counterstained with hematoxylin. Sections were mounted onto slides with Permount (SP15-500, Thermo Fisher Scientific, USA) for review.