Pierce bicinchoninic acid bca reagent

The whole-cell lysates or kidney lysates were collected using a buffer containing 2% SDS buffer with 1% protease inhibitor cocktail and 1% benzonase (Sigma, St. Louis, MO, USA) [23 (link),32 (link)]. The protein concentration was measured with the Pierce Bicinchoninic Acid (BCA) reagent (ThermoFisher Scientific, Waltham, MA, USA). An equal amount, usually 20 μg, of protein was loaded for SDS-PAGE and transferred onto PVDF membrane. The membranes were then incubated sequentially with a blocking solution containing 5% nonfat milk, the primary antibody incubation in 4 °C overnight, and finally horseradish peroxidase-conjugated secondary antibody. The antigens on the blots were revealed using the enhanced chemiluminescence (ECL) kit from Bio-Rad Laboratories (Hercules, CA, USA) to record signals by MyECL Imager (ThermoFisher Scientific, Waltham, MA, USA) or KwikQuant Imager (Kindle Biosciences, Greenwich, CT, USA). The densitometry of the blots was performed with ImageJ software (https://imagej.nih.gov/ij/download.html, accessed on 19 August 2021).

Dr. A. Kihara kindly supplied mouse embryonal carcinoma F9-0 cells and F9-2 cells (S1PL knocked-out) as well as F9-4 cells (S1PL overexpressed) (Hokkaido University, Japan). Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (v/v) FBS and 1% penicillin–streptomycin in 0.1% gelatin-coated dishes. Cells were cultured at 37 °C in a humidified 5% CO₂ atmosphere and routinely subcultured every other day using a solution of trypsin–EDTA from Life Technologies, Inc. (Gaithersburg, MD, USA). Cells were washed twice with ice-cold PBS and then scraped in 1 mL of S1PL extraction buffer (5 mM Mops, 1.0 mM EDTA, 0.25 M sucrose, 1.0 mM PMSF), Then, 1× protease inhibitor and 10% (v/v) glycerol at pH 7.4 were sonicated for 10 s. We removed cell debris via low-speed centrifugation (1000× g for 5 min), and the protein concentration of the supernatant was determined by using the Pierce bicinchoninic acid (BCA) reagent (Thermo Fisher Scientific, Rockford, IL, USA)