Switching head group selectivity in mammalian sphingolipid biosynthesis. A: HeLa cells were transfected with empty vector (EV), SMS1-HA, or SMS1CPE-HA, fixed, and then double-labeled with antibodies against the HA-tag (green) and Golgi marker, GM130 (red). Note that both SMS enzymes localize to the Golgi. Scale bar, 10 μm. B: Immunoblots of KBM7-derived WT and SMS1-null cells (SMS1KO) transfected with empty vector (EV), SMS1-HA, or SMS1CPE-HA were stained with antibodies against the HA-tag and β-actin. C: KBM7-derived WT and SMS1KO cells transfected with EV, SMS1-HA, or SMS1CPE-HA were metabolically labeled with [14C]ethanolamine for 24 h and then subjected to lipid extraction, TLC analysis, and autoradiography. In some extracts, glycerolipids were deacylated by mild alkaline hydrolysis (hydr. +) prior to TLC analysis. Note that only SMS1CPE-expressing cells produce a radiolabeled lipid resistant to alkaline hydrolysis and with an Rf of CPE. Metabolic labeling of insect Sf21 cells, which produce bulk amounts of CPE, served as control. D: SM and CPE levels in lipid extracts of KBM7-derived WT and SMS1KO cells expressing SMS1-HA or SMS1CPE-HA were determined by LC-MS/MS and expressed as mole percent of total phospholipid analyzed. E: Levels of SM and CPE species in KBM7-SMS1KO cells expressing SMS1-HA or SMS1CPE-HA were determined as in (D). Data shown in (D) and (E) are representative of two independent experiments.