Mammalian cells synthesize only trace amounts of CPE despite the ER residency of SMSr. (A) TLC analysis of reaction products formed when lysates of control (untransfected [untransf]) or hSMSr-V5–expressing HeLa cells were incubated with NBD-Cer. (B) TLC analysis of reaction products formed when NBD-Cer was incubated with lysates of HeLa cells expressing hSMSr-V5 in the presence or absence of externally added PE or PC. (C) Expression of hSMSr-V5 stimulates CPES activity in HeLa cells. CPES and SMS activity levels were determined by TLC analysis of reaction products formed when cell lysates were incubated with NBD-Cer and expressed relative to control cells. (D) CPES and SMS activity levels were determined in lysates of HeLa cells treated for 7 d with siRNA-targeting LA (si LA) or siRNA-targeting hSMSr (si hSMSr) or with nonsilencing siRNA (si NS) as in C and expressed relative to si LA–treated cells. (E) Confocal sections of HeLa cells were transfected with hSMSr-V5 or an SAM-deficient truncation mutant, hSMSrΔSAM-V5, and immunolabeled for V5. Note that removal of SAM causes hSMSr-V5 to redistribute from the ER to the Golgi. Bars, 5 µm. (F) Human Caco-2, Hep-G2, HeLa, hSMSrΔSAM-V5-expressing HeLa, and Drosophila S2 cells were labeled with [¹⁴C]ethanolamine for 48 h and subjected to lipid extraction, TLC analysis, and autoradiography. In some extracts, glycerolipids were deacylated by mild alkaline hydrolysis (hydr; +). (G) Chinese hamster CHO-K1, CHO-K1–derived LY-A (CERT mutant), and LY-A/human CERT cells (mutant expressing human CERT) were labeled with [³H]sphingosine for 24 h and subjected to lipid extraction, TLC analysis, and autoradiography. Error bars indicate SD; n = 3.