Figure 4.

Bulk production of CPE in insect cells is independent of SMSr. (A) TLC analysis of reaction products formed when NBD-Cer was incubated with lysates of Drosophila S2 cells expressing dSMSr-V5, active site mutants of dSMSr-V5 (H401A and D405E), hSMSr-V5, active site mutant of hSMSr-V5 (D348E), or SAM-deficient hSMSr-V5 (hSMSrΔSAM-V5). (B) S2 cells treated with ds GFP or dSMSr (ds dSMSr #1 or #2) for 7 d were metabolically labeled with NBD-Cer (intact cells) or lysed and incubated with NBD-Cer for 2 h (cell lysates). CPES activity levels were determined by TLC analysis and expressed relative to mock-treated cells. Error bars indicate SD; n = 3. (C) Immunoblots of S2 cells treated with dsRNA as in B were stained for dSMSr and dGolgin245. Note that the anti-dSMSr antibody cross reacts weakly with a dSMSr-unrelated protein (asterisk). (D) S2 cells treated with dsRNA as in B were metabolically labeled with [¹⁴C]ethanolamine ([¹⁴C]eth) for 2 h and subjected to TLC analysis and autoradiography. (E) TLC analysis of products formed in S2 (top) or HeLa cell lysates (bottom) when incubated with 50 µM NBD-Cer in the presence or absence of 500 µM CDP-ethanolamine (CDP-ethanol), 5 mM MgCl₂, or 10 mM MnCl₂. (F) TLC analysis of products formed in lysates of dsRNA-treated S2 cells incubated with NBD-Cer and MnCl₂ in the presence or absence of CDP-ethanolamine as in E.