FIGURE 4.

dCCS4 is required for CDP-Eth-dependent CPE production in Drosophila S2 cells. A, immunoblots of Drosophila S2 cells transfected with V5-tagged dCCS1, dCCS2, dCCS3, or dCCS4 and then treated with dsRNA targeting individual dCCS proteins (ds dCCS1–4). Cells treated with dsRNA targeting GFP (ds GFP) served as control. Blots were stained with anti-V5 and anti-dGolgin245 antibodies. B, lysates of Drosophila S2 cells treated with dsRNA as in A were incubated with NBD-Cer in the presence of CDP-Eth, MnCl₂, and UDP-glucose. The amount of NBD-CPE formed was determined by TLC analysis, normalized against NBD-GlcCer levels, and then expressed as the percentage of control (ds GFP-treated cells). Error bars: S.D., n = 3. C, lysates of Drosophila S2 cells depleted for dCCS4 (ds dCCS4) or mock-depleted for GFP (ds GFP) were incubated with CDP-[¹⁴C]ethanolamine and then subjected to lipid extraction, TLC analysis, and autoradiography. Levels of [¹⁴C]CPE were normalized against [¹⁴C]PE levels and expressed as the percentage of control (ds GFP-treated cells). Error bars: range, n = 2. D, Drosophila S2 cells depleted for dCCS4 (ds dCCS4) or mock-depleted for GFP (ds GFP) were metabolically labeled for 2 h at 27 °C with [¹⁴C]ethanolamine and then subjected to lipid extraction, TLC analysis, and autoradiography. Levels of ¹⁴C-labeled CPE were normalized against [¹⁴C]PE levels and expressed as percentage of control (ds GFP-treated) cells. Error bars: S.D., n = 3.