d-cer-C6 treatment causes permanent segregation of a Golgi-resident enzyme from its substrate. (A) Scheme of the trapping procedure using rapamycin (Rap.)-mediated dimerization of FRB and FKBP domains. The domain structure of the chimeric constructs TGN46-FRB-GFP and ST-FKBP-RFP is shown relative to the Golgi membrane. Transmembrane (TM), FRB, and FKBP domains and the N and C termini of the proteins are indicated. (B) HeLa cells expressing TGN46-FRB-GFP and ST-FKBP-RFP were treated with 20 µM l-cer-C6 or 20 µM d-cer-C6 for 4 h, after which 100 µM cycloheximide and DMSO or 500 nM rapamycin was added to the culture media for an additional 2 h. Cells were then fixed for fluorescence microscopy. (C) Quantitation of the relative colocalization of TGN46-FRB-GFP and ST-FKBP-RFP in the experiment shown in B, as measured by the Pearson’s correlation coefficient between the green and red channels. Bars show the mean values ± SEM of ≥10 cells counted from four independent experiments. (D) HeLa cells expressing TGN46-FRB-GFP and ST-FKBP-RFP were treated with DMSO or 500 nM rapamycin for 2 h, after which 100 µM cycloheximide and 20 µM l-cer-C6 or 20 µM d-cer-C6 were added to the culture media for an additional 4 h. Cells were then fixed, and the localization of TGN46-FRB-GFP and ST-FKBP-RFP was monitored by fluorescence microscopy. (E) Quantitation of the relative colocalization of TGN46-FRB-GFP and ST-FKBP-RFP in the experiment shown in D, as measured by the Pearson’s correlation coefficient between the green and red channels. Bars show the mean values ± SEM of ≥10 cells counted from four independent experiments. **, P < 0.01. Bars, 5 µm.