Figure 7
In vivo acylation of 1-NBD-GPC in Chlamydia-infected cells. Cells were grown and labeled as described in the legend of Fig. 6. Cells were either infected after labeling of the cells (pre-labeling conditions; images shown in Fig. 6) or were infected before addition of 1-NBD-GPC (post-labeling conditions; images shown in Figure S2). Cells were collected, washed, and lysed. The lysates were centrifuged at 8,000 g to generate pellet (P8) and supernatant (S8) fractions that represented the organelle and the cytosolic fractions enriched in EBs/RBs of infected cells, respectively. Lipids were extracted from the fractions obtained from pre-labeled cells (panel A) and post-labeled cells (panel B) and separated by TLC. Fluorescence quantification is shown in panel C. Labeled lipids obtained from pre-labeled cells were treated with bee venom PLA2 and analyzed (S8 + bvPLA2 in panel A). 1-NBD-GPC, which was used as a migration standard, was run on the TLC plate shown on panel A. The fluorescence of the lipids obtained from the samples was weaker than that of the standards, and the gamma intensity of the standard lane was adjusted to match that of the samples. For clarity, the image manipulation is indicated by presenting the standard lane as a separate section of the TLC plate on the left (NBD-LPC lane).
