FIGURE 7.
Disruption of the OPPC domain affects protein segregation. PC12 cells that had been treated with NGF for 48 h were treated with 100 μm RHC80267 in the bottom lane of A and F–J (blue in B–E) or with vehicle in the top lane of A and F–J (red in B–E) for 3 h. These cell preparations were analyzed as follows. A, in situ PLA1 activity assay by PED-A1 shown in green. B, remodeling assay with TMF from RHC80267-treated cells (blue) or from control cells (red). C, quantification of the remodeled products in B. The percentage of each remodeled product in TMF derived from RHC80267 treated cells (blue) or from control cells (red) is shown. D, RP-HPLC and MALDI-TOF-MS analysis of TMF lipids (red, +NGF; blue, +NGF with RHC80267). The lipids in fraction 30 of the C8 column (Fig. 2) of each sample were analyzed with MALDI-TOF-MS with 6.25 pmol of dimyristoylphosphatidylcholine as a calibration standard for quantification. E, OPPC content per protein in TMF (red, +NGF; blue, +NGF with RHC80267). Data are means from four independent experiments evaluated using two-tailed Student's t test (*, p < 0.02). Error bars, S.D. Immunofluorescent staining with mAb#15 (F; green), with antibody to Thy-I (G; green) and with antibody to DAT (H; red; double staining the same cell in G). I, dopamine uptake assay using a fluorescent dopamine analog shown in green. J, immunofluorescent staining with antibody to Gαo (green). The thick arrows and the thin arrows point to the presence and the absence of the activity, respectively, in each image. Bar, 40 μm.