FIGURE 6.

NGF stimulates remodeling at the sn-1 site of phospholipids by tip membrane. A–C, the purity and identity of each of DPPE-NBD (A), 1-palmitoyl-2-hydroxyphosphatidylethanolamine-NBD (B), and 1-oleoyl-2-hydroxyphosphatidylethanolamine-NBD (C) standard was verified with MALDI-TOF-MS analysis in negative ion mode with PNA as a matrix. D–F, after incubation of DPPE-NBD with the cells, its remodeled products in TMF were analyzed by RP-HPLC with a C8 column. D, standard molecules (1-palmitoyl-2-hydroxy-PE-NBD (green; 3.2 min), POPE-NBD (12.5 min), and DOPE-NBD (13.6 min)). E, the substrate DPPE-NBD (11.6 min). F, the remodeled products recovered in TMF fraction. Black, −NGF; red, +NGF. G, amounts of each remodeled product were quantified, and the percentages in total phospholipid-NBD (retention time between 7.5 and 20 min) were shown (white, substrate DPPE; black, TMF lipid without NGF stimulation; red, TMF lipid with NGF stimulation). H–J, the peaks 3 at the retention time 12.5 min in F were recovered and further analyzed by PLA₂ treatment. The acyl chains at the sn-1 site of PLA₂-treated products were analyzed with RP-HPLC by a C18 column. H, standard molecules (1-palmitoyl-2-hydroxy-PE-NBD (green; 7.0 min) and 1-oleoyl-2-hydroxy-PE-NBD (purple; 7.9 min)). I, the PLA₂ digests of the peaks 3 in F (black, −NGF; red, +NGF). J, amount of each lysophospholipid was quantified, and the contents of 1-oleoyl-phospholipids in peak 3 in F are shown (black, −NGF; red, +NGF). K, OPPE-NBD content per protein in TMF and in whole cell extract. Data in G, J, and K are means from six independent experiments evaluated using two-tailed Student's t test (*, p < 0.02). Error bars, S.D. a.u., arbitrary units.