FIGURE 2.
mAb#15 recognizes OPPC at the tips of PC12 processes. A, the tip membrane fractions were prepared from NGF-stimulated PC12 cells or unstimulated cells. The purification of the tips was examined by co-purification of Gαo. Whole cell protein (10 μg each) and TMFs (purified from 80-mg cell pellets) were analyzed by SDS-PAGE and Coomassie staining (top) and by Western blot with antibody to Gαo (bottom). B, lipid in the TMFs (black, −NGF; red, +NGF) was separated by RP-HPLC. Top, elution profile of optical density at 205 nm. Bottom, ELISA with mAb#15 on the eluted fractions. The lipids in fraction 30 in B from unstimulated PC12 (C) or from NGF-stimulated PC12 (D) were analyzed by MALDI-TOF-MS with DHB-lithium mix in positive ion mode. In E, instead of DHB-Li, DHB-Na was used as the matrix for ionization. The peak at m/z 766.6 was observed only with DHB-Li in D, indicating that this was a Li+ ion adduct. F, the cation at m/z 766.6 in D was analyzed by fragmentation with collision-induced dissociation. G–I, positioning of the acyl chains was analyzed by comparing fragmentation profiles of the cation at m/z 766.6 in (D and H) with that of OPPC (G) and with that of POPC (I). Spectra between m/z 400 and 550 are presented. The mass loss of 59 corresponds to the loss of a trimethylamine fragment (17).