DAT proteins are present in the membranes of PC12 cells and are elevated by 2–7 day NGFβ treatment. A. Cell lysate immunoblots with Ab to the second extracellular loop of DAT, showing an increase in DAT protein levels due to a 20 ng/ml NGFβ treatment over a time course of 2 (NGF 2d), 4 (NGF 4d) and 7 (NGF 7d) days, compared to controls without NGFβ treatment (2d, 4d, and 7d). Representative of 2 experiments. B. Staining of nonpermeablized fixed cells with the same DAT e2 Ab. Fluorescent images viewed with an FITC filter were photographed. Vector Red appears as orange-red signal on a background of yellow-green autofluorescence (typically seen with this cell fixation protocol). Note the staining of cell bodies, especially at the growth cones (overexposed), and the smaller amount of staining on processes of these NGFβ-differentiated cells. The bar represents 2 μm. C. DAT levels are stimulated ~8-fold on day 2 of NGFβ treatment, as demonstrated by the plate immunoassay of fixed cells using the same e2 DAT-specific Ab; 2° Ab conjugated to alkaline phosphatase was used to generate paranitrophenol (pNp) colorimetric signals, which were normalized to the cell number determined in each well by the crystal violet (CV) assay. Symbols are +NGF (■); -NGF (○). Controls: NGF-treated cells probed with a nonspecific IgG Ab is labeled as "+NGF IgG" (▼) "-NGF IgG" IgG (△); clathrin (◇); no primary Ab (no 1° Ab) and no primary or secondary Ab (no 1°, no 2° Abs) are at the origin. A very low clathrin Ab signal under these nonpermeabilizing conditions demonstrated the lack of inadvertent permeabilization of the cells in these assays. This graph represents the average values from 3 experiments ± S.E.M.