Fig. 1. Immunoprecipitation of PS1 in these transfectants. Immunoprecipitation of PS1 protein and subsequent Western blot analysis with an anti-PS1 NTF antibody were performed in mock, wild type (wt), and mutant (mu) PS1 transfected cells as described in Materials and methods. Note that in wt and mu cells, full-length PS1 at 50 kDa and N-terminal fragment (NTF) of PS1 at 30 kDa were clearly detected. On the other hand, in mock-transfected cells, no obvious PS1 holoprotein was detected.

Fig. 2. Cell death assay. Cell death assay against hydrogen peroxide on these transfectants was performed to elucidate the sensitivity to the oxidative stress. Cells were serum-starved for 30 min prior to the treatment and then treated with hydrogen peroxide as described in Materials and methods. After the staining of the cells with trypan blue solution, we counted both stained and unstained cell numbers. The figure shows the percentage of live cells. Note that vulnerability to H₂O₂ was observed in mu cells compared with mock-transfected cells with statistical significance (P < 0.04). These experiments were performed on five different cell preparations.

Fig. 3. Immunoblot analysis of Trk-immunoprecipitates prepared from transfected cells. Immunoprecipitation of the Trk protein and subsequent immunoblot analysis of these Trk-immunoprecipitates with an anti-phosphotyrosine and α-Trk were performed in mock, wild type (wt), and mutant (mu) PS1-transfected cells as described in Materials and methods. Note that autophosphorylation of Trk in response to NGF-treatment was clearly observed in mock-transfected cells. However, no obvious enhancement of autophosphorylation level of the Trk protein in response to NGF treatment was observed in mu and wt cells. We also examined MAPK phosphorylation response to NGF in these transfectants. In wt and mu cells, no obvious MAPK phosphorylation was observed, whereas NGF clearly induced MAPK phosphorylation in mock-transfected cells.

Fig. 4. Immunocomplex kinase assay of Trk-immunoprecipitates prepared from these transfectants. Trk-immunoprecipitates were prepared from these transfectants as described in Materials and methods. These Trk immunoprecipitates were subjected to immunocomplex kinase assay employing radioactive ATP as described previously [17] and [18]. Autophosphorylation of Trk protein in response to NGF was clearly observed in mock-transfected cells, whereas no obvious response was observed in wt and mu cells.

Fig. 5. Confocal laser microscopic observation. Each transfectant was cultured on coverslips coated with collagen/poly-l-lysine described in Materials and methods. Cells were fixed with paraformaldehyde and processed for the observation with confocal laser microscope. The data showed that in mock-transfected cells, Trk was distributed to cell surface with patchy staining pattern in association with somewhat nuclear staining (1). On the other hand, in the wt (2) and mu PS1 (I143T) (3) transfected cells, Trk was mainly distributed in the cytosol and nucleus and no obvious staining was observed in the plasma membrane. On the other hand, the intensity of PS1-like immunoreactivity is much weaker in mock-transfected cells than those of wild and mutant PS1 transfectants.

Fig. 6. Subcellular fractionation of Trk protein in these transfectants. To substantiate the data on confocal laser microscope, we examined the subcellular distribution of the Trk protein by performing subcellular fractionation of the homogenates prepared from these transfectants as described in Materials and methods. In mock-transfected cells, Trk was mainly located in the membrane fraction. On the other hand, in wt and mu cells, Trk was abnormally located in the cytoplasm and in nuclear fraction.