by Slunt HH, Thinakaran G, Van Koch C, Lo ACY, Tanzi RE, Sisodia SS. nerve growth factor regulation of APP processing. The mitogen-activated protein Folinic acid kinase cascade may provide a novel target for altering catabolic processing of APP. Human embryonic kidney (HEK) 293 cells were transiently transfected with pCMV695, an expression vector for APP695 (Selkoe et al., 1988), pCMV, an expression vector for bacterial -galactosidase (Clontech Laboratories), and either pCDNAK97A, an expression vector for kinase-inactive MEK, or the expression vector alone using a high-efficiency calcium phosphate transfection protocol (Chen and Okayama, 1987) as described previously (Raymond et al., 1996). Transfection efficiency was assessed by staining for -galactosidase and determining the percentage of positively stained cells according to the method of Raymond et al. (1996). HEK 293 cells stably transfected with Folinic acid a construct carrying the Alzheimers disease-linked double (Swedish) mutation (K695sw), known to secrete elevated levels of both A40 and A42 (Citron et al., 1996), were cultured in DMEM supplemented in 10% fetal calf serum. HEK 293 cells were cultured in MEM supplemented with 10% fetal calf serum as Folinic acid described previously (Raymond et al., 1996). Rat pheochromocytoma (PC12) cells were cultured in DMEM supplemented with 10% horse serum and 5% fetal calf serum. One day before stimulation, HEK 293 cells or PC12 cells were exposed to culture media containing charcoal-inactivated calf serum at the same percentage used previously for cell maintenance. All cell lines were exposed to drugs for 15 min. PC12 cells were exposed to drugs in DMEM according to the method ofBuxbaum et al. (1990). HEK 293 cells were exposed to drugs in MEM supplemented with 1 mg/ml glucose, whereas K695sw cells were exposed to drugs in DMEM. Timed pregnant Sprague Dawley rats were anesthetized with halothane at 18 d of gestation, and the cerebral cortex was removed from rat embryos and dissociated using a method described previously (Murphy et al., 1992). Culture maintenance and drug exposure were performed using the method of Fiore et Folinic acid al. (1993) with minor modifications. In brief, before drug treatment, cells were washed once with 1 ml HBSS and preexposed to PD 98059 or drug vehicle for 1 hr. Both PD 98059 and phorbol esters were diluted from 10 mm stocks, made up in dimethylsulfoxide. After drug exposure, the medium was centrifuged for 10 min at 16,000 to remove cellular debris. For APPs detection, the medium was subsequently desalted and concentrated by centrifugation in the presence of protease inhibitors (17 g/ml phenylmethanesulfonyl fluoride, 2 g/ml leupeptin, 10 g/ml aprotinin, and 2 g/ml pepstatin) according the method ofMills and Reiner (1996). APP was detected by Western blot analysis using an anti-APP N-terminal antibody (anti-PreA4 monoclonal antibody, Boehringer Mannheim, Laval, Quebec, Canada) or WO-2, a monoclonal antibody generated against the first 16 amino acids of the N-terminal region of A (Ida et al., 1996; anti-1C16) as described previously (Mills and Reiner, 1996). All Western blots were probed first with the anti-PreA4 monoclonal antibody (22C11). In some experiments, membranes were subsequently stripped Folinic acid of antibodies and reprobed with the APP-selective antibody WO-2 to prevent detection of secreted APLP (Slunt et al., 1994). For CDKN2AIP A detection, proteins were precipitated by trichloroacetic acid according to the method of Hames (1981). A was separated by Tris/Tricene SDS-PAGE according to the method of Klafki et al. (1996) and detected by Western blot analysis according to the method of Ida et al. (1996) using the monoclonal antibody WO-2. After densitometric measurements, ANOVA followed by Fishers analysis was used to determine the significance of observed differences. Data are expressed as mean.
