Fw: 59-GGATCCCACCTCGTGCAGATG -39 Rev: 59-GGAGTAATTCAGGTGGAG -39 Probe size: 232 bp -Right arm: Fw: 59-actgggtggaaacacctgag-39 Rev: 59-gagagaggagcctgcagaga-39 Probe size: 542 bp. One mg of PCR probe was labelled with 5 mL of 32P-dCTP (3000 Ci/mmol, ICN) and purified through a Push Column (Stratagene) according to the manufacturer’s protocol. Mem- branes, containing the cleaved genomic DNA, were hybridized at 65uC and subsequently washed 4 times in SSC buffer (Sigma). Film was exposed to the hybridised membranes at 280uC and then developed. Immunoblot analysis Whole mouse brain was dounce homogenized (1:10 w/v) in tissue homogenization buffer (20 mM Tris-base pH 7.4, 250 mM sucrose, 1 mM EDTA, 1 mM EGTA plus protease (Roche, Complete) and phosphatase inhibitors. For detection of full length APP or APP CTF’s, the lysates were spun at 1,000 g for 15 min and an equal amount whole protein homogenates were loaded for either 4–20% SDS-PAGE or 13% tris-tricine SDS-PAGE respectively and transferred onto nitrocellulose membranes for detection using AbD (Zymed). For detection of sAPPa and b (IBL antibody #27724 & #27722, respectively) an additional 45 min spin at 100,000 g was used to remove membranes prior to SDS- PAGE. Finally, in order enhance the signal for detection of both sAPPb and APP CTF’s, the nitrocellulose membranes were subject to epitope retrieval, prior to blocking, through incubation with boiling PBS-T and subsequent cooling to room temperature. Reverse Transcriptase-PCR and Real Time Quantitative PCR Analysis Mouse brain mRNA was extracted with Trizol reagent (Invitrogen). Briefly, one mouse hemisphere was shock frozen, weighed and homogenized in 4 volumes of Trizol reagent with an electric dounce homogenizer, 3630’’ in ice. The suspension was cleared of debris and membranes by centrifugation, and nucleic acids were separated by chloroform extraction and ethanol precipitation. The mix was applied to RNeasy columns, and RNA purified with RNeasy Protect Kit (Qiagen) according to the manufacturers’ protocols, including on-column DNase digestion (Qiagen). One mg of RNA, quantified with the Nanodrop (Thermo Scientific), was reverse-transcribed to cDNA using random primers and the SuperScript III First-Strand Synthesis System for RT-PCR kit (Invitrogen). Real time PCR was based on the TaqMan technology, using 200 ng of cDNA and mouse APP, Beta Actin and beta-2-microglobulin inventoried assays (Mm01344172_m1, Mm00607939_s1 and m00437762_m1, Ap- plied Biosystems) according to the manufacturer’s protocols, in 20 mL volume and in 96well plate format. The threshold cycles (Ct) for the endogenous controls mRNA (b-actin and b-2- microglobulin) and the target (APP) signal were determined and the relative RNA quantification was calculated using the comparative DDCt method. Each experiment was conducted in triplicate. Data analysis was conducted according to Applied Biosystems references and protocols, and using student-t test. Neuronal cultures Neuronal cultures were performed as described previously [15,16] from E16-17 fetuses. APP Processing and Tyrosine682 Mouse Dermal Fibroblasts To culture mouse dermal fibroblasts (MDFs), skin was removed from mouse tails, soaked in 70% ethanol, washed in PBS, diced into small pieces and incubated at 37uC overnight in CO2 incubator in DMEM containing 20% FBS, supplemented with penicillin/streptomycin and 1.6 mg/ml collagenase II. On the next day, clumps were removed by passing through a nylon mesh, and the material was centrifuged at 1000 rpm for 5 min to collect the cells. The collected cells were maintained in DMEM containing 20% FBS and penicillin/streptomycin. Biotinylation and streptavidin precipitation For biotinylation experiments, MDFs were washed three times with cold PBS plus Ca++ and Mg++ (PBS-CM) and labelled for 30 min on ice in 0.5 mg/ml sulfo-NHS-SS-biotin (Pierce) dissolved in PBS-CM. Free biotinylation reagent was removed by washing three times with PBS-CM containing 0.1% BSA. The cells were lysed in the RIPA buffer. The lysates were cleared by centrifuging at 20,000 g for 10 min, and were mixed with streptavidin agarose beads (Sigma S1638). After collecting unbound lysate, the beads were washed four times with the RIPA buffer, and were boiled in 26 SDS buffer. Comparable volume of the samples were subjected to western blot. Synaptosomes For synaptosomes, mouse brain was homogenized in H buffer [5 mM Hepes/NaOH pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.32 M sucrose, plus protease (PI) and phosphatase (PhI) inhibitors] at 10% (w/v) and centrifuged at 800 g for 10 min. The supernatant (S1) was separated to supernatant (S2) and pellet (P2) by spinning at 9,200 g for 15 min. P2 was lysed on ice for 30 min in H buffer containing 35.6 mM sucrose. The lysed P2 was separated to supernatant (LS1) and pellet (LP1) by spinning at 25,000 g for 20 min. S2 was separated to supernatants (S3) and pellet (P3) by spinning at 165,000 g for 2 hrs, respectively. P3 was suspended in H buffer containing 0.32 M sucrose by sonication. Synaptosomes fractions represent: S1, postnuclear supernatant; S2, cytosol, soluble proteins and light membrane; P2, crude synaptosomal fraction; S3, soluble fraction; P3, light membrane abundant in Golgi and ER; LS1, crude synaptic soluble; LP1, synaptic membrane fraction. Ab40 ELISA DEA extraction of Ab from brain lysates was carried out as previously described [17]. Prior to ELISA, DEA extracts were further purified with Oasis HLB sample extraction cartridges (Waters, WAT094226) to decrease background artifacts which otherwise prevent detection of endogenous wild-type Ab40 in mice [18]. Ab40 ELISA kit (IBL America, discontinued product) was used according to manufacturer’s protocol with equal quantities of protein loaded. Co-immunoprecipitation Whole mouse brain lysate was centrifuged at 9000 g for 15 min and the resulting supernatant at 100,000 g for 45 min. The supernatant was removed and the membrane pellet re-suspended overnight in IP buffer (20 mM Hepes pH 8.0, 10% glycerol, 137 mM NaCl, 0.5% Triton-X-100, 2 mM EDTA) Any remain- ing debris was removed with centrifugation at 9000 g for 15 min and the membrane enriched supernatant diluted to 1 mg/ml in IP buffer. 500 ug of membrane protein was used for each IP. Supernatants were pre-cleared with 30 ul protein A/G (Pierce) for 30 min. 2 mg of either monoclonal anti-APP, non-specific mouse PLoS ONE | www.plosone.org 3 November 2010 | Volume 5 | Issue 11 | e15503