EDTA), centrifuged at 21,000 g for 5 min at 4uC, and cleared supernatant retained as the total fly Ab42 sample. Alternatively, soluble and insoluble pools of Ab42 were extracted using a protocol adapted from previously published methods [80]: 200 fly heads were homogenised in 200 ml tissue homogenisation buffer (250mM sucrose, 20mM Tris base, 1mM EDTA, 1mM EGTA, protease inhibitor cocktail (Sigma) then mixed further with 200ml DEA buffer (0.4% DEA, 100mM NaCl and protease inhibitor cocktail). Samples were centrifuged at 135,000 g for 1 hour at 4uC (Beckman Optima Max centrifuge, TLA120.1 rotor), and supernatant retained as the cytosolic, soluble Ab42 fraction. Pellets were further resuspended in 400ml ice-cold formic acid (70%), and sonicated for 2630 sec on ice. Samples were re-centrifuged at 135,000 g for 1 hour at 4uC, then 210 ml of supernatant diluted with 4ml FA neutralisation buffer (1M Tris base, 0.5M Na2HPO4, 0.05% NaN3) and retained as the insoluble, formic acid-extractable Ab42 fraction. Total, soluble or insoluble Ab42 content was measured in Arctic mutant Ab42 flies and controls using the hAmyloid b42 ELISA kit (HS) (The Genetics Company, Switzerland). Total Ab42 samples were diluted 1:100, and soluble versus insoluble Ab42 samples diluted 1:10 in sample/standard dilution buffer, then ELISA performed according to the manufacturers’ instructions. Protein extracts were quantified using the Bradford protein assay (Bio-Rad protein assay reagent; Bio-Rad laboratories Ltd (UK)) and the amount of Ab42 in each sample expressed as a ratio of the total protein content (pmoles/g total protein). Data are expressed as the mean 6 SEM obtained in three independent experiments for each genotype. ANOVAs and Tukey’s-HSD post-hoc analyses were performed using JMP 7.0 software. Analysis of tau phosphorylation by Phos-tag SDS-PAGE Fly heads were homogenised in Mes buffer (100 mM Mes, pH 6.5, 1 M NaCl, 0.5 mM MgCl2, 1 mM EGTA, 10 mM NaF, Protease inhibitor cocktail [Sigma, P8340] and Phosphatase inhibitor cocktail 2 [Sigma, P5726]), centrifuged at 20,000 g for 30 minutes at 4uC, then supernatants adjusted to 0.5% b- mercaptoethanol, boiled for 5 minutes and re-centrifuged. Cleared supernatants were then retained as heat-stable soluble tau. Control samples were dephosphorylated by incubating with l-protein phosphatase (NEB, P0753) for 3 hours at 30uC. To detect phosphorylated and non-phosphorylated tau, samples were separated by SDS-PAGE, using Phos-tag AAL-107 (FMS laboratory) according to the manufacturers’ instructions. Western blotting was then performed using a non-phosphorylation dependent Drosophila rabbit anti-tau antibody (1:5000). Quantita- tion was performed using Image J software (National Institutes of Health). Phosphorylated and non-phosphorylated tau was ex- pressed as a percentage of the total amount of tau present in each sample. ANOVA and Tukey’s-HSD post-hoc analyses were performed using JMP 7.0 software. Western blotting Drosophila heads were pooled and homogenised in 26Laemmli sample buffer containing b-mercaptoethanol and boiled for 10 mins. Proteins were separated on SDS polyacrylamide gels and blotted onto nitrocellulose. Membranes were incubated in a blocking solution containing 5% milk proteins either in TBST for 1hr at room temperature for Tau blot or in PBST for 20 min for Sgg blot, then probed with primary antibodies overnight at 4uC. Mouse anti-actin antibody (Santacruz) was used at a 1 in 1000 dilution. Tau antibody (rabbit) was a kind gift from Nick Lowe, and used at a 1 in 2000 dilution. Rabbit anti-phospho S262 and S356 (Abcam) were used at a dilution of 1:1000 in 5% BSA TBST. Quantitation was performed using Image J software. Rabbit anti- GSK-3 Inhibition Ameliorates Ab Pathology phospho S9/S21 GSK-3 antibody (AB 9331 Cell Signaling) at a 1 in 250 dilution was detected using an anti-rabbit 800 nm flourophore conjugate (Rockland, USA). Monoclonal antibody, pan Sgg mouse monoclonal (4G1G) at a 1 in 2000 dilution was a kind gift from Marc Bourouis, which was subsequently detected using a 680 nm anti-mouse flourophore conjugate (Invitrogen, UK). Membranes were sequentially scanned at 700 and 800 nm using a Licor Odyssey infrared scanner. Densitometric measure- ments were taken in both wavelengths. Relative phospho-serine 9 Sgg levels were determined by dividing the signal at 800 nm by that obtained at 700 nm. Details of secondary antibodies and Odyssey analysis have been previously described in [69]. Supporting Information Figure S1 Neuronal electrophysiology of flies over-expressing Arctic Ab42 peptide in adulthood. Representative traces for (A) TTM and DLM response latencies and (B) TTM responses to high frequency stimulation (100 Hz) measured in elavGS/+;UAS- ArcAb42/+ flies fed with + or 2 RU486 medium at days 16 and 28. TTM and DLM response latency was increased in Arctic Ab42 over-expressing flies at day 28, but not at day 16 (marked with arrows). Vertical scale bars, 50 mV (TTM) and 60mV (DLM) for response latencies, 20 mV for following at 100Hz; horizontal, 2 ms for response latencies, 20ms for following at 100 Hz. Found at: doi:10.1371/journal.pgen.1001087.s001 (0.11 MB DOC) Figure S2 Neuronal cell loss is not evident in flies over- expressing Arctic Ab42 peptide in adulthood. Cell loss was quantified at day 21 in elavGS/+;UAS-ArcAb42/+ flies, fed with + or 2 RU486 medium and maintained at 27 degrees, by counting the number of cell bodies per brain hemisphere. No significant difference was observed when the two genotypes were compared (student’s t-test), N = 7 per genotype. Found at: doi:10.1371/journal.pgen.1001087.s002 (0.22 MB DOC) Figure S3 Investigating tau phosphorylation. (A) Western blot analyses of tau phosphorylation using phos-tag polyacrylamide gels. Tau showed no changes in phosphorylation in flies expressing Ab42 (UAS-ArcAb42/GFP;elavGS/+) in comparison to their 2RU controls, and when the Ab42 expressing flies were fed lithium (+RU +Li). (B) Phospho-Ser262 and Ser356 human tau antibodies specifically detect tau in fly head homogenates. The 55 kDa tau band is reduced in tau lof/TM6 and absent in tau lof/ tau lof flies compared to w1118 controls. Found at: doi:10.1371/journal.pgen.1001087.s003 (1.23 MB DOC) Figure S4 Full-length Tau (P10636), the longest CNS Tau isoform (P10636-8) and Drosophila Tau were aligned by Clustal W alignment. *Shaded in sky and light blue are sites conserved between human and Drosophila tau that are phosphorylated by GSK-3 in vitro. The two Ser sites 262 and 356 (light blue) had available human tau antibodies that detect those conserved sites in Drosophila. ** Shaded in red are likely GSK-3 sites and Abeta sites (212 and 214 for Abeta) that we had available antibodies for and tested in Drosophila, but did not work since they are not conserved. ***Shaded in yellow is an Abeta site (Ser 422) that is conserved in Drosophila, which we had an antibody for; Ser 262 is also a likely Abeta site. Found at: doi:10.1371/journal.pgen.1001087.s004 (0.03 MB DOC) Figure S5 Reduction in tau levels does not affect amyloid levels of Arctic Ab42 flies. Protein levels of UAS-ArcAb42/+;elavGS Tau EP3203/Tau Dfc to UAS-ArcAb42/+;elavGS Tau EP3203/ PLoS Genetics | www.plosgenetics.org 16 September 2010 | Volume 6 | Issue 9 | e1001087