Total tau

For coimmunoprecipitation experiments, 200 fly heads expressing UAS-PAR-1-WT-Myc transgene driven by GMR-GAL4 were homogenized in lysis buffer (50 mM Tris-HCl pH 8.0%, 1% Triton X-100, 150 mM NaCl, 2 mM Na₃VO₄,10 mM NaF, 10% glycerol, protease inhibitors), and the supernatant of lysate was obtained after centrifugation at 12,000 × g for 30 minutes at 4 °C. The supernatant was pre-cleared by incubation with protein A/G agarose (Pierce) for 1 hour at 4 °C. The supernatant was incubated with antiPAR-1 antibodies for 2 hours at 4 °C and then incubated with protein A/G agarose for 12 hours at 4 °C. Beads were washed 5 times with lysis buffer or PBS and boiled in SDS sample buffer. The samples were separated by gel electrophoresis and analyzed by western blot analysis. For western blot analysis, the following primary antibodies were used: anti-PAR-1 (1:8000), anti-phospho-PAR-1 (1:1000); anti-GFP (1:2,000, Abcam); anti-Myc (1:1,000, Millipore); anti-tau (pS262) (1:2,000, Life technology); total tau (1:2,000, Dako); and anti-GAPDH (1:2,000, Abclone). All primary antibodies were diluted in 0.5% BSA in TBST (tris-buffered saline, 0.1% tween 20) and incubated with the membrane at 4 °C overnight. All secondary antibodies (Jackson ImmunoResearch Laboratories) were incubated for 1 hour at room temperature.