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L., Li B. in controlling tau toxicity. These findings suggest that serine-proline/threonine-proline sites cooperate to mediate neurodegeneration in vivo. Intro Phosphorylation of the microtubule-associated protein tau is elevated in neurodegenerative disease cells, and tau-positive neurofibrillary pathology is definitely characteristic of several disorders, including Alzheimer’s disease and additional tauopathies COTI-2 (Feany and Dickson, 1996 ; Lee tauopathy model (Wittmann (Fulga tauopathy model to investigate how phosphorylation of individual SP/TP sites effects tau-induced neurotoxicity. We generated phosphorylation-incompetent forms of tau in which solitary phosphorylation sites, or a pair of sites, were replaced by alanines. Our results exposed that although SP/TP sites are essential mediators of neurotoxicity, there is no solitary site whose phosphorylation is essential for neurodegeneration. These findings suggest that SP/TP sites function collectively to promote neurotoxicity in vivo. MATERIALS AND METHODS Constructs, Genetics, and Stocks The and transgenic lines have been explained previously (Wittmann Stock Center (Indiana University or college, Bloomington, IN) (medium at 25C. Flies were analyzed at 1 to 3 d of age. Western Blot Mind from adult flies at 1 d posteclosion were homogenized in Laemmli buffer (Sigma-Aldrich, St. Louis, MO), boiled for 10 min at 100C, and centrifuged at 13,000 for 2 min to remove insoluble debris. Proteins were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) by using a 10% separating gel (Cambrex, East Rutherford, NJ), transferred to nitrocellulose (Bio-Rad, Hercules, CA), clogged in 5% milk in phosphate-buffered saline with 0.1% Tween 20, and immunoblotted using one of the following antibodies: polyclonal anti-C terminal tau (Dako North America, Carpinteria, CA) at 1:106 dilution; AT8 (Innogenetics, Zw?nderecht, Antwerp, Belgium) at 1:1000 dilution; Tau1 (Chemicon International, Temecula, CA) at 1:10,000 dilution; TG3 (P. Davies, Bronx, NY) at 1:100 dilution; PHF1 (P. Davies) at 1:500 dilution; pSer212 (BioSource International. Camarillo, CA) at 1:40,000; pSer214 (BioSource International) at 1:40,000; AT270 (Pierce-Endogen, Rockford, IL) at 1:100,000 dilution; and AT100 (Innogenetics) at 1:1000 dilution. For each blot, the same quantity of take flight heads was used for each genotype, Ponceau S staining were performed to ensure equivalent total protein loading and even SOCS2 transfer, and blots were reprobed for total tau (Dako North America). Western blots were repeated a minimum of three times with different animals, and representative blots are demonstrated. The appropriate anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibody was applied and signals were recognized by chemiluminescence (Pierce-Endogen). Histology Mind from adult flies at 1 to 3 d posteclosion were fixed in Formalin, inlayed in paraffin, COTI-2 and 4-m frontal sections were prepared. Serial sections were cut through the entire brain and placed on a single glass slip, and immunostaining was performed as explained previously (Khurana head homogenates were prepared in 1 lambda phosphatase buffer (New England Biolabs) comprising a protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN) COTI-2 and 200 g of total protein were incubated with 800 U of lambda protein phosphatase for 3 h at 37C. Homogenates were then subjected to SDS-PAGE and immunoblot analysis as explained above. RESULTS Generation of Individual SP/TP Transgenic Tau Mutants To determine the relative contribution of individual SP/TP COTI-2 phosphorylation sites to tau toxicity, we generated eight transgenic lines expressing mutant forms of human being tau in which the following serine and/or threonine residues were replaced by alanine: tau111/153 (T111A, T153A); tau175/181 (T175A, T181A); tau199/217 (S199A, T217A); tau202/205 (S202A, T205A); tau212 (T212A); tau231/235 (T231A, S235A); tau396/404 (S396A, S404A); and tau422 (S422A) (Number 1). Many of these sites, when phosphorylated, are identified by a group of monoclonal antibodies that preferentially react with tau found in individuals with neurodegenerative disorders. These antibodies include AT8, AT100, AT270, TG3, and PHF1, and the phosphoepitopes identified by each of them are schematized in Number 1. Although not an SP site, we also generated tau214 (S214A), because Thr212 and Ser214 create the phosphoepitope identified by the.