Ravikumar B, Vacher C, Berger Z, Davies JE, Luo S, Oroz LG, Scaravilli F, Easton DF, Duden R, O'Kane CJ, Rubinsztein DC. Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease. Nat Genet. 2004 Jun;36(6):585-95. PubMed Abstract

REAGENTS/MATERIAL:

Cell lines used for culturing and transfection were COS-7 and PC12. Transgenic mice expressing N-terminal mutant huntingtin fragments (HD-N171-N82Q) were also used. The Drosophila melanogaster yw;gmr-Q120 line was obtained from G. Jackson (UCLA) and was crossed to a w[1118] stock isogenized for the X chromosome and two major autosomes.

Primary antibodies for Western Blot included antibodies to GFP (Clontech), mTOR, phosphorylated mTOR (Ser2448), S6K1, phosphorylated S6K1 (Thr389; 1A5), 4E-BP1 and phosphorylated 4E-BP1 (Thr37) from Cell Signaling Technology; antibody to huntingtin EM48 (Chemicon); and antibody to tubulin (Sigma).

For histochemical analysis, primary antibodies included antibodies to mTOR, phosphorylated mTOR (Ser2448), 4E-BP1, phosphorylated S6 (Ser235/236; Cell Signaling Technologies), LC3 (gift from T. Yoshimori, National Institute of Genetics, Japan), huntingtin and ubiquitin (both from Chemicon).