Cabin DE, Shimazu K, Murphy D, Cole NB, Gottschalk W, McIlwain KL, Orrison B, Chen A, Ellis CE, Paylor R, Lu B, Nussbaum RL. Synaptic vesicle depletion correlates with attenuated synaptic responses to prolonged repetitive stimulation in mice lacking alpha-synuclein. J Neurosci. 2002 Oct 15;22(20):8797-807. PubMed Abstract

REAGENTS/MATERIAL:

Alpha-synuclein deficient knockout mice were generated.

Mouse brains were analyzed by Western Blot using polyclonal anti-synuclein which recognizes both alpha and beta. Total mouse protein was run on two dimentional electrophoresis using pH 4-8 ampholines and 10% PAGE gel and then Western blotted using anti-alpha-synuclein (Transduction Labs.) with detection by HPR and chemiluminescence (Amersham Biosciences).

Synaptosomal protein or post nuclear supernantant from cultured hippocampal neurons were analyzed by Western blot using the following antibodies: anti-synapsin I, anti-synaptophysin, anti-synaptotagmin, anti-rab3a, and anti-amphiphysin (Stressgen); anti-VAMP-2 (vesicle-associated membrane protein-2)and anti-SNAP-25 (synaptosomal-associated protein) from Santa Cruz; Anti-flotillin from Transduction Labs and anti-SV2 (ATCC). Quantitating Western blot was done by making a standard curve with recombinant alpha-synuclein, electrophoresing it on SDS-PAGE and immunoblotting with anti-synuclein 202 (Zymed). The bands were scanned by densitometry and quantitated using ImageQuant software (Molecular Dynamics).