Full-length parkin cDNA and its antisense construct were transfected into human dopaminergic neuroblastoma cells (BE(2)-M17). Resulting clones were screened by Western Blot with monoclonal antibody to alpha-synuclein (Clone 42, Transduction Labs)and rabbit polyclonal antibody to the C terminus of parkin (Cell Signaling), developed with peroxidase-labeled secondary antibodies (Jackson Immunochemicals) and chemiluminescence substrates (Amersham). Wildtype or mutant alpha-synuclein stable cell lines previously generated were also screened with both antibodies.
Cell viability assessed by MTT assay.
Stable cells lines transfected with GFP construct and treated with lactacystin (Calbiochem). Western blot used to detect GFP-CL1 fusion protein with antibody GFP (Clontech).
Primary mouse midbrain explants of neurons were prepared and exposed to MG132 or lactacystin. Toxicity was assesed by staining for tyrosine hydroxylase with polyclonal anti-TH (Chemicon) and MAP2 (microtubule-associated protein)(clone Ap-20, Sigma), secondary antibody was goat anti-rabbit conjugated to Alexafluor488 (Molecular Probes) and goat anti-mouse conjugated to AlexaFluor 568.
cDNAs from either alpha-synuclein or parkin were cloned into pHSVPrPUC and packaged into recombinant viral particles. MOI of 10 was used with primary cells.