ABSTRACT
Wild-type (wt) and A53T-α-synuclein-and βsynuclein-expressing TSM1 cells have been described elsewhere.12,13 The XTT assay, caspase activity measurements and p53 transcriptional activity have been described in detail.14,15
Transient transfections were performed in TSM1 neurons with DAC30 containing 2 µg of cDNA encoding either α-synuclein or β-synuclein alone or in combination. Cells were used 48 h posttransfection. For the detection of α-and βsynucleins, equal amounts of protein (50 µg) were separated on Tris-tricine gels, Western blotted and probed with the anti-α-and anti-βsynuclein rabbit polyclonal antibodies (Affiniti Laboratories). Active caspase 3, p53 and β-tubulin immunoreactivities were analyzed by Western blot performed by means of antiactive caspase 3 (rabbit polyclonal, R&D System), anti-p53 (mouse monoclonal, Santa Cruz), and anti-β-tubulin (Sigma, Saint Quentin-Fallavier). Immunological complexes were revealed, as previously described.14,15
Viability of TSM1 neuronal cells was examined by the XTT assay. Clearly, wt-α-synuclein protects TSM1 neurons from staurosporineinduced toxicity (Figure 10.1a) and etoposideinduced toxicity (not shown). Conversely, the A53T-α-synuclein-expressing TSM1 neurons appear more susceptible to toxic stimuli (Figure 10.1a). Staurosporine induces caspase 3 activation in a time dependent (Figure 10.1b) and dose-dependent (Figure 10.1c) manner.
