In vitro incubation of lysates with 2 mM DSP followed by βME reduction allows optimal αSyn detection.

A. HEL intact cells (in vivo) or lysates (in vitro) were incubated with DMSO only (-) as well as gradients of 0.5, 2.0 and 8.0 mM DSP and DTBP, respectively. Cytosols (post-100,000 g) were boiled in sample buffer/5% βME and blotted with αSyn antibody 2F12. Identical exposures of the same blot are shown; film was cut at dotted line. B. HEL cell cytosols (post-100,000 g) at three different protein concentrations (2.4, 3.3., 4.5 µg/µL) were incubated with DMSO only (-) as well as a gradient of 0.5, 2.0 and 8.0 mM DSP and DSG. Samples were normalized to 2.4 µg after quenching, then boiled in sample buffer plus 5% βME and blotted with αSyn antibodies Syn1 and 15G7 as well as an antibody to GAPDH. C. HEL cell cytosols (post-100,000 g) were treated with DMSO-only (-) or DSP, quenched, boiled in sample buffer plus 5% βME and analyzed by blotting for αSyn (mAb Syn1 and pAb C20), Calmodulin, DJ-1, Ran, 14-3-3 and β-actin.