Western Blot Analysis of Protein Expression

Western analyses were run as described (42 (link)) with the following changes: Purified mouse anti-ASyn antibody clone 42 (BD Biosciences) was diluted at 1/1000, rabbit anti-Hsp70 antibody (Enzo Life Science, Inc, ADI-SPA-812) and mouse monoclonal antibody G3.1 anti Hsp27 antibody (Enzo Life Science, Inc) were diluted at 1/1000, and mouse anti-actin antibody clone AC15, (Sigma-Aldrich) was diluted at 1/35,000. Membranes were then washed four times for 10 min each in PBS 0.1% Tween (PBST) and incubated with secondary antibody donkey anti-mouse infrared 680 and goat anti-rabbit infrared 800 (LI-COR) diluted at 1/10,000 in Odyssey PBS Blocking Buffer (LI-COR, Inc) with 0.2% Tween 20 followed by washing four times for 10 min each in PBST. Membranes were scanned and quantitated using the Odyssey CLx Imaging System (LI-COR). Hsp70 was visualized in the 800 nm fluorescent channel and actin, αSyn, Hsp27, and the molecular weight markers visualized at 700 nm. As previously described (42 (link)), proteins are cross-linked to blots by 30 min treatment with 0.4% PFA in PBS prior to staining to enhance ASyn binding (72 ) to ensure visualization of all ASyn.