Rabbit anti hsp70 antibody

Cell lysis and Western blotting were performed as described previously [31 (link)] The following antibodies were used: a polyclonal rabbit anti-Hsp104 antibody (Thermo Scientific Project NJ1240S), anti-Pgk1 monoclonal antibody (Molecular Probes, Carlsbad, CA, USA), and rabbit anti-Hsp70 antibody (Enzo SPA 757). HPR conjugated goat anti-rabbit or goat anti-mouse secondary antibodies (Thermo Fisher, Waltham, MA, USA) and SuperSignal Plus Chemiluminescent Substrate (Thermo Scientific) were used to develop the Western blot. Amersham Imager 600 was used to scan the blot and the intensities were quantified using the ImageJ-win64.

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.