Goat anti hsp60

Cell lysates were prepared with lysis buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na₃VO₄, 1 μg/ml leupeptin. 1 mM PMSF was added immediately prior to use. The protein concentration was measured by DC protein assay (Bio-Rad, Irvine, CA). Quick neuron nuclear extract preparation: the cells were rinsed with PBS once, and plates placed on ice and 1 ml of ice cold Buffer A (25 mM Hepes pH 7.0, 25 mM KCl, 0.05 mM EDTA, 5 mM MgCl₂, 10% glycerol, 0.1% NP-40, 1 mM DTT) added. The plate was scraped and cells were transferred into an Eppendorf tube, which was centrifuged and rinsed once with Buffer A (no NP-40). The pellet was resuspended in PBS, and 2x SDS PAGE sample buffer added, prior to boiling for 10 min at 95°C. The following primary antibodies and dilutions were used: antibodies against the major glycolysis enzymes were from Cell Signaling sold as glycolysis antibody sampler kits (#8337&12866); all were used at 1:1000; Rabbit anti-TFAM (Cell Signaling) used at 1:1000; Rabbit anti-CS, IDH2, PGC-1α and ATP5O (Abcam, Cambridge, United Kingdom) used at 1:1000, and mouse anti-Sucla2 and goat anti-Hsp60 (Santa Cruz Biotechnology) used at 1:1000. Immunoblotting results were analyzed by Odyssey Imager (Licor, Lincoln, NE) scanning.

Striatal cultures were fixed, blocked, and incubated as described [13 (link)]. Primary antibodies include mouse anti-glutamate (1:5000; ImmunoStar, Inc., Hudson, WI) and goat anti-HSP60 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA). Secondary antibodies include goat anti-mouse Cy3 and donkey anti-goat Alexa 488 (1:300, Jackson ImmunoResearch, West Grove, PA).