Immunoblot and dot-blot analysis of mice and human brain tissues were performed as previously described (Lee et al., 2002 (link); Li et al., 2004b (link); Li et al., 2005 (link); Martin et al., 2006 (link); Wang et al., 2008 (link)). For semi-quantitative analysis of protein expression, the chemiluminescence signal associated with antibody binding (Pierce, Rockford, IL) was captured using the Biorad Molecular Imager ChemiDoc XRS+ System system (Biorad, Hercules, CA) or on X-ray films. The intensities of the immunoreactive bands were determined using the Quantity One software (Biorad, Hercules, CA). For dot-blot analysis, lysates were spotted directly on the nitrocellulose membrane and let it dry completely. Immunoreactivity was visualized using chemiluminescence detection (Pierce, Rockford, IL) after incubations with the appropriate horseradish peroxidaseconjugated secondary antibody, using a CCD based Biorad Molecular Imager ChemiDoc XRS+ System (Biorad, Hercules, CA) or X-ray films. The intensities of the immunoreactive bands were determined by densitometry and the Quantity One software (Biorad, Hercules, CA).
Following antibodies were used: grp78, grp94, PDI, calnexin, syntaxin 6 (Stressgen, Ann Arbor, MI); p-eIF2α, eIF2α, caspase 9, caspase 3, synaptotagmin (Cell signaling Technology, Danvers, MA); syn-1, cytochrome C (BD Transduction Laboratories, Franklin Lakes, NJ); ATF4, CHOP, p38, caspase 12, ubiquitin, cathepsin D (Santa Cruz Biotechnology, Santa Cruz, CA); VDAC, NeuN, calnexin (Abcam, Cambridge, MA); pser129-αS (Fujiwara et al., 2002 (link)); syn303 (Duda et al., 2000 (link)); βS (Oncogene, Boston MA).