Grp78

Samples from the CPu or from cultured astrocytes were solubilized in buffer containing 1% NP40, 0.1% sodium dodecyl sulfate, and 0.2% deoxycholate and were subjected to western blotting with the following antibodies: tyrosine hydroxylase (TH; EMD Millipore, Billerica, MA, USA), glial fibrillary acidic protein (GFAP; Dako, Glostrup, Denmark), GRP78 (StressGen, Victoria, British Columbia, Canada), heme oxygenase-1 (HO-1; Abcam, Cambridge, UK), and β-actin (Sigma). Primary antibody binding was visualized using the ECL system (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA).

Immunohistochemical analysis was performed as previously described [27 (link)]. In brief, brains were removed from mice after perfusion with 4% paraformaldehyde and postfixed in the same fixative for 4 hours at 4°C. After being cryoprotected in 30% sucrose, brains were cut in serial coronal 10 μm-thick sections containing the CPu (from Bregma +1.34 mm to Bregma +0.26 mm) and the midbrain covering the whole SNpc (from Bregma −2.80 mm to Bregma −3.80 mm) on a cryostat. Brain sections were mounted in series on ten slides (around ten sections were mounted on each slide). One out of these ten slides, representing a set of sections 100 μm apart, was processed for immunohistochemistry with the following antibodies: TH, GFAP, GRP78, HO-1, Ub (StressGen), α-synuclein (BD, Franklin Lakes, NJ, USA), and LC3B (Cell Signaling Technology, Danvers, MA, USA). In some cases, the cell nuclei were visualized with 4′,6-diamidino-2-phenylindole (DAPI; Sigma). Alexa488-conjugated (Thermo Fisher Scientific, Rockford, IL, USA) or Cy3-conjugated (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) secondary antibody was used for visualization of immunolabeling.