Quantitative PCR and Immunoblot Analysis of Brain Protein Expression

For real-time quantitative PCR (qPCR), brain tissues were homogenized in Trizol and total RNA was extracted using the Aurum Total RNA kit for fatty and fibrous tissue (Bio-Rad, Hercules, CA, USA). qPCR were performed as previously described (Martins-Silva et al., 2011 (link)). Primer sequences: STIP1-F, 5′-GCCAAGAAAGGAGACTACCAG-3′; STIP1-R, 5′-TCATAGGTTCGTTTGGCTTCC-3′; HsP90-F, 5′-CCACCCTGCTCTGTACTACT-3′; HsP90-R, 5′-CCAGGGCATCTGAAGCATTA-3′; HsP70-R, 5′-ACCTTGACAGTAATCGGTGC-3′; HsP70-F, 5′-CTCCCGGTGTGGTCTAGAAA-3′; PRP-F, 5′-GAACCATTTCAACCGAGCTG-3′; PRP-R, 5′-CATAGTCACAAAGAGGGCCAG-3′; Actin-F, 5′-TGGAATCCTGTGGCATCCATGA-3′; and Actin-R, 5′-AATGCCTGGGTACATGGTGGTA-3′. Immunoblot analysis was carried out as described previously (Beraldo et al., 2013 (link)). The antibodies used were anti-STIP1 (1:5000, in-house antibody generated by Bethyl Laboratories Montgomery, USA using recombinant STIP1) (Beraldo et al., 2013 (link)), anti-Hsp90 (1:1000), anti-Hsp70 (1:1000), anti-Hsp90α (1:1000), anti Hsp90β (1:1000) (Cell Signaling, Danvers, USA) and anti-PrP 8H4 (1:2000) (Abcam, Cambrige, UK).