Immunoblot detection of PrPres from various tissues of 22L scrapie-infected anchorless PrP tg44 mice and WT mice.

<p>Blots were developed using D13 anti-PrP <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000232#ppat.1000232-Matsunaga1" target="_blank">[26]</a>. No PrPres was detected in uninoculated tg44 or WT mice. For tg44 mice (A), lane 1, mock infected tg44 mouse brain, 1.0 mg tissue equivalents. Lanes 2–6 were loaded with 0.25–0.5 mg tissue equivalents and developed using an enhanced chemiluminescence exposure of 5 seconds, and lanes 7–13 were loaded with 1.0 mg tissue equivalents and exposed for 1–2 minutes. Lane 11, liver was negative, and lane 12 was the only positive liver of nine tested. In tg44 mice, spinal cord, spleen, lymph node, intestines, kidney, and lung were also positive, and eye and skin were negative (data not shown). Note different PrPres banding pattern seen in tg44 mice: predominant band is the 18 kD unglycosylated form. Next in abundance is the 22kD monoglycosylated form. The 25kD diglycosylated form is barely visible in lanes 5, 6, 9, and 10. Dimeric PrP bands at 36kD are seen most clearly in lanes 3, 5, 6, and 9. For WT mice (B), lane 1 was loaded with 0.25 mg tissue equivalents while lanes 2–8 were loaded with 1.0 mg tissue equivalents. Blot was exposed for 2 minutes to search for possible weak bands in lanes 2–8. Lane 1 was overexposed, but showed usual PrPres banding pattern for 22L mouse scrapie brain. Results are representative of tissues from at least five mice analyzed in each group.