APP and PrP^C expression in MBs during culture.

(A) Real-time PCR analysis shows APP and PRNP gene expression at different times during culture. Expression during MB generation and differentiation was normalized with iPSCs expression, which was used as the baseline. (1) iPSCs; (2) embryoid bodies, (EBs); (3) embryoid bodies after neural induction (NI); (4) MBs after 19 days of culture (d19); (5) 2-month old MBs (2m); (6) 4-month old MBs (4m). (B) Western Blot analysis of APP and PrPC using specific antibodies: PrPC antibody SAF32 is specific for the octapeptide repeat region at the N-terminal site which contains the octarepeat region; full-length diglycosylated PrPC appears at ∼ 35 kDa (arrow) whereas unglycosylated and truncated forms appear at a lower molecular weight. 22C11 antibody recognizes amino acid sequence 66–81 and allows identification of a full-length APP695 isoform at ∼ 100 kDa (arrow) as well as an N-truncated form of APP protein. The Bradford method was used to measure the protein load (5 μg of total protein per well); α-tubulin (∼ 55 kDa) was used as loading control. (4) MBs after 19 days of culture (d19); (5) 2-month old MBs (2m); (6) 4-month old MBs (4m); (>6) 7-month old MBs (7m). (C) Tissue sections of 1 month- (left panel) and 3-month old MBs (right panel), immunostained with specific antibodies recognizing PrPC (SAF32 antibody, specific for an N-terminal epitope of protein, upper panel) and Aβ peptides (BAM10 antibody, lower panel).