Figure 2.
Temporal regulation of APP and its proteolytic fragments in mouse primary neurons. a, Expression of full-length APP increases over time in mouse primary neurons. Sister cultures of neurons plated in 10 cm dishes (1.5 × 106 cells per dish) were collected directly in 2× Laemmli sample buffer, boiled for 5 min, and sonicated before loading on a 10-20% Tris-tricine gel for analysis. Nitrocellulose membranes were probed with C8 antibody. b, The same samples as in a were probed for APP CTFs and AICD with antibody C8. Note that there is an associated increase in C83 that corresponds to the AICD spike at DIV 6 and 7 (for additional analysis, see Fig. 4). As a migration control, C60 (far left lane) migrated slightly higher than the AICD. The identities of the various CTFs are based on previous studies (Buxbaum et al., 1998a) and on c. The asterisk represents a nonspecific band that appears intermittently depending on the bleed date of the polyclonal C8 antibody being used. c, Confirmation of the phosphorylation of APP CTFs using λ-protein phosphatase. Lysates from DIV 5 and DIV 13 neurons were incubated in reaction buffer without (-) or with (+) λ-PP for 1 h. Analysis on 10-20% Tris-tricine gels with C8 Western blotting resolved the various species. The asterisk represents a nonspecific band as in b. d, Comigration of the APP CTFs with C83 and C99 identifies these proteins among the five CTF bands. e, Quantitation of relative levels of C83, C89, and C99 at DIV 5 and DIV 13, time points before and after the transient appearance of AICD, respectively. Western blots from four independent batches were quantified by densitometry after dephosphorylation with λ-PP. Left, Relative amounts of total CTFs at DIV 5 versus DIV 13. Middle, Within the DIV 5 CTFs, there is ∼1.5 times more C83 and C89 than C99. Right, This same ratio of C83, C89, and C99 levels is obtained at DIV 13 (although there are twofold more total CTFs compared with DIV 5).