Figure 3.

Increased Aβ levels in 22C11-treated cultured rat PHNs correlate with increased BACE1 protein levels and not with increased γ-secretase activity. Treatment of PHNs for 8 h with a 100 ng/ml solution of 22C11 did not significantly affect PS1 cleavage compared with PBS-treated PHNs. a, Representative Western blots showing no significant changes in the levels of the N-terminal fragment of PS1 in 22C11-treated PHNs compared with PBS-treated PHNs. b–d, Treatment of PHNs for 8 h with a 100 ng/ml solution of 22C11 did not significantly affect γ-secretase activity in 22C11-treated PHNs compared with PBS-treated PHNs. b, Scheme depicting the in vitro γ-secretase activity assay: solubilized membranes from C100-myc overexpressing HEK293 cells were mixed with solubilized 22C11-treated (100 ng/ml; 8 h) or PBS-treated PHNs and incubated for 2 h, resulting in the formation of AICD-myc fragments detectable by Western blot. c, Representative Western blots showing no observable difference in AICD-myc production in 22C11-treated compared with PBS-treated PHNs. d, Densitometric analysis of the AICD-myc levels was quantified from three independent experiments, expressed as the relative mean ± SEM signal intensity (ns). e–g, Treatment of PHNs for 8 h with a 100 ng/ml solution of 22C11 resulted a significant increase in BACE1 protein levels with a concomitant increase in secreted APPβ fragments. e, Representative Western blots showing increased levels of BACE1 and sAPPβ in 22C11-treated compared with PBS-treated PHNs. f, g, Densitometric analysis of BACE1 levels was quantified from at least three independent experiments and expressed as the mean ± SEM band intensity (***p < 0.001 for BACE1; *p = 0.05 for sAPPβ).