Figure 6.
Conditional deletion of all three Mint proteins in neuronal cultures decreases the accumulation of APP-CTFs induced with the γ-secretase inhibitor DAPT. Quantitative analysis of APP-CTF and full-length APP levels in neurons containing or lacking Mints as a function of time after addition of γ-secretase inhibitory DAPT. A, Top, Representative immunoblots for APP-CTFs with GDI, a ubiquitously expressed protein used as internal loading control from control neurons infected with CreΔ and Mint-deficient neurons infected with Cre after DAPT treatment for 0, 3, 6, 12, and 24 h. Bottom, Summary graph of APP-C99 levels as relative values normalized to GDI. Mint-deficient neurons (Cre) carrying the transgene (tg+) showed a significant decrease in accumulation of APP-C99 levels induced with DAPT compared with control neurons (CreΔ). B, Summary graph showing total APP-C83 and APP-C89 levels of CreΔ and Cre after DAPT treatment. C, Representative immunoblots and quantitative analysis of full-length APP levels from cultured neurons expressing transgenic mutant human APP and presenilin (tg+) containing (CreΔ) or lacking Mint1, Mint2, and Mint3 (Cre). Bottom, Plot showed no changes in full-length APP levels. D, Quantitative analysis of secreted sAPP levels from conditioned media of cultured neurons expressing transgenic mutant human APP and presenilin (tg+) containing (CreΔ) or lacking all three Mint proteins (Cre) and probed with anti-6E10, anti-sAPPα, and anti-sAPPβ antibodies and normalized against total protein and plotted as a percentage of wild-type control (CreΔ). All summary graphs depict means ± SEMs (n = 3 independent cultures performed in triplicates). *p < 0.05, **p < 0.01, ***p < 0.001.