Figure 3
Lysosomal inhibition attenuates the effect of UCHL1 on APP and UCHL1 interacts with APP and increases free ubiquitin levels in the cell.
(A, B) Haw cells were treated with 5 μM MG132, 100 μM chloroquine or control solutions for 6 h. MG132 treatment reduced mature APP level, whereas chloroquine treatment enhanced it. N = 3, *p<0.05, **p<0.01 by ANOVA followed by post-hoc Newman-Keuls test. (C, D) 20E2 cells were treated MG132, chloroquine or control solutions. N = 3, *p<0.05 by ANOVA. (E, F) Haw cells were transfected with pz-UCHL1-st or control plasmid for 48 h and then treated with chloroquine. Chloroquine treatment attenuated the difference in APP level between UCHL1 and control groups. N = 3, *p<0.05, **p<0.01 by ANOVA. (G, H) The experiments in (E) were replicated in 20E2 cells. N = 3, *p<0.05, **p<0.01 by ANOVA. Values represent mean ± SEM. (I) Haw cells were transfected with pZ-UCHL1-st and then treated with 100 μM chloroquine. Cell lysates were immunoprecipitated with C20 antibody and then immunoblotted with anti-UCHL1 antibody BH7 (Novus Bio). Interaction between UCHL1 and APP was detected and the interaction was enhanced with chloroquine. (J) Haw cells were co-transfected with pZ-UCHL1-st and ubiquitin expression plasmid pCW7. APP was detected by C20 antibody and ubiquitin detected by rabbit anti-ubiquitin antibody (Calbiochem). (K) Overexpression of UCHL1 significantly reduced mature APP level. Overexpression of ubiquitin further downregulated mature APP. N = 3, *p<0.05, **p<0.01 by ANOVA. (L) Overexpression of UCHL1 upregulated ubiquitin level. N = 3, *p<0.05, **p<0.01 by ANOVA. Values represent mean ± SEM.
