Figure 5.

APP accumulation and Aβ generation by suppression of HRD1 expression. a, Induction of APP accumulation by HRD1 siRNA. SH-SY5Y cells stably expressing APP-FLAG were transiently transfected with NC or HRD1 siRNA. The total cell lysates were analyzed by Western blotting with the indicated antibodies. GRP78 and GRP94 were detected with anti-KDEL antibody. b, CHX assay. SH-SY5Y cells stably expressing APP-FLAG were transfected with NC or HRD1 siRNA. At 48 h after transfection, the cells were treated with 20 μg/ml CHX for the indicated periods. Total cell lysates were analyzed by Western blotting using anti-FLAG M2 antibody. Levels of APP at each time point were plotted relative to the amount present at time 0 (mean ± SEM; n = 5). Asterisk represents a significant difference (Student's t test, *p < 0.05). c, d, Aβ levels were measured by a sandwich ELISA using the culture medium of a. Results are expressed as a ratio of the total amount of Aβ peptides (pg) to the total amount of protein from whole-cell extracts (mg) (mean ± SEM; n = 4). Statistical analysis was performed with ANOVA followed by Bonferroni correction (control and NC vs HRD1; *p < 0.05, **p < 0.01).