Figure 3

Downregulation of APP-BP1 by siRNAs in primary neurons affected APP processing. A. APP-BP1 was suppressed by APP-BP1 siRNAs but not by missense siRNAs. Primary neurons were infected with 0.5 IU of siRNA virus per cell. Equal amount of proteins extracted by RIPA buffer was analyzed by western blotting for APP-BP1 expression. The same blot was reprobed with a mouse anti-γ-tubulin antibody. No obvious changes in γ-tubulin levels were detected. BP1siRNA, APP-BP1siRNA; mis-siRNA, misssense siRNA. B. Suppression of APP-BP1 by siRNAs caused increases in exogenous human APP. Primary neurons were infected with or without APP and siRNA constructs. APP was probed with the antibody 369. The same membrane was reprobed for γ-tubulin with a mouse anti-γ-tubulin for loading controls. In each sample, 10 μg of proteins was analyzed. The data is representative of three-independent experiments. C. APP-BP1 siRNA inhibited APP-CTF processing in primary neurons expressing human APP. APP-CTFs were resolved on 16% Tris-Tricine gels. The blot was probed with 369. A representative of 4 independent experiments was shown. The intervening lanes between mis-siRNA and L685459 were cut off. D. APP-BP1 siRNA expression significantly blocked APP processing. Quantitative western blot analyses of 3 (APP) or 4 (CTF) independent experiments were carried out using two-tail t-Test (APP: BP1siRNA vs mis-siRNA, p < 0.003; CTF: BP1siRNA vs mis-siRNA, p < 0.001).