(A) Immunoblot of full-length APP and α-tubulin in cell lysates, and sAPPα and sAPPβ in conditioned media, from CHO-APPwt cell. RIPA lysates (20 µg) and 20 µL of conditioned media were resolved in 7.5% Tris-glycine SDS-gels, blotted and probed with 5685 (fl-APP), Ban50 (sAPPα), and α-tubulin antibodies. While the level of sAPPα was reduced by BMS-561392 in a dose-dependent manner, the sAPPβ level in the media remained unchanged. Consistent levels of fl-APP and tubulin confirmed a lack of BMS-561392 toxicity. (B) Immunoblot and quantitation of APP C-terminal fragments (CTFs). RIPA lysates (500 µg) were immunoprecipitated with 5685 antibody, resolved in a 16.5% Tris-tricine SDS-gel, blotted and probed with the same antibody. Note that the levels of CTFβ, a pan-precursor protein of Aβ, remain unchanged after BMS-561392 treatment. Quantification was based on the means of three independent experiments +/− standard error measurements. P<.01*, two-tailed Student’s t-test; n.s.*, not significant, two-tailed Student’s t-test, P = .150; n.s.** = not significant, one-way ANOVA, P = 0.598. #Results are shown in arbitrary unit (DMSO treatment as 1.0). (C) Aβ40 and 42 in conditioned media after BMS-561392 treatment. Quantification was based on the mean of six independent experiments +/− standard error measurements. n.s.* = not significant, one-way ANOVA, P = 0.902; n.s.** = not significant, one-way ANOVA, P = 0.874. #Results are normalized to fl-APP levels. (D) Reduction of sAPPα secretion with no concomitant change in Aβ secretion in primary neurons derived from Tg2576 mice cultured for 7 days with or without BMS-561392 for 24 hrs. RIPA lysates (20 µg) were in 7.5% Tris-glycine SDS-gels, blotted and probed with 5685 (fl-APP), Ban50 (sAPPα), C5A4/2 (sAPPβ), and α-tubulin antibodies. Quantification was based on the means of three independent experiments +/− standard error measurements. n.s.* = not significant, one-way ANOVA, P = 0.943; n.s.** = not significant, one-way ANOVA, P = 0.996. #Results are normalized to fl-APP levels.

(A) Immunoblot of full-length APP and α-tubulin in cell lysates, and sAPPα and sAPPβ in conditioned media, from CHO-APPwt cells. (B) Quantitation of sAPPα level in conditioned media, measured by densitometry of a 2B3 Western blot. (C) Aβ40 and Aβ42 levels in conditioned media. PMA activates α-secretase activity and reduces Aβ secretion, and BMS-561392 reverses the effect of PMA on CHO695wt cells. Quantification based on the means of three independent experiments +/− standard error measurements. P<0.05¹ ~ P<0.05⁶; two-tailed Student’s t-test. #Results are normalized to fl-APP levels. sAPPα levels are shown in arbitrary units (average of DMSO only treatments as 1.0).

(A) BMS-561392 reduces sAPPα level in Tg2576 mouse hippocampi, but TAPI-I fails to do so. RIPA lysates (20 µg) of drug-treated mouse cortices and hippocampi were resolved in 7.5% Tris-glycine SDS-gels, blotted and probed with 5685 (fl-APP), 2B3 (sAPPα), and α-tubulin antibodies. (B) Quantification based on the average of n = 5 for each group in 6 independent immunoblots +/− standard error measurements. n.s.^a = not significant, one-way ANOVA, P = 0.734; n.s.^b = not significant, unpaired two-tailed Student’s t-test, P = 0.236; P<0.0001⁺ ; unpaired two-tailed Student’s t-test. #Results are normalized to fl-APP levels and shown in arbitrary units (control as 1.0). (C) Aβ40 and Aβ42 levels in brain lysates and CSF showed no statistically significant change. n.s.¹ ~ n.s.⁶ = not significant, one-way ANOVA, P = 0.735, 0.590, 0.647, 0.480, 0.550, and 0.606, respectively.

CHO cells expressing human APP695wt and pro-TNFα were treated with BMS-561392 and TAPI-I for 24 hrs. (A) Inhibition of TNFα secretion by TAPI-I and BMS-561392. 50% inhibition with respect to DMSO controls was achieved with 0.90 µM TAPI-I and 0.15 µM BMS-561392. (B) Inhibition of sAPPα secretion by TAPI-I and BMS-561392 from CHO cells expressing APPwt and APPswe. 50% inhibition with respect to DMSO controls was achieved with 59.1 µM TAPI-I and 4.47 µM BMS-561392 on CHO-APPwt cells, and 0.23 µM BMS-561392 on CHO-APPswe cells. Data are expressed as the mean of three independent experiments +/− standard error measurements. * = two-tailed Student’s t-test, P<.01; ** and *** = two-tailed Student’s t-test, P<.0001.

(A) Immunoblot of full-length APP and α-tubulin in cell lysates, and sAPPα and sAPPβ in conditioned media, from CHO-APPwt cells. RIPA lysates (20 µg) and 20 µL of conditioned media were resolved in 7.5% Tris-glycine SDS-gels, blotted and probed with 5685 (fl-APP), Ban50 (sAPPα), and α-tubulin antibodies. (B) Quantification of sAPPα, Aβ40, and Aβ42 levels in conditioned media by sandwich ELISAs. BACE inhibitor potently decreases the secretion of Aβ, but does not induce higher sAPPα secretion. Quantification was based on the means of three independent experiments +/− standard error measurements. n.s., not significant, one-way ANOVA, P = 0.945. #Results are normalized to fl-APP levels. sAPPα levels are shown in arbitrary units (average of DMSO treatments as 1.0).

(A) Immunoblot of full-length APP and α-tubulin in cell lysates, and sAPPα and sAPPβ in conditioned media, from CHO-APPwt and CHO-APP-TGN cells. APP-TGN migrated slower because of the addition of the furin sequence (Skovronsky et al., 2000;Huse et al., 2002;Chyung et al., 1997). RIPA lysates (20 µg) and 20 µL of conditioned media were resolved in 7.5% Tris-glycine SDS-gels, blotted and probed with 5685 (fl-APP), Ban50 (sAPPα), and α-tubulin antibodies. (B) Quantification of sAPPα in conditioned media. Unlike CHO-APPwt cells, BACE inhibitor increases sAPPα secretion from CHO-APP-TGN cells. Quantification are means of three independent experiments +/− standard error measurements. n.s., not significant, two-tailed Student’s t-test, P = 0.210; P<0.005, paired two-tailed Student’s t-test. #Results are normalized to fl-APP levels in each cell line. sAPPα levels are shown in arbitrary units (DMSO treatment as 1.0).

(A) BMS-561392 reduces sAPPα level in drug-treated SJL/B6 F1 mouse cortices and hippocampi, while Aβ40 and Aβ42 levels in brain lysates and CSF were unchanged. Fifty µg of DEA/NaCl lysates (for sAPPα) and RIPA lysates(for fl-APP and α-tubulin) of drug-treated mouse cortices and hippocampi were resolved in 7.5% Tris-glycine SDS-gels, blotted and probed with 5685 (fl-APP), 2B3 (sAPPα), and α-tubulin antibodies. (B) Quantification based on the average of n = 6 for each group +/− standard error measurements. P<0.05⁺, P<0.05⁺⁺, unpaired two-tailed Student’s t-test. #Results are normalized to fl-APP levels and shown in arbitrary units (control as 1.0). (C) Aβ40 and Aβ42 levels in brain lysates and CSF were unchanged. n.s.¹ ~ n.s.⁶ = not significant, unpaired two-tailed Student’s t-test, P = 0.877, 0.482, 0.633, 0.574, 0.928, and 0.785, respectively.