Figure 4

EVP-0015962 does not alter processing of APP or other γ-secretase substrates. A. Effect of EVP-0015962 treatment on the ε-cleavage of APP β-CTF, measured in a cell-free assay with Aph1A_L containing γ-secretase complexes. Representative Western blot of AICD fragments, for EVP-0015962 (1 nM to 100 μM) and DAPT (10 μM). DMSO is the control lane. N = 2 experiments. B. Full length APP (flAPP), α-CTF, and β-CTF from H4-APP₇₅₁ cell lysates, analyzed by Western blot after a 16-h treatment with 1 nM to 10 μM of LY-411,575 or EVP-0015962. N = 1 experiment. C and D. Effect of EVP-0015962 treatment (1 nM to 30 μM) on the S3 cleavage of NotchΔE to form NICD, measured in transfected HEK293 cells. C. Representative Western blot of NICD fragments fluorescently labeled with cleaved Notch1 antibody (green) and Myc-tagged antibody (red). The NICD fragments are detected as yellow in an overlay of both antibodies. DMSO is the control lane and Empty vector is a lysate from cells without the NotchΔE construct. D. NICD intensity histogram corresponding to the Western blot in E. NICD is quantified as a percentage of total Notch. N = 2 experiments. E and F. Effect of 24 h of treatment with 100 nM to 10 μM of LY-411,575 or EVP-0015962 on the production of EphA4 CTF in cell lysates from rat primary neocortical cultures. E. Western blot of full length (FL) EphA4 (upper band) and EphA4 CTF (lower band). F. Ratio of EphA4 CTF to full length EphA4 corresponding to the Western blot in E. N = 1 experiment.