Washout experiments with LY450139 in SH-SY5Y cells. Inhibition with high concentration of γ-secretase inhibitor LY450139 leads to substrate accumulation and decreased Aβ secretion but no rebound is detected after washing out the inhibitor. SH-SY5Y APPwt cells were treated with vehicle (0.1% DMSO) or 300 nM LY450139 for 24 h, after which the medium was collected for Aβ40 (A) or Aβ42 (B) measurements (black bars). The media was replaced with fresh medium for another 24 h, and again collected for Aβ40 (A) or Aβ42 (B) measurements (grey bars). C. All three bands detected with 6E10 antibody increased in density in the cells treated with 3, 30 or 300 nM LY450139 indicating substrate accumulation. D. Densitometric quantification of data in C as means ± SEM, n = 2.