CK1 inhibitor D4476 does not inhibit Notch cleavage. N2A cells transiently expressing N-terminally truncated mNotchΔE-myc were incubated with the indicated concentrations of DAPT, L-685,458, D4476, or Gleevec for 3 h. Cell lysates (20 μg) were analyzed by Bis·Tris acrylamide gel electrophoresis and Western blotting by using anti-cleaved Notch (A) and anti-actin antibodies (C), or by Aβ40 ELISA (B). DAPT and L-685,458 are γ-secretase inhibitors known to inhibit βCTF and Notch cleavage; Gleevec is known to inhibit βCTF but not Notch cleavage; D4476 is a CK1 inhibitor. Data for at least three experiments (mean ± SEM) were compared (GraphPad Prism ver. 4.0) with the control condition (no drug). NT, nontransfected. ∗∗, P < 0.01; one-tailed Student's t test, 95% significance level.