FIGURE 3.

PS1 L435F and neighboring mutations markedly impair γ- secretase-dependent proteolytic cleavage of APP and Notch. To assess cleavage activity, WT and mutant PS1 were co-expressed with APP C99-myc or NotchΔE-myc by transient transfection in PS-null MEFs, and full-length and cleaved substrates were detected by Western analysis. Con (A, B) and vector (C) indicate transfection with empty rather than PS1-expressing vector. Representative expression of PS1 for the same series of experiments is shown in Fig. 2A. A, APP C99 and AICD were detected with antibody recognizing the myc epitope tag (*, nonspecific background band). IB, immunoblot; α-tub, α-tubulin. B, γ-secretase-dependent Notch cleavage was detected by Western analysis with antibody recognizing cleaved Notch ICD (top panel). Full-length NotchΔE and NICD were detected with anti-myc antibody (middle panel). C, quantification of AICD and NICD production by PS1 mutants is shown. AICD and NICD levels were normalized to α-tubulin and expressed as % of WT levels. Data are the means of three independent experiments (*, p < 0.05 versus AICD or NICD level in cells expressing WT PS1; n = 3). ICD, intracellular domain.