FIGURE 2.

Characterization of fibroblasts heterozygous (Pen-2^+/−) or homozygous (Pen-2^−/−) for the Pen-2 deletion gene and of Pen-2^−/− fibroblasts expressing the cysteine-less Pen-2 mutant. A, 4–12% BisTris SDS-PAGE/Western blot analysis of Pen-2^+/+, Pen-2^+/−, and Pen-2^−/− fibroblasts derived from E9.5 mice. 20 μg of solubilized membrane protein was applied per lane. Antibody 9C3 was used to stain immature (immat) and mature (mat) NCT, and B19 was used to stain full-length presenilin1 (FL PS1) and presenilin1 N-terminal fragments (PS1 NTF) and Mab5232 for the C-terminal fragments (PS1 CTF). Antibody B63 was used to stain full-length APP (FL APP) and APP C-terminal fragments (APP CTF) and antibody B80 for Aph-1a. Actin, loading control. B, Blue Native PAGE/Western blot analysis of Pen-2^+/+, Pen-2^+/−, and Pen-2^−/− DDM extracted membranes (5 μg/lane) stained with antibodies against PS1 CTF, NCT, and Aph-1a as in A. Full γ-secretase complex is absent in Pen-2^−/− fibroblasts. The trimeric complex in Pen-2^−/− fibroblasts is composed of FL PS1, NCT, and Aph-1a. *, artificial complex because of detergent-dependent dissociation, composed of PS1 CTF, Aph-1a, and NCT (59). C, Blue Native PAGE/Western blot analysis (5 μg/lane) of membrane fractions of Pen2^−/− fibroblasts reconstituted with cysteine-less Pen-2 (CL Pen-2) or wild type Pen-2 (WT Pen-2) and Pen-2^+/+ or Pen-2^−/− fibroblasts. The cysteine-less Pen-2 mutant is incorporated in the mature γ-secretase complex to the same extent as the wild type Pen-2. *, artificial complex (A). D, SDS-PAGE/Western blot analysis of membranes of the same fibroblasts as in C and using the same antibodies as in A. The cysteine-less Pen-2 mutant rescues NCT maturation, PS1 endoproteolysis, and APP CTF cleavage to the same extent as wild type Pen-2.