FIGURE 7.

Cross-linking studies reveal close proximity of the loop of Pen-2 to PS1 CTF. Membrane fractions of Pen-2^−/− fibroblasts rescued with cysteine-less (CL) Pen-2 or cysteine-less Pen-2 E49C were treated with the heterobifunctional cysteine-amine cross-linker SPDP (spacer arm, 6.8 Å). Preblocking of the free sulfhydryl groups with N-ethylmaleimide was performed as negative control. As additional negative control, cross-linker was omitted in the sample (DMSO). Protein extracts were separated in SDS-PAGE under nonreducing conditions on a 4–12% BisTris gel and visualized in Western blot with antibodies against γ-secretase components. A, band with a molecular mass of ∼26 kDa was observed with Pen-2 antibody (arrow). B, same band of 26 kDa was observed with PS1 CTF antibody (arrow).