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. 2009 May 8;284(19):13013–13022. doi: 10.1074/jbc.M807653200

FIGURE 5.

FIGURE 5.

Endoproteolysis, Aβ, and NICD production by F411Y/S438P mutant in mouse embryonic fibroblasts. A, PS double knock-out cells were transiently transfected with C1–99pcDNA, PS1pcDNA3.1/Zeo, or F411Y/S438PpcDNA3. 1/Zeo, and siRNA for NCT or control siRNA (cont. siRNA), as indicated. Cells were recovered and analyzed by immunoblotting with specific antibodies. The endoproteolysis of PS1 was detected by the production of PS1 CTF. B, NCT knock-out (KO) cells or wild type (WT) fibroblast cells were transiently transfected with C1–99pcDNA, PS1pcDNA3.1/Zeo, or F411Y/S438PpcDNA3.1/Zeo as indicated. Cells and media were recovered and analyzed by immunoblotting with specific antibodies (human PS1-specific antibody (PS1L) was used for PS1). Aβ was detected in media. Synthetic Aβ40 (30 pg) was loaded as indicated. C, NCT knock-out cells (KO) or wild type (WT) fibroblast cells were transiently transfected with pCS2+mNΔEmyc, PS1pcDNA3.1/Zeo, or F411Y/S438PpcDNA3.1/Zeo as indicated. Membrane-bound mNotch-1 (NΔE) was expressed from the pCS2+mNΔEmyc vector. 48 h after transfection, cells were incubated with 10 μm lactacystin for 4 h, recovered, and analyzed by immunoblotting with specific antibodies. The asterisks indicate nonspecific bands.