1. Dictyostelium has PS-dependent γ-secretase activity that processes human APP to release Aβ peptides.
Dictyostelium has PS-dependent γ-secretase activity that processes human APP to release Aβ peptides. (A) Diagrams of human APP α, β and γ proteolytic cleavage sites, and processed fragments; shown are FL (full-length), ΔN (N-terminal deletion), α- and β-CTFs, Aβ40 peptide, and AICD regions. (B) Media conditioned by growing WT and ps1-null cells that express ΔN-APP (a truncated human APP) were analyzed for levels of Aβ40 and Aβ42 peptides by quantitative ELISA. Fresh media and media conditioned by native WT cells were used as negative controls. Bars indicate standard errors that are derived from two independent experiments, each with two replicates. (C)Protein samples were collected from growing native WT Dictyostelium, or WT and ps1-null Dictyostelium that express ΔN-APP. ΔN-APP expression and processed fragments (arrows) were determined by immunoblot assay using α-APP C-terminus. (D) Protein samples were collected from native and APP-expressing CHO cells untreated or incubated with DAPT, and from growing native WT Dictyostelium or WT, aph1-null, ps1;ps2-null and ps1-null Dictyostelium that express ΔN-APP. APP expression and processing was determined by immunoblot assay using α-APP C-terminus. (E) Protein samples were collected from growing or 16-hour-developed WT, aph1-null, nct-null, ps2-null, and aph1;ps2-null Dictyostelium that express ΔN-APP. ΔN-APP expression and processing was determined by immunoblot assay using α-APP C-terminus. (F) Total cell fractions (T), membrane fractions (M) and cytosolic fractions (C) were prepared from growing WT, aph1-null and nct-null Dictyostelium that express ΔN-APP. ΔN-APP and α- and β-CTFs in WT and γ-secretase-null mutants were detected by immunoblotting with α-APP C-terminus. Positions of molecular weight markers, APP, ΔN-APP and α- and β-CTFs are indicated.
Vanessa C. McMains et al. Dis. Model. Mech. 2010;3:
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