Figure 7.

Modulations of PIP2 levels correlate with C99 cleavage levels. (A) Schematic of the AlphaScreen Assay. Biotinylated liposome binds to Streptavidin-coated donor beads, and the nickel chelated acceptor beads bind to His-tagged peptide. The interaction between His-tagged peptides and biotinylated liposome brings the two beads into close proximity to generate a light emission signal. (B) Validation of PIP2 binding by C99 polybasic peptide. The PIP2 lipid content varies from 0 to 10%. Underneath is the sequence of His-tagged C99 polybasic peptide. (C) Competition assay of C99/Notch1 polybasic peptides in presence of 4% PIP2. The IC₅₀ values were obtained from curve fitting using the GraphPad Prism competitive inhibitor model. (D) Modulations of PIP2 levels by the PLC pathway inhibitor edelfosine (EDEL) and the PLC activator m-3M3FBS (M3M) led to changes in total C99 cleavage. Cleavage was measured by the Epsilon-Cleavage assay. (E) Modulations of PIP2 levels affect Aβ₄₀ levels and Aβ₄₀/Aβ₄₂ cleavage ratios as determined by AlphaLISA assay. Absolute concentrations were calculated using a previous calibration graph. Error bars=SEM, n=3, P-values (two-tailed Student's t-test versus WT): ^*P<0.05; ^**P<0.01, ^***P<0.001.