Extended Data Fig. 2: Effect of IFITM3 knockdown and knockout on γ-secretase.
From: The innate immunity protein IFITM3 modulates γ-secretase in Alzheimer’s disease
a, Quantification of western blotting (Fig. 2a) showed that IFITM3 knockdown did not change protein expression levels of APP, NCT and PS1-NTF in HEK-APP wild-type cells (n = 3). b, Schematic representation of cell-free γ-secretase assay. γ-secretase is incubated with a recombinant APP substrate in the presence of 0.25% CHAPSO. Cleaved Aβ40 and Aβ42 species are measured with cleavage specific antibodies and AlphaLISA technology. c, Schematic model showing different GSM and GSI binding sites in γ-secretase: E2012 (imidazole GSM), GSM-1 (acid GSM), and L458 (transition state analogue inhibitor, GSI). d–f, Comparison of IC50 values of GSM-25 (EV: n = 9, KO: n = 8) for Aβ40 (****P < 0.0001) and Aβ42 (ns) (d), GSM-1 (n = 6) for Aβ40 (**P = 0.0039) and Aβ42 (ns) (e), and L458 (n = 3) for Aβ40 (ns) and Aβ42 (ns) (f) cleavages in the U138 EV or KO cell lines (n ≥ 3). g, IFITM3 knockdown (KD) does not affect expression of γ-secretase subunits. IFITM3 was knocked down by siRNA (6 pmol, n = 3) in HEK-Notch∆E cells and scramble siRNA (SC, n = 3) was used as a negative control. Cell lysates were probed by antibodies against NCT, PS1-NTF and IFITM3. β-Actin was used as a loading control. h, Effect of IFITM3 knockdown on γ-secretase activity. IFITM3 knockdown increased γ-secretase cleaved product NICD, analysed by western blotting. Cell lysates were probed by antibodies against c-Myc (NotchΔE) and NICD and a representative quantification of NICD (n = 8, ***P = 0.001) is shown (bottom). i, Cell-based NICD AlphaLISA assay (left) revealed an increase in NICD production with IFITM3 knockdown. Quantification of NICD (n = 8, ***P = 0.001) is shown in the right panel. j, Effect of IFITM3 knockout on γ-secretase activity. Knockout cells lines have increased γ-secretase activity as compared to the EV cell line. The NICD cleavage in vitro was measured by AlphaLISA assay (n = 3, **P = 0.0096). All western blotting images and graphs are representative of three independent experiments. Data are mean ± s.d. P values were determined by two-sided Student’s t-test.
