X11–Munc18 regulates APP–BACE1 interaction and β cleavage in N2a cells. (a) Effects of truncated X11 mutants on APP–syntaxin 1 and APP–BACE1 copatching. X11 interacts with Munc18–syntaxin 1 through MID and APP through the phosphotyrosine-binding domain. MID expression reduced APP–syntaxin 1, suggesting a requirement of X11–Munc18 for APP–syntaxin 1 copatching. APP–BACE1 copatching was promoted by MID and inhibited by CID, suggesting an inhibitory effect of Munc18 and a positive role of CID on the APP–BACE1 interaction. Copatching values were normalized against the control without X11 mutant expression. Error bars represent the SD based on three independent experiments (n = 30–45). (b) Effects of truncated X11 mutants on α/β cleavage. The extracellular domain of VSVG-tagged APP released into the medium by endogenous BACE1 or α-secretase was precipitated with anti-VSVG and quantified with end-specific antibodies. The effects of X11 mutants on β cleavage were consistent with their effects on APP–BACE1 interaction as shown in panel a. Ratios of sAPPβ to sAPPα were normalized against the control without X11 mutant expression. Data are means + SD based on three to four independent experiments (n = 9 or 12). (c) Effects of truncated mutants of X11 on interactions between X11 and Munc18 in N2a cells. The effects of MID and MID–CID on protein interactions were tested by co-IP analysis. Venus-X11 with MID or MID–CID was expressed, and the cell lysate was used for IP with anti-GFP. The X11–Munc18–syntaxin 1 interaction was diminished by MID and MID–CID, and the X11–CASK interaction was abolished by MID–CID. *, P < 0.05; **, P < 0.01.