Fig 2: TMEM30A interacts with ßCTF in AD model mice, which follows by lipid flippase dysfunction(A) The membrane fractions from WT and Tg (A7) mice brain at three- or 6-month-old applied to co-immunoprecipitation analysis using TMEM30A antibody. Na+/K+-ATPase was used as a loading control of membrane fractions.(B and C) Quantification of the complex formation of (B) TMEM30A and ßCTF or (C) TMEM30A and ATP8A1 (N = 3, n = 3, mean ± SEM, two-tailed Student’s t-test, *p < 0.05, **p < 0.01).(D) Quantification of Evectin-2 localization in membrane fractions (N = 3, n = 3, mean ± SEM, two-tailed Student’s t-test, **p < 0.01).(E) The membrane fractions from WT and KI (AppNL-G-F/NL-G-F) mice brain at 3-month-old applied to co-immunoprecipitation analysis using TMEM30A antibody.(F and G) Quantification of the complex formation of (F) TMEM30A and ßCTF or (G) TMEM30A and ATP8A1. (N = 3, n = 3, mean ± SEM, two-tailed Student’s t-test, *p < 0.05, ***p < 0.001).(H) Quantification of Evectin-2 localization in membrane fractions (N = 3, n = 3, mean ± SEM, two-tailed Student’s t-test, p = 0.08).

Fig 3: Calpain activity is inhibited by 2-APB and DPBA, but not DP3A.a Platelets were treated with the calpain inhibitor, calpeptin (140 µM), 2-APB (100 µM) or the vehicle (DMSO) prior to stimulation with A23187 as indicated, then lysed. Proteins were separated by SDS-PAGE. Talin, ATP8A1, PTP1C and caspase-3 were detected with specific antibodies. The membranes were then stripped and re-probed with anti-CD41-antibody or anti-GADPH antibody, as indicated. A23187-triggered protein cleavage was inhibited by calpeptin and partially inhibited by 2-APB. The blots are representative of five independent experiments. b Platelets were treated with the indicated analogues prior to stimulation with A23187. The blots are representative of five independent experiments.