PS externalization and cytosolic Ca²⁺ level in tMEFs. (A) WT, SMS1-KO, and SMS2-KO tMEFs were stained with FITC–annexin V, and then analyzed with flow cytometry. The fluorescence of FITC–annexin V was quantified as mean fluorescent intensity (MFI). (B) Intracellular Ca²⁺ levels were measured with Fura2-AM and calcium imaging. The fluorescence was measured with Argus (aHamamtsu Photonics, Hamamatsu, Japan) and is presented as the 340 nm/380 nm fluorescence ratio (R_340nm/380nm). Scale bars, 50 µm. (C) The DRM fraction was extracted with lysis buffer containing 1% Brij 58 (Sigma-Aldrich, St. Louis, MO) and OptiPrep (Axis-Shield Alere Technologies, Oslo, Norway) discontinuous gradients (5% and 30%). Fractions were collected from the top to the bottom of the gradient, and the DRM fractions (fractions 8-10) were analyzed by western blot analysis with antibodies against TMEM16F and the lipid raft marker flotillin-1. (D) Western blotting of TMEM16F protein in WT and SMS1-KO platelets was performed. (E) TMEM16F-KO tMEFs were established from SMS1-KO tMEFs by using CRISPER/Cas9 plasmids constructed based on lentiCRISPR-V2. The sequence of TMEM16F is shown at the top with the target sequence and the protospacer adjacent motif (PAM) sequence. Insertions are shown in red, and deletions are shown as black dashes. The change in length caused by each indel mutation is listed to the left of each sequence (+, insertion; −, deletion). TMEM16F protein in the SMS1-KO/TMEM16F-KO (1KO/16FKO) and SMS1-KO/vector (1KO/vec) tMEFs was detected by western blot analysis. Arrows indicate TMEM16F protein. (F) Cell surface PS was stained with FITC–annexin V and then analyzed with flow cytometry. The fluorescence was quantified as MFI. (G) Cytosolic Ca²⁺ levels were measured with Fura2-AM and are presented as the R_340nm/380nm. Scale bars, 50 µm. Values show the mean ± standard deviation. **P < .005.