Fig. 1.

Constitutive phospholipid scrambling by mXkr8. (A and B) Cold temperature- and Ca²⁺-dependent exposure of PtdSer by mXkr8 in Ba/F3 cells. (A) TMEM16F^−/−Ba/F3 (16F^−/−Ba/F3) and its transformants expressing mXkr8 (16F^−/−BaF-Xkr8) were incubated at 4 °C (blue) or 20 °C (red) for 15 min with Cy5-annexin V and then analyzed by flow cytometry. (B) 16F^−/−Ba/F3 and 16F^−/−BaF-Xkr8 cells were incubated with (red) or without (green) BAPTA-AM and then stained at 4 °C with Cy5-annexin V. Annexin V-staining profiles in the PI-negative population are shown. (C) Ca²⁺-dependent internalization of NBD-PC by mXkr8 in Ba/F3 cells. (Left) 16F^−/−BaF-Xkr8 cells were incubated in annexin V buffer with NBD-PC and SYTOX red at 4 °C or 20 °C for the indicated times, treated with BSA, and subjected to flow cytometry. The incorporated NBD-PC in the SYTOX red-negative population is expressed as mean fluorescence intensity (MFI). (Right) 16F^−/−Ba/F3 and 16F^−/−BaF-Xkr8 cells were incubated with or without BAPTA-AM and then incubated at 20 °C for 8 min with NBD-PC in annexin V buffer containing SYTOX red, and the incorporated NBD-PC was determined as described above. The experiments were carried out in triplicate, and average MFI values were plotted with SE (bars).