Fig. 5
C-terminus of ATP11C is required for inhibition of flippase activities by PKC activation. a–c Ba/F3 cells stably expressing HA-tagged ATP11A, ATP11C, and ATP11AAC chimeric proteins were treated with vehicle alone (a), 400 nM PMA (b), or 1 μM A23187 in the presence of CaCl2 (c) for 15 min. The cells were fixed and immunostained with anti-HA and anti-ATP1A1 antibodies, followed by incubation with Cy3-conjugated anti-rat and Alexa Fluor 488-conjugated anti-rabbit secondary antibodies. Scale bars, 5 μm. d–f Ba/F3 cells were treated with vehicle alone (white bars), 400 nM PMA (black bars), or 400 nM PMA and 2 μM of BIM-1 (gray bars) for 15 min. The cells were then washed with flippase assay buffer and incubated at 15 °C for 5 min with NBD-PS (d), or 15 min with NBD-PE (e) or NBD-PC (f). After extraction with fatty acid-free BSA, the residual fluorescence intensity associated with the cells was determined by flow cytometry. Fold increase of NBD-lipids uptake is shown relative to that in Mock-treated parental Ba/F3 cells (−). Graphs display averages from three independent experiments ± SD. **p < 0.005; ***p < 0.001
