Fig. 3.

Exogenous expression of human ATP11C (WT) but not ATP11C(E184Q) rescued PS-flipping activity in UPS-1 cells. A: UPS-1 cells (-) infected with empty retrovirus vector (vector) or recombinant virus vector encoding HA-tagged ATP11C(WT) or ATP11C(E184Q) were incubated with the indicated NBD-lipids at 15°C for 15 min (10 min for NBD-PS). After extraction with fatty acid-free BSA, the residual fluorescence intensity associated with the cells was determined by flow cytometry. Graphs show the results from three independent experiments (averages ± SD). B: Histogram of a representative experiment showing the differences in fluorescence intensity among UPS-1 (dashed lines), UPS-1 infected with empty retrovirus vector (gray lines), or UPS-1 infected with retrovirus vector encoding ATP11C (blue lines) or ATP11C(E184Q) (red lines) in the presence of the indicated NBD-lipids. C: Expression levels of ATP11C and ATP11C(E184Q) in UPS-1 cells were analyzed by immunoblotting with antibodies against HA and β-tubulin (as an internal control).