FIGURE 3.

Cell surface expression of P4-ATPases in HeLa stable cell lines. HeLa cell lines stably expressing HA-tagged P4-ATPase were established by plasmid transfection followed by selection in the presence of antibiotics. Two or three independent clones of cells expressing ATP8B1(WT), ATP8B2(WT), ATP11A(WT), or ATP11C(WT) or one clone expressing ATP8B1(E234Q), ATP11A(E186Q), or ATP11C(E184Q) was examined. A and C, the total expression level of the P4-ATPase in each cell line was analyzed by immunoblotting with anti-HA and anti-TfnR antibodies (as an internal control). 4% of the input of the biotinylation reaction was loaded in each lane. Arrow, ATP8B2; open arrow, ATP8B1. B and D, the cell surface level of the P4-ATPase in each cell line was analyzed after surface biotinylation. Proteins precipitated with streptavidin-agarose beads were subjected to immunoblot analysis for HA and TfnR (as an internal control). The numbers show the relative expression level of proteins, which were normalized with the level of the internal control, TfnR, and were used for normalizing the enzymatic activities shown in Fig. 4 (J and K). In A and B, open and solid arrows indicate positions of ATP8B1 and ATP8B2, respectively. In A–D, middle panels show overexposures. WT(vi), cell lines established by infection of recombinant retrovirus for each P4-ATPase.