Fig. 3. Deglycosylated Na⁺/K⁺-ATPase ß-subunits can still interact with the catalytic -subunits. (A) Hep G2 cells treated with tunicamycin (TM) or untreated were subjected to immunoprecipitation (IP) experiments using either anti--subunit antibody (anti-) or anti-ß-subunit antibody (anti-ß). The resulting immunoprecipitates were analyzed by western blotting. The positions of the -subunit and the fully glycosylated (^***ß), intermediately glycosylated (^**ß), and the core (^*ß) proteins of the ß-subunit are indicated. (B) The interactions between the exogenous FLAG-tagged ß-subunit (the wild-type and three deglycosylated mutant constructs) and the endogenous protein were tested by IP-western analyses; co-transfected EYFP--tubulin were used as a control. (C) The localization of - and ß-subunits before and after tunicamycin treatment was visualized by IF using antibodies specific to either - or ß-subunit (green), together with F-actin staining (red). Note the basolateral only (arrowheads) and the apical presence (double arrowheads) of both subunits in the control (CTL) and tunicamycin-treated (TM) cells, respectively. (D) Hep G2 cells transfected with EGFP-labelled ß-subunit (wild-type or N124Q mutant, green), were stained for endogenous -subunit (blue) and F-actin (red). Note both wild-type ß-subunit and -subunit were found only along the basolateral membrane (arrowheads) in the wild-type transfectants, whereas a portion of the N124Q ß-subunit and the endogenous -subunit was aberrantly translocated to the apical domain (double arrowheads) in Hep G2 cell transfected with N124Q ß-subunit. Bars, 10 µm.