*4.5. Subcellular Localization of rAbcc6 in HEK293 Cells*

The localization of rAbcc6 in intact HEK293 cells was detected as described previously [19]. Briefly, HEK293 cells were seeded and grown for 2 days on 4 well µ-Slides (ibiTreat 1.5 polymer coverslip, 80426, Ibidi) coated with poly-D-lysine. The cells were fixed in 4% PFA and subsequently in −20 ◦C cold methanol for 5 min each and then samples were blocked with Protein Block (Fisher Scientific) for 60 min. Samples were then incubated for 60 min with the polyclonal rabbit anti-rAbcc6 antibody K14 diluted 1:100 and the mouse monoclonal anti-Na+/K<sup>+</sup> -ATPase antibody (ab7671, Abcam, Cambridge, UK) diluted 1:250. Then samples were incubated for 60 min with Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (A11008, Fisher Scientific, Waltham, MA, USA) and Alexa Fluor 568 conjugated goat anti-mouse antibody (A11004, Fisher Scientific, Waltham, MA, USA), both diluted 1:1000. The samples were subsequently incubated with 300 nM DAPI for 5 min to stain nuclei. The subcellular localization of wild-type and mutant forms of rAbcc6 was then analyzed using a Nikon (Tokyo, Japan) Eclipse Ti two point-scanning laser confocal microscope equipped with a Nikon A1R+. A Plan Fluor 40× Oil DIC H N2 objective with a 1× optical zoom was used with 405.5, 490.0 and 561.3 nm excitation and 450/50, 525/50 and 595/50 nm emission filters, respectively. Images were acquired with the pinhole set to 1 airy unit.
