*4.3. Cell Culture and Generation of Mutant Cell Lines*

Cell culture and generation of mutant cell lines were performed as described previously [14,19]. Briefly, HEK293 cells overproducing wild-type rAbcc6 (HEK293-rAbcc6) and control, untransfected cells (HEK293-control) were cultured at 37 ◦C in a 5% CO<sup>2</sup> atmosphere under humidifying conditions in DMEM (HyClone, GE Healthcare, Chicago, IL, USA) with 100 U pen/strep per mL (Gibco, Waltham, MA, USA) supplemented with 5% FBS (Fisher Scientific, Waltham, MA, USA). rAbcc6 mutants cloned into the pQCXIP expression vector were transfected into HEK293 cells using the calcium phosphate precipitation method. Transfected cells were selected in medium containing 2 µM puromycin. Cell lines were established from clones showing high expression of the respective rAbcc6 mutants. Of note, several clones were generated for each rAbcc6 mutation and these subclones behaved very similarly with respect to PPi accumulation in the culture medium.
