All cell lines were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) with high glucose, GlutaMAX, and sodium pyruvate (ThermoFisher 10569) with 10% fetal bovine serum (FBS) at 37°C and 5% CO2. Parent HEK293T, Flp-In T-REx 293, and HeLa T-REx cell lines were authenticated by STR profiling. ATP13A1 knockout cell lines were generated by transfecting Flp-In T-REx HeLa (Fig. 2D and fig. S5 -9 ) or Flp-In T-REx 293 (Fig. 2A -C and fig. S3 -4 ) cells with pX459 plasmids containing target guide RNAs using Lipofectamine 2000 or TransIT293 (Mirus Bio), respectively, according to manufacturer’s instructions. After 24 hr, transfected cells were selected with 2 mg/mL puromycin for 48 hr. Single clones were isolated and knockouts validated by immunoblotting and amplicon sequencing.
To establish stable doxycycline-inducible cell lines expressing either FLAG-tagged tail-anchored proteins or FLAG-tagged ATP13A1, wildtype or ATP13A1 knockout Flp-In T-REx HeLa or 293 cells were co-transfected with a 1:1 ratio of pOG44 and pcDNA5/FRT/TO vector containing the desired insert using Lipofectamine 2000/Lipofectamine 3000 or TransIT293 (Mirus Bio), respectively. After 24 hr, cells were selected with 10 μg/mL Blasticidin and 150-300 μg/mL Hygromycin for at least two weeks, and expression was validated by induction with 10 ng/mL doxycycline for 24-48 hr and immunoblotting.
To establish stable doxycycline-inducible cell lines expressing either FLAG-tagged tail-anchored proteins or FLAG-tagged ATP13A1, wildtype or ATP13A1 knockout Flp-In T-REx HeLa or 293 cells were co-transfected with a 1:1 ratio of pOG44 and pcDNA5/FRT/TO vector containing the desired insert using Lipofectamine 2000/Lipofectamine 3000 or TransIT293 (Mirus Bio), respectively. After 24 hr, cells were selected with 10 μg/mL Blasticidin and 150-300 μg/mL Hygromycin for at least two weeks, and expression was validated by induction with 10 ng/mL doxycycline for 24-48 hr and immunoblotting.