Lipofectamine 3000

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.

Cells were plated on acid-washed coverslips and either transfected with plasmids (using Lipofectamine 3000 or Mirus) or infected with PKAc variant lentivirus 48 h before harvest. Cells were fixed in 4% PFA for 15 min at RT and washed 3x in PBS. Coverslips were moved to humidity chamber and blocked for 1 h at RT in 3% BSA, 0.3% Triton X-100. Primary antibodies were diluted in blocking solution and applied to coverslips overnight at 4°C. Coverslips were washed 3x with PBS, incubated with fluorescent secondaries (1:1000) and DAPI (~1:10,000), and washed 3x in PBS again before mounting. Images were taken on either a Keyence BZ-X710 microscope or a GE Healthcare OMX and processed/analyzed using ImageJ analysis software (FIJI).