Constructs and generation of stable cell lines

Constructs were purchased from IDT (gBlocks Gene Fragments) and subcloned into pcDNA5/frt/to (Thermo Fisher Scientific, # V652020).

For the generation of V5-FLAG-GCase lines, the tag was positioned at the N-terminus, three aa after the cleavage site of the leader sequence. These three amino acids are repeated after the tag to ensure that the GBA1 sequence is intact.

To ensure proper cleavage, the V5-FLAG tag was inserted 12 bp after the cleavage site, and this 9 bp were repeated after the tag. To avoid interference with proper protein folding, we employed the short V5 sequence (IPNPLLGLD).

Site-directed mutagenesis was performed according to the manufacturer’s protocol (Agilent, QuickChange II XL), and base pair exchange was confirmed by Sanger sequencing.

Flp-In 293 T-Rex cells(Thermo Fisher Scientific, #R78007) were grown in media composed of Dulbecco’s modified Eagle’s medium (DMEM, Sigma–Aldrich), 10% fetal bovine serum

(Gibco), 1% GlutaMax (Gibco) supplemented with 100 μg/ml Zeocin

(InvivoGen) and 15μg/ml blasticidin (InvivoGen).

Inducible Flp-In T-Rex 293 cells were generated according to the manufacturer’s protocol

(Thermo Fisher Scientific).

The selection was performed with DMEM supplemented with 15μg/ml blasticidin and 100

μg/ml hygromycin B Gold (InvivoGen) 48 h after transfection and continued until the

expression of the gene of interest was induced by treating the cells with 50 ng/ml doxycycline hyclate (Sigma–Aldrich) for 48 h.