Transfection and Subcellular Localization

Twenty-four hours prior to transfection, the cells were seeded onto 12-well plates. For transfections, 450 ng of the respective AGA expression plasmid and 50 ng of pRL-TK encoding renilla luciferase for normalization (Promega, Mannheim, Germany) were transfected using MACSfectin™ (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s protocol. After 24 h, the cells were transferred into 6-well plates and harvested 48 h post-transfection using 250 µL passive lysis buffer (Promega). Fibroblasts were electroporated using the Neon™ Transfection System (Invitrogen) according to the manufacturer’s instruction. 1 × 10⁶ cells in a total volume of 100 µL were transfected with 1 µg plasmid (1200 V, 40 ms, 1 pulse). Cells that were used for lysotracker stainings were cotransfected with 250 ng pEGFP-N1 plasmid (Clontech, Takara, Saint-Germain-en-Laye, France) for the detection of transfected cells. Transfected cells were either seeded into 6 well plates and used for enzyme activity measurements or onto coverslips and used for staining with Lysotracker as described [20 (link)].