Enzyme expression in Expi293 cells and enzyme uptake assays

Expi293 cells in suspension were cotransfected with the cDNAs of cathepsins B, C, D, L, and Z, along with the GNPTAB S1‐S3 mutant construct, as described [13]. Media was collected aseptically 2–3 days post‐transfection for use in uptake assays. For cell uptake experiments, GNPTAB ^−/− HeLa cells were plated on a 12‐well plate at ~ 80% confluence 1 day prior to the uptake experiment. The total protein concentration of the media containing the various secreted cathepsins was measured using the Bradford Assay (Bio‐Rad) and determined to be roughly equivalent. Fifty microlitre aliquots of this media containing each enzyme from the producing cells were then added to the GNPTAB ^−/− HeLa cells in a final volume of 500 μL. The cells were incubated with the media for 24 h before being collected and processed for immunoblotting.