Fig. 1. Increase in protein expression and activity of CTSD in differentiated keratinocyte cultures. (A) Protein expression of CTSD isoforms in the primary and differentiated keratinocytes was determined in cell lysates by western blotting using anti-CTSD antibodies. There was an increase in the prepro and enzymatically active pro forms in differentiated keratinocytes. (B) CTSD activity was measured by an in vitro enzyme assay of keratinocyte lysates using parathyroid hormone (PTH) as a CTSD-specific substrate. The amount of PTH in the absence of sample protein was used as a control. The level of PTH protein was determined by western blotting using anti-PTH mAb (peptide 1-34) and quantified by two-dimensional laser scanning densitometry (Molecular Dynamics Personal Densitometer). CTSD activity, calculated as the amount of PTH cleaved/hour, was increased in differentiated keratinocytes.