Examination of phosphorylation status of 32- and 34-kDa cathepsin L by treatment with alkaline phosphatase.

<p>An aliquot of the cell fraction, lysosomal fraction (34-kDa cathepsin L), or culture medium (32-kDa cathepsin L) was mixed with or without ALP in 0.5 mM Tris-HCl buffer (pH 9.3 at 25°C) containing 0.1 mM leupeptin, 0.1 mM MgCl₂, 10 μM ZnCl₂, and 0.1 mM spermidine, and incubated for 30 min at 37°C. Gelatin zymography was performed for each incubation mixture. Gelatin zymograms of the cell, lysosomal, and medium fractions with (lane 2) or without (lane 1) ALP treatment are shown. Lane 3 shows the chromatogram of a mixture of samples from lanes 1 and 2.