Fig 2. The mature form of HYAL1 co-distributes with lysosomal markers after osteoclast fractionation by differential centrifugation.

Osteoclasts differentiated from RAW264.7 cells were fractionated into five fractions (N, M, L, P and S) following de Duve's fractionation scheme. (A) The activities of β-galactosidase and alkaline α-glucosidase were detected by fluorometric assay to establish the distribution of lysosomes and of the ER, respectively. The graph shows the relative specific activity (ratio of the percentage of activity of the enzyme in a given fraction to the percentage of proteins in this fraction), which is indicative of the enrichment factor of the enzyme in the fractions, plotted against the percentage of proteins in each fraction. (B-C) The distribution of cathepsin K and HYAL1 was analyzed by western blotting (reducing conditions). Equal amounts of proteins were loaded for each fraction, except for S, which was diluted 1:2 compared to the other fractions. The mature and precursor forms are highlighted by open and closed arrowheads, respectively. Of note, in panel B, a longer exposure time is shown for the upper part of the blot to help visualization of cathepsin K proforms.