Man 6 p

As previously described [26 (link)], the presence of Man-6-P moieties on HYAL1 and cathepsin K was assessed using CI-MPR affinity chromatography [40 (link)]. Briefly, cell lysates and conditioned media (collected after a 5 h culture period in the absence of serum) were loaded on 1 mL columns made of CI-MPRs immobilized on a sepharose 4B matrix (a generous gift from P. Lobel, Center for Advanced Biotechnology and Medicine, Piscataway, USA). After washes and elution of non-specifically bound proteins with 5mM of glucose 6-phosphate (Gluc-6-P, Sigma-Aldrich), the mannose 6-phosphorylated proteins were eluted with 5 mM of Man-6-P (Sigma-Aldrich). The collected fractions were pooled two by two, precipitated with trichloroacetic acid and resolved by SDS-PAGE to detect HYAL1 and cathepsin K using western blotting.

The western blotting experiments were performed as previously described [26 (link)]. The following antibodies were used: mouse monoclonal anti-GAPDH (1:4 000 dilution, G8795, Sigma-Aldrich), anti-HYAL1 (1:1 000 dilution, 1D10, produced by hybridoma cells generously provided by B. Triggs-Raine, University of Manitoba, Winnipeg, Canada) and anti-cathepsin K (1:1 000 dilution, MAB3324, Millipore), as well as goat polyclonal anti-TRAP (1:1 000 dilution, SC-30833, Santa Cruz Biotechnology). When conditioned media were prepared, the cells were cultured for 5 h in serum-free medium prior to lysis of the cells in PBS—Triton X-100 1% supplemented with protease inhibitors (cOmplete, mini protease inhibitors cocktail, Roche). When indicated, cells were incubated for 24 h with either 15 mg/mL of mannan (Sigma-Aldrich) or 5 mM of Man-6-P (Sigma-Aldrich). In Fig 5C, cell extracts and conditioned media were treated with PNGase F (New England Biolabs) according to the manufacturer's instructions. Of note, the specificity of the anti-HYAL1 antibody was validated by an absence of signal in osteoclasts differentiated from BMM of Hyal1 -/- mice (S1A Fig). HYAL1 signals were quantified using the ImageJ (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 1997–2015).