Protein assay kit

The addition of more than 0.1% Triton X-100 and 0.05% sodium cholate to rice GCase was essential for stabilizing the enzyme. However, protein concentrations in enzyme solutions containing Triton X-100 are not correctly measured by the Protein Assay Kit (Bio-Rad), because Triton X-100 in the solution also causes the blue color change that proteins cause. Therefore, the protein concentration of rice GCase was measured by HPLC analysis on a TSKgel Octyl-80Ts column (4.6 mm i.d. × 15 cm; Tosoh) eluted with a linear gradient of 0 to 60% acetonitrile in 0.05% trifluoroacetic acid (TFA) at 0.8 ml/min. The peak was monitored with an ultraviolet detector at 280 nm. The protein concentration of rice GCase was determined by comparison with the peak area for recombinant human GCase (Imiglucerase), for which the concentration was previously determined by the Protein Assay Kit (Bio-Rad) using bovine serum albumin as the standard (39 (link)).

MPO and EPO assay protocols were adapted from a previous protocol [29 (link), 42 (link)]. Briefly, mouse lung samples were homogenized in 500 µl of 50 mM HEPES (Invitrogen, Burlington, ON, Canada) and then re-homogenized in 500 µl of 0.5% cetyltrimethyl ammonium chloride solution. Diluted MPO standards from human leukocytes (Sigma-Aldrich, St. Louis, MO, USA) and mouse lung samples were added to 96-well plate. The MPO substrate (3, 3’, 5, 5’-tetramethylbenzidine) was then added, followed by use of 1 M H2SO4 to terminate the reaction. The plate was read at 450 nm OD using NOVOstar software (Bio-Rad). Total protein concentrations in each sample was quantified using a protein assay kit (Bio-Rad). The data are expressed as units of MPO per mg of lung protein.
To assess EPO levels, samples and EPO standards were added to 96-well plates. Stop solution was added after two-minute incubation with eosinophil peroxidase assay substrate solution (3 mM O-phenylenediamine). EPO levels were read at 490 nm OD using NOVOstar software (Bio-Rad). Total protein concentration in each sample was quantified using a protein assay kit (Bio-Rad). Data were expressed as the units of EPO per mg of lung protein.

Bovine deep zone articular chondrocytes or mouse limbs were collected and lysed in Lysis Buffer (containing 50 mM Tris^. HCl, pH 7.4, 1% NP-40, 0.5% Sodium deoxycholate, 1% SDS, 150 mM NaCl, 2 mM EDTA, 50 mM NaF, protease inhibitor cocktail (Roche)). The cell lysate were incubated on ice for 15 min, and centrifuged at 3000 × g, at 4 °C to remove cell debris. Protein concentration was measured with a Bio-Rad Protein Assay Kit (BIO-RAD, Cat#: 500-0006). The protein was denatured at 70 °C for 10 min with LDS Sample Buffer (Invitrogen, Cat#: NP0007) and Sample Reducing Agent (Invitrogen, Cat#: NP0009). General SDS-PAGE processing procedures were followed. The blots were visualized by the enhanced chemiluminescence detection method, employing the Pierce ECL Western Blotting Substrate (Pierce, Cat#: 32106). All primary antibodies used for Western blots are listed in Supplementary Table 2.