4 methylumbelliferyl β d glucuronide hydrate

The HA-degrading activity of HYAL1 was analyzed by "native" and "renatured protein" zymography as detailed in Puissant et al. [26 (link)]. Signals were quantified using the ImageJ software.
The enzymatic activity of lysosomal acid hydrolases and ER marker alkaline α-glucosidase was measured with the following 4-methylumbelliferyl-coupled specific substrates (Sigma-Aldrich): 4-methylumbelliferyl-β-D-galactopyranoside (β-galactosidase), 4-methylumbelliferyl-β-D-glucuronide hydrate (β-glucuronidase), 4-methylumbelliferyl-N-acetyl-β-D-glucosaminide (β-hexosaminidase) and 4-methylumbelliferyl-α-D-glucopyranoside (alkaline α-glucosidase). The samples were incubated at 37°C with 5 mM of substrate in a 50 mM citrate buffer, pH 4.5 containing 0.05% Triton X-100, except for the alkaline α-glucosidase activity assay. In this case, the reaction was conducted using 1 mM of substrate diluted in a 0.1 M glycine-NaOH solution (pH 9) containing 0.05% Triton X-100. After 6 h of reaction, a 0.1 M glycine-NaOH solution (pH 10.3) was added to stop the enzymatic activities and the fluorescence was measured at 495 nm.

DR5::GUS staining in Arabidopsis samples was conducted as previously described (Ma et al., 2014 (link)). Samples were stained in solution containing 50 mM NaPO₄, 0.4 mM K₃Fe(CN)₆, 0.4 mM K₄Fe(CN)₆, 1 mM X-gluc, and 0.1% Triton X-100 incubated at 37°C for 8 h in the dark. After GUS staining, the stained samples were washed with 70% ethanol three times and then microscopically imaged. GUS activities were measured as previously described (Liu et al., 2015 (link)). Briefly, seedlings of each sample were collected in Eppendorf tubes (100 mg per measurement) and homogenized with steel beads in buffer (50 mM potassium phosphate buffer, pH 7, 1 mM EDTA, 0.1% SDS, and 0.1% Triton X-100). The supernatant was used for measurement after centrifuging at 12,000 rpm for 15 min at 4°C and the protein concentrations were quantified using a Micro BCA Protein Assay Kit (Vigorous). Enzyme activity was calibrated by the concentration of 4-methylumbelliferone (Sigma-Aldrich). The fluorescence of the samples was measured on 96-well plates on a Fluoroskan Ascent FL fluorometer (excitation wavelength of 365 nm and emission wavelength of 455 nm) after incubating with 4-methylumbelliferyl-β-D-glucuronide hydrate (Sigma-Aldrich). Measurements were read when stopped by adding stop buffer (0.2 M Na₂CO₃) at 0, 15, 45, and 60 min, and the standard curve was fitted.