Subtilisin

Thirty microliter Flag-tagged AHR, AHR mutants and negative control BSA in 100 μL PBS was incubated with vehicle (DMSO) and Leflunomide at the indicated concentrations (5.0 and 10.0 μM) for 18 h at 4 °C, respectively. The samples were then digested with subtilisin (Sigma-Aldrich) at room temperature for 20 min. The reactions were stopped by adding SDS loading buffer and boiling for 5 min. Samples were loaded onto a 12% acrylamide SDS-PAGE gel and then stained with coomassie brilliant blue to visualize the banding pattern^{27 (link)}.

Assay for inhibitory activity against protease was performed as previously described with modification (23 (link)–27 (link)). Trypsin, α-chymotyrpsin, pronase, subtilisin, thrombin were purchased from Sigma-Aldrich (St. Louis, USA), and proteinase K and papain were purchased from Sangon Biotech (Shanghai, China). SPINK7 (100 μg) was preincubated with 10 μg protease in 100 μL Fluoro assay buffer (100 mM Tris-HCl and 20 mM CaCl₂, pH 7.5) for 30 min at 37°C. Then, 100 μL FITC-casein substrate buffer (G-Biosciences, USA) was added and incubated at 37°C in the dark for 1 h. The fluorescence was measured at 485/535 nm (excitation/emission). Protease inhibition by SPINK7 was assessed using the following formula: % residual activity = (residual enzyme activity/enzyme activity without inhibitor) × 100. Three independent replicates were performed for each experiment. Student’s t test was used to evaluate statistical significance. These data were analyzed by GraphPad Prism 5 software.

Mass spectrometry-based analyses were performed with a Micromass QTof Global Ultima instrument (Waters, Mildford, MA, USA), as previously described [45 (link)]. Intact masses were determined by direct infusion; peptides obtained from digestion with trypsin (ProteoExtract Trypsin Digestion Kit, Calbiochem, Gibbstown, NJ, USA) or subtilisin (Sigma-Aldrich, St. Louis, MO, USA) were separated by capillary HPLC online coupled to a mass spectrometer. For de novo sequencing, the ProteinLynx Global Server, software version 2.2.5 (Waters), was used. N-terminal sequencing by Edman degradation was done by the protein sequencing service of the Division of Clinical Biochemistry at the University of Innsbruck, Austria.

In total, 34 mulberry varieties, including 30 cultivated species and 4 wild species, were cultivated at Hengkou Sericulture Research Base, Ankang University, China. Mulberry leaves were picked on 19 August 2020. The maturity of the mulberry leaves was approximately the same. Trypsin, chymoTrypsin, subtilisin and N-acetyl-D,L-phenylalanine-β-Naphthalene ester (APNE) were purchased from Sigma. Elastase was obtained from Sangon Biotech.

To quantify microtubule binding by Cy5-labeled DPRs to microtubules with or without C-terminal tubulin tails, TRITC-labeled microtubules were first polymerized as described above. Microtubules resuspended in BRB80 with 20 μM taxol were treated with a 1:50 (w/w) ratio of subtilisin (Sigma-Aldrich) to tubulin, or a buffer only control, for ~16 hours. Digestion was quenched with 5 mM PMSF and confirmed by gel electrophoresis and Coomassie staining. Microtubules were then pelleted, washed, and resuspended in fresh BRB80 with taxol before application to imaging chambers. Cy5-labeled DPRs in kinesin motility buffer were added to the chambers to a final concentration of 10 μM. For analysis, five microtubules were selected randomly from three images acquired from two independent chamber preparations. Mean Cy5 fluorescent intensity was then calculated using Fiji. Background signal was first subtracted with a rolling ball radius of 50 pixels to generate uniform fluorescent intensity across the region. Enrichment of fluorescence on microtubules was calculated by subtracting mean background intensity from microtubule-associated intensity. Background intensity was calculated from five lines per image equivalent to the median length of microtubules assessed, which were positioned in microtubule-free areas close to the selected microtubules.