Coomassie blue r 250

gelatin zymography was performed in order to determine the activity of MMP-2 and MMP-9. Briefly, the protein samples (40 µg) were separated by 10% SDS-PAGE containing 1 mg/ml gelatin (Sigma-Aldrich) at 4°C. Following electrophoresis, the gel was incubated with 2.5% Triton X-10 0 (Amresco LLC) in distilled water with gentle agitation for 40 min at room temperature. Subsequently, the gel was further incubated in developing buffer (50 mM Tris-HCl, 0.2 M NaCl, 5 mM CaCl₂, 1 µM ZnCl₂ and 0.02% Brij35) overnight with gentle agitation. The gel was then stained with Coomassie blue R-250 (Amresco LLC) for 30 min. Following incubation with the staining solution, the gel was washed with a destaining solution (2.5% Triton X-100, 50 mmol/l Tris-HCl, 5 mmol/l CaCl₂, 1 µmol/l ZnCl₂; pH 7.6) until the bands became visible against the blue background. The incubation time was optimized depending on the enzyme activity. The quantitative analysis of the gelatin zymography was carried out using the Gel Documentation & Analysis system (Beijing Liuyi Instrument Factory, Beijing, China).

TSP or purified proteins were separated using 10–12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under the reducing condition, and the proteins on the gels were either stained with 0.25% Coomassie blue R-250 (AMRESCO, cat. no: 6104-59-2, Zottegem, Belgium) solution or analyzed by Western blotting using mouse anti-His (Qiagen, Valenica, CA, USA), mouse anti-LIF (abcam, cat. no., ab34427, Cambridge, UK), mouse anti-GFP (Clontech, cat. No., 632381, CA, USA), rat anti-HA (Roche, cat. No., 1583 816, Basel, Switzerland), mouse anti-IL6 (abcam, cat. no., ab9324, Cambridge, UK), and anti-CBD (Bioapp, Pohang, Korea) antibodies. The horseradish peroxidase (HRP)-conjugated sheep anti-mouse IgG (Bethyl, cat. no., A90-146P, TX, USA), and goat anti-rat IgG (Bethyl, cat. no., A110-105P, TX, USA) were used as secondary antibody. Bands on the immunoblots were detected using chemiluminescence detection reagents (GE healthcare, Illinois, USA), and images were captured with a LAS 4000 image capture system (Fujifilm, Tokyo, Japan).

Unless otherwise specified, all reagents were purchased from Millipore-Sigma (Oakville, ON, Canada). The BCA protein assay reagent (bicinchoninic acid), NuPAGE® 4–12% Bis-Tris gels, NuPAGE® MES SDS running buffer, NuPAGE® sample reducing agent, NuPAGE® antioxidant, NuPAGE® LDS sample buffer, prestained protein standard, methanol, sodium chloride, and Corning clear polystyrene 96-well microplates were purchased from ThermoFisher Scientific (Nepean, ON). Glacial acetic acid, glycerol, sodium phosphate dibasic, and trizma base were from Bioshop Canada (Burlington, ON, Canada). Bromophenol blue, Coomassie blue R250, potassium chloride, potassium phosphate monobasic, and sodium dodecyl sulfate were from VWR (Mississauga, ON, Canada). Petri dishes (150 mm) were purchased from Ultident Scientific (St. Laurent, QC, Canada).

Plasmin-digested TTR V30M samples (15 μg) were loaded into a 15% polyacrylamide SDS-PAGE gel. Samples were then transferred onto a PVDF membrane (Bio-Rad, Hercules, CA, USA) and proteins were further stained with Coomassie blue R-250 (VWR International, Radnor, PA, USA). Membranes were allowed to dry and the bands below to the TTR monomer, corresponding to TTR fragments (band 1 and band 2) were excised for N-terminal sequencing analysis (Edman degradation method) using an ABI Procise Protein Sequencer, an ABI Microgradient Pump System, and an ABI Programmable Absorbance Detector (Applied Biosystems Inc., Waltham, MA, USA).