Western blot analysis

The wild-type and RFP⁺ cells were lysed in denaturing lysis buffer (50 mM Tris-HCl pH 7.5, 1% SDS, 5 mM EDTA, 10 mM β-mercaptoethanol [BME], 1 mM PMSF, and 15 U/mL DNase) in the presence of protease/phosphatase inhibitor cocktails (Thermo Scientific) and mixed by vortexing for 3 s at maximum speed, and samples were heated at 95°C for 5 min. Suspension was then diluted in NP-40 lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, and 5 mM EDTA) and mixed gently. The lysed suspension was passed through a 26-gauge needle attached to a 1-mL syringe and incubated on ice for 5 min. Samples were centrifuged for 15 min at 16,000 × g at 4°C. The supernatants were transferred to a fresh microcentrifuge tube, and protein concentrations were determined with a BCA Protein Assay Kit (Pierce, Thermo Scientific). 40 μg of total protein was boiled at 95°C for 5 min and loaded into SDS-PAGE gels (Bio-Rad) and then transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad). Membrane was incubated with anti-uricase (sc-166070, Santa Cruz Biotechnology) or anti-catalase (#21260-1-AP, Proteintech-Thermo Scientific) or anti-b-actin (sc-47778, Santa Cruz Biotechnology). Membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies. Signal was detected after incubation with a Clarity Western ECL Substrate kit (Bio-Rad) and recorded with a ChemiDoc Imaging System (Bio-Rad).