Chemiluminescent imaging system

The phosphorylations of extracellular signal-regulated kinases (ERK) and inhibitor of nuclear factor kappa B (IκBα) which enhanced by rMgPa induced eCypA were detected by western blotting. Firstly, CypA-siRNA, CD147-siRNA group, ERK and NF-κB inhibitor preincubation group were setted up followed by treatment of cells with 20 μg/m rMgPa for 24 h. The total proteins were extracted, separated and then transferred onto the membrane. The membranes were blocked and then incubated with rabbit anti-ERK (1:1000, CUSABIO, CSB-PA002421, China), anti-phospho-ERK (1:1000, CUSABIO, CSB-PA000749, China), anti-p-IκBα (1:2000, Abcam, ab133462, United Kingdom), and mouse anti-IκBα (1:2000, Abcam, ab12134, United Kingdom) antibodies respectively overnight at 4°C. The transferred membranes were washed with TBST and incubated with HRP-conjugated goat anti-rabbit IgG (1:1000, Proteintech, BL003A, United States) or HRP-conjugated goat anti-mouse IgG antibody (1:1000, Beijing ComWin Biotech, China) at 37°C for 60 min. The proteins were visualized with a chemiluminescent imaging system (Syngene, United States). Meanwhile, the mRNA and protein levels of inflammatory cytokines were verified by qRT-PCR (see section “Quantitative Reverse Transcriptase Polymerase Chain Reaction”) and ELISA (see section “Quantitative Reverse Transcriptase Polymerase Chain Reaction”), respectively.

SV40-immortalized human urothelial cells were washed with cold PBS three times and were then lysed with ice-cold radio immunoprecipitation assay (RIPA) buffer (Beyotime, P0013B, China) supplemented with phenylmethane sulfonyl fluoride (PMSF) followed by centrifugation at 4°C for 15 min. The supernatants were harvested by centrifugation and the protein concentrations were measured using the bicinchoninic acid (BCA) assay (Beyotime, P0010, China). SDS-PAGE was used to fractionate 20 μg of boiled protein and transferred to a polyvinylidene fluoride (PVDF) membrane, which was blocked with 5% skimmed milk and then incubated with rabbit anti-CypA (1:1000, Abcam, ab41684, United Kingdom), mouse anti-CD147 (1:1000, Abcam, ab666, United Kingdom), and rabbit anti-GAPDH (1:2000, BOSTER, BA2913, China) antibodies respectively overnight at 4°C. The membranes were then washed with tris-bufferes saline with Tween-20 (TBST) six times and incubated with corresponding horse-radish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:1000, Proteintech, BL003A, United States) and HRP-conjugated goat anti-mouse IgG (1:1000, ComWin Biotech, CW0102, China) for 60 min at 37°C. The protein expression was visualized using the chemiluminescent imaging system (Syngene, United States) and quantified with the Image J analysis software.