Hrp conjugated goat anti mouse igg

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.

SV Total RNA Isolation System, Access reverse transcription polymerase chain reaction (RT-PCR) System, and T4 DNA ligase were purchased from Promega (Madison, WI, USA). The restriction enzymes of NcoI and XhoI were purchased from NEB (Ipswich, MA, USA). Competent cell NovaBlue, pTriEx-1.1 Hygro vector, and GeneJuice® transfection reagent were supplied by Novagen (Billerica, MA, USA). Nickel–nitrilotriacetic acid (Ni–NTA) His Bindresin was purchased from Qiagen (Hilden, Germany). HEK293 and horseradish peroxidase (HRP)-β-agonists were supplied by Beijing Kwinbon Biotechnology Co., Ltd (Beijing, China). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco (Grand Island, NY, USA). Anti-His monoclonal antibody and HRP-conjugated goat anti-mouse IgG were obtained from Beijing ComWin Biotech Co., Ltd (Beijing, China).
CBL, SAL, and ractopamine (RAC) were purchased from Sigma-Aldrich (St. Louis, MO, USA). HRP-β-agonists were gifts from Beijing Kwinbon Biotechnology Co., Ltd (Beijing, China). All chemicals were of analytical grade without any further purification.