Mouse anti cd147

The transfected A375 cells or the A375 cells without transfection were grown on glass coverslips. After washing with PBS, both the transfected A375 cells or the A375 cells without transfection were fixed, permeabilized and were incubated with 1% BSA in PBS (adjusted pH value to 7.2–7.4) for 1 h at room temperature. The cells were then incubated with with mouse anti-Myc (Santa Cruz, 1:100) and rabbit anti-Flag (Sigma,1:100) or mouse anti-CD147 (Abcam, 1:100) and rabbit anti-RING1 (Santa Cruz,1:50) in a humidified box for 4°C overnight. Immunolabeling was visualized by incubation in Alexa Fluor-conjugated secondary antibodies (Invitrogen, 1:200) for 1 h at room temperature. The slides were covered with Vectashieldr Mounting medium containing DAPI (Vector Laboratories, CA, USA) and viewed under an inverted fluorescence microscope (Leica, Solms, Germany).

Binding between Mac-1 and CD147 was evaluated using a modified enzyme-linked immunosorbent assay (ELISA). In a 96-well plate, wells were coated with recombinant Mac-1 (R&D Systems, Minneapolis, MN, USA) or bovine serum albumin (BSA) as the control. Recombinant CD147 was added in increasing concentrations (0–20 µg/mL) for 1 h. After removing CD147 and gentle washing, the wells were incubated with an anti-CD147 antibody (mouse-anti CD147, Abcam, Cambridge, UK) followed by a biotinylated secondary antibody (polyclonal rabbit anti mouse, Dako, Glostrup, Denmark) and a streptavidin/HRP complex (Life technologies, Carlsbad, CA, USA). The binding was detected using 3,3′,5,5′-tetramethylbenzidine (Serva, Heidelberg, Germany). The reaction was stopped using 1 M H₂SO₄. The absorption was measured using an ELISA plate reader at 450 nm with a reference value of 570 nm. Measurements from 6 independent experiments were analyzed.