Mouse anti anxa2 monoclonal antibody

EBL cells were seeded in 24-well plates at a density of 2×10⁵ cells. After overnight growth, monolayers were washed with PBS and incubated with mouse anti-ANXA2 monoclonal antibody at different concentration (0.1 μg/ml, 1.5 μg/ml, 3 μg/ml, Santa Cruz Biotechnology, Dallas, USA) or HRP-conjugated goat anti-mouse IgG monoclonal antibody as negative control (3 μg/ml, Millipore, Massachusetts, USA) for 1 h at 4°C according to the literature (28 (link)). Then, 2 × 10⁸ CFU/ml M. bovis was added and maintained for 30 min. The cells were washed three times with PBS to remove unbound mycoplasmas and lysed by pure sterile water. The suspension was serially diluted and plated on PPLO agar plate to count adhered bacteria. Similarly, the effects of anti-ANXA2 antibody on M. bovis invasion to EBL cells was assessed by gentamicin invasion assay at a concentration of 3 μg/ml after 12 h post infection as described above. Each treatment was repeated three time in triplicate.

To see the effects of M. bovis on location of ANXA2, the plasma membrane and cytosolic proteins of the mock- and M. bovis- infected EBL cells at 12 hpi were isolated using Minute™ plasma membrane protein isolation and cell fractionation kit (Invent Biotechnologies, Plymouth, Minnesota, USA) as manufacturer’s recommendations. The abundance of ANXA2 on the cell surface and in the cytoplasm was tested by the western blot assay as described above. The following appropriate primary antibodies were used for immunodetection: mouse anti-ANXA2 monoclonal antibody (SantaCruz Biotechnology, Dallas, USA), mouse anti-Na⁺/K⁺ ATPase (sigma-Aldrich, St louis, MO, USA) and rabbit anti-tubulin (Abcam, Cambridge, UK). After incubation overnight at 4 °C, the blots were exposed to the appropriated secondary antibody. For indirect immunoflurorescence assay, cells were immunoblotted with rabbit anti-ANXA2 Ab (1:200) and with mouse anti-M. bovis 1c11 mAb (1:1000), followed with fluorescein isothiocyanate (FITC)-labled goat anti-mouse IgG (Invitrogen) and Cy3 Red-labeled goat anti-rabbit IgG (Invitrogen) as secondary antibody. DAPI was used to stain the cell nuclei (Beyotime Technology, China). Finally, the slides were covered and examined under a confocal laser fluorescence microscope (Olympus FV1000 and IX81, Tokyo, Japan).