Mouse anti na k atpase

The following anti-human CFTR antibodies were used: mouse monoclonal, clone 596 (Cystic Fibrosis Foundation Therapeutics, Inc.; Chapel Hill, NC) and clone M3A7 (Millipore; Billerica, MA), and rabbit polyclonal Ab-737 (Assay Biotechnology Inc. San Francisco, CA). Other antibodies used were rabbit anti-LMTK2 (Sigma-Aldrich) and anti-LMTK2 kinase domain (Cocalico Biologicals Inc., Reamstown, PA), mouse anti-Na,K-ATPase (Millipore), anti-FLAG M2 (Sigma-Aldrich), and anti-ezrin (BD Biosciences, San Jose, CA), and horseradish peroxide-conjugated goat anti-mouse, goat anti-rabbit, secondary antibodies (Bio-Rad). All antibodies were used at the concentrations recommended by the manufacturer. The following reagents were used: Complete Protease Inhibitor Mixture and PhosSTOP phosphatase inhibitor mixture tablets (Roche Applied Sciences, Indianapolis, IN), the adenylate cyclase activator forskolin and the cAMP phosphodiesterase inhibitor IBMX (3-isobutyl-1-methylxanthine) (Sigma-Aldrich), and the inhibitor of protein serine/threonine phosphatases calyculin A (Cell Signaling Technology, Inc.; Danvers, MA).

These experiments were performed as described previously (27 (link)). Primary antibodies were as follows: rabbit anti-PRRT2 (1:500, Sigma-Aldrich), mouse anti-actin (1:1000, Sigma-Aldrich), mouse anti-Na⁺/K⁺ATPase (1:1000, Millipore), and mouse anti-dynamin I (1:1000, Millipore).

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).