Figure 6

Annexin A2 is a cellular receptor of M. bovis LppA. A LppA-immunoprecipitated proteins identified by immunoprecipitation-mass spectrometry (IP-MS) assays. Yellow arrowheads indicate LppA; red arrowheads indicate host proteins that interact with LppA. B Co-immunoprecipitation analysis. Lysates from 293T cells (+) transfected with pCAGGS-FLAG-N-LppA and pCAGGS-HA-N-ANXA2 plasmids were immunoprecipitated using an anti-Flag antibody and analyzed using western blotting with antibodies to FLAG and HA. Lysates from 293T cells (−) not transfected with pCAGGS-FLAG-N-LppA or pCAGGS-HA-N-ANXA2 plasmids were used as the control group. Whole-cell lysates (WCL) of 293T were used as the input. C Co-localization of the LppA and ANXA2 proteins was analyzed using confocal microscopy. 293T cells were co-transfected with pCAGGS-FLAG-N-LppA and pCAGGS-HA-N-ANXA2 plasmids and labeled with mouse anti-HA and rabbit anti-Flag, followed by Alexa-Fluor-conjugated donkey anti-mouse (488) and donkey anti-rabbit (647) secondary antibodies. The cells were then observed using a confocal microscope. Green represents LppA, red represents ANXA2, and blue represents the nuclear stain 4′,6-diamidino-2-phenylindole (DAPI); co-localization assay suggests that LppA is almost completely coincident with ANXA2.