FIG 1 .

CARDS toxin binds to A549 cell membrane-associated AnxA2. (A) Identification of AnxA2 bound to CARDS toxin. Membrane-enriched fractions of A549 cells were incubated with Ni-NTA alone or CARDS toxin coupled to Ni-NTA. Ni-NTA-bound membrane proteins (lane 1) or CARDS toxin-coupled Ni-NTA-bound membrane proteins (lane 2) were separated on NuPAGE (4 to 12% gradient) gels and stained with Coomassie brilliant blue G-250. Mass spectrometry analysis was performed on eluted proteins. The short dashed arrows point to protein bands that were identified as FL or processed/degraded CARDS toxin, and the long solid arrow points to AnxA2. The capital boldface letters in the AnxA2 sequence are AnxA2-specific amino acids identified by mass spectrometry. The molecular masses (in kilodaltons) of molecular mass markers are indicated to the left of the gel. (B) Immunoblot confirmation of AnxA2 bound to CARDS toxin during pulldown assay. Eluted proteins from panel A were resolved on 4 to 12% NuPAGE gels, transferred to nitrocellulose membranes, and probed with anti-AnxA2 monoclonal antibody. Eluted proteins from control uncoupled Ni-NTA beads (lane 1) show no immunoreactivity, whereas eluted proteins from CARDS toxin-coupled Ni-NTA beads (lane 2) demonstrate clear immunoreactivity at ~36-kDa range.