Figure 2. AnxA2 binds NHERF2.

(A) Measured BiFC signal intensities by FACS in COS-7 cells. Results are means ± S.D. for three independent experiments (10000 cells/experiment) expressed as percentages of the signal intensity obtained with an equivalent amount (500 ng) of the pVenus plasmid. (B) ELISA quantification of binding of AnxA2 and NHERF2. A range of concentrations of purified AnxA2–HA were added to microtitre plate wells coated with purified NHERF2 or BSA. Binding was detected with horseradish-peroxidase-conjugated anti-HA antibodies (left-hand panel). A range of concentrations of purified NHERF2 were added to microtitre plate wells coated with purified AnxA2–HA or BSA. Binding was detected with rabbit polyclonal anti-NHERF2 antibodies followed by horseradish-peroxidase-conjugated anti-rabbit antibodies (right-hand panel). O.D. = absorbance. (C) Protein overlay assay showing binding of NHERF2 to AnxA2. Purified proteins AnxA2–HA, NHERF2 and BSA were separated by SDS/PAGE and stained with Coomassie Blue (left-hand panel) or immobilized on to PVDF membranes and incubated with purified NHERF2 (right-hand panel). Bound NHERF2 was detected by immunostaining with rabbit polyclonal anti-NHERF2 antibodies followed by horseradish-peroxidase-conjugated anti-rabbit antibodies, showing specific interaction of NHERF2 with AnxA2 either as monomer or as stable dimer. Molecular masses are indicated in kDa. (D) NHERF2 co-immunoprecipitates with AnxA2. HA–NHERF2 was immunoprecipitated with mouse anti-HA antibody from HeLa or HeLa-NHERF2 cells. Western blots revealed that AnxA2 was immunoprecipitated with NHERF2 in HeLa-NHERF2 cells, but not in HeLa cells, whereas equivalent levels of AnxA2 were found in the whole-cell extracts. Equivalent protein loading was confirmed by Western blotting with anti-tubulin antibody in cell extracts and anti-(mouse IgG) antibody in co-IP samples.