FIGURE 5:

Annexins interact with proAREG and ADAM17 on the cell surface. (A) Schematic representation of a result of the pulldown assay shown in Figure S3. V5-tagged AREG derivatives were pulled down by GST-annexins. The binding properties were estimated based on the relative intensity of the bands as compared with the proAREG band, and are indicated as ++ (>50%), + (10–50%), and − (<10%). (B and C) Effects of the membrane-impermeable cross-linking agent on the association between proAREG, annexins, and ADAM17. HT1080 cells were transiently transfected with proAREG-V5 or ADAM17-V5, with or without an ANX-Flag expression vector. Cells were incubated in the absence or presence of DTSSP; this was followed by immunoprecipitation with an anti-Flag antibody. Western blotting was carried out using an anti-V5 antibody. The upper and lower bands in the ADAM17 immunoblot indicate the proform and mature ADAM17, respectively (Sahin et al., 2004).