FIGURE 4:

Multiple annexin family members regulate proAREG shedding. (A) HT1080 cells stably expressing AP-AREG were incubated with siRNA and transiently transfected with the proAREG-Myc and ADAM17-V5 expression vectors. Cells were treated with the membrane-impermeable cross-linking agent DTSSP; this was followed by immunoprecipitation with an anti-Myc antibody. Western blotting was carried out using an anti-V5 antibody. The intensity of the ADAM17-V5 bands is represented as the fold-change relative to control siRNA-transfected cells. (B) The effect of siRNA on the subcellular localization of proAREG-YFP. The ucF-proAREG-YFP cells were transfected with siRNA and stimulated with 20 nM TPA for 30 min. Scale bar: 20 μm. (C) AP activity was measured in the conditioned medium of HT1080 cells stably expressing AP-AREG. Cells were cotransfected with combinations of siRNA and were stimulated with 20 nM TPA for 30 min. Data represent the mean ± SEM; **p < 0.01. (D) Prevention of the interaction between proAREG and ANXA2 by ANXA8. HT1080 cells were transfected with expression vectors encoding proAREG and ANXA2-V5 together with empty or ANXA8-Flag expression vectors. Cell lysates were immunoprecipitated with an anti-V5 antibody, and the coprecipitated proAREG was detected using an anti–AREG-N antibody.