Figure 2: PACS-2 regulates ADAM17-mediated shedding.

(a) AP-HB-EGF was transiently expressed in control and Pacs2^−/− MEFs. Cells were PMA-stimulated, and the cell medium was analysed for AP activity. To overcome differences in transfection efficiency, the fraction of shed AP-HB-EGF was calculated as AP-HB-EGF released to the medium divided by the total amount of AP-HB-EGF in the medium and cell lysate. The fold change in AP-HB-EGF release was then calculated by setting the unstimulated negative control for each experiment to 1, normalizing the other raw data to this value and finally calculating the average of all individual experiments. The PMA-stimulated fold increase in AP-HB-EGF shedding was depicted for each cell line. Data were compiled from four individual experiments, each performed in triplicate. (b) AP-HB-EGF was expressed in Pacs2^−/− MEFs together with green fluorescent protein (GFP) or PACS-2-HA and shedding analysed as in a. Data were compiled from three individual experiments, each performed in triplicate. (c–e) siRNA-transfected MDA-MB-231 cells were stimulated with PMA (c,d) or TNF-α (e). The medium was analysed by ELISA for shed HB-EGF (c) or TGF-α (d,e), shown as concentrations in pg ml⁻¹. In all cases, data were compiled from three individual experiments, each performed in triplicate. (f) Representative western blots showing knockdown in cells used for ELISA. On blots, # denotes a nonspecific band. Graphs show mean values±s.e.m. Data were analysed by analysis of variance or unpaired two-tailed Student’s t-test, as appropriate. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.