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Fig. 6. PMA stimulation of cells causes the translocation of TACE to the cell surface in an ERK phosphorylation-dependent fashion. (A) HeLa cells transfected with 1 µg pcDNA3.1+ (Lane U) or pWT.TACE-HA were pre-incubated with 10 µM U0126 or carrier (DMSO) for 30 minutes, 24 hours after transfection. Cells were stimulated with 1 µM PMA at 37°C for the time points shown (in minutes) before cell surface proteins were biotinylated and TACE immunoprecipitated using anti-HA antibodies (C-15). Immune complexes were washed and analysed by western blotting with streptavidin-HRP (top panel). Equal volumes of whole cell lysates (3% input) were also analysed following biotinylation by western blotting using an anti-HA antibody to detect pro-TACE (lower panel). HeLa cells were transfected with 1 µg pWT.TACE-HA (B) and pT735A.TACE-HA (C) and 24 hours after transfection treated with 10 µM U0126 or carrier (DMSO) at 37°C for 30 minutes. Cells were stimulated at 37°C with 1 µM PMA for the times stated (or carrier for 10 minutes) before cell surface proteins were biotinylated and TACE immunoprecipitated then analysed by western blotting with streptavidin-HRP. Whole cell lysates (3% input) were also analysed by western blotting for the expression of pro-TACE (lower panels in B and C). The arrowheads indicate mature TACE (100 kDa).
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