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. 2016 Oct 12;6:35067. doi: 10.1038/srep35067

Figure 4. Inhibition of ADAM17 internalisation.

Figure 4

(A–G) As inhibitor against the clathrin-dependent internalisation 5 μM ikarugamycin (IK) were used. To inhibit caveolin-dependent internalisation cells were treated with 200 μM genistein (GE). Each depicted flow cytometry graph is one representative experiment of a set of n = 3. (A) Flow-cytometry analysis of HEK293 and HeLa cells, which were left unstimulated, were stimulated with 100 nM PMA or were stimulated with 100 nM PMA and treated either with genistein or with ikarugamycin 1 hour before stimulation. (B) HEK293 cells were left untransfected or were transfected with DN-AP180_BFP2. Afterwards, cells were stimulated with PMA for 5 minutes or 60 minutes, or left unstimulated and harvested for flow cytometry analysis. Each depicted flow cytometry graph is one representative experiment of a set of n = 4. Right panel: Comparison of relative MFI (median fluorescence intensity) between untransfected and with DN-AP180 transfected cells. The MFIs were normalised to the respective 5 minutes stimulated sample. (C) Pulse chase experiments: HEK293 cells were incubated with inhibitors like described before. After 1 hour surface proteins were biotinylated and cells were immediately harvested (unstim (0 h)), or were stimulated with 100 nM PMA or left unstimulated in addition to the indicated treatment with inhibitors for 2 hours. The lysates, which were used for the Streptavidin-Sepharose precipitation were immunoblotted as additional input control. (D,E) Western blots of cell-surface fractions of HEK293 cells, which were either left unstimulated, stimulated with 100 nM PMA (D) or stimulated with 30 μM TRAP-6 (E). An additional set of cells were treated with ikarugamycin and were stimulated like described before 1 hour after treatment with the inhibitor. Cell-surface proteins were biotinylated at given time points after stimulation. The lysates, which were used for the Streptavidin-Sepharose precipitation were immunoblotted as additional input control. (F) Flow-cytometry analysis of HEK293 cells, which were left unstimulated and were treated with ikarugamycin for indicated times. (G) Flow-cytometry analysis of HEK293 cells, which were treated with ikarugamycin for 60-minute prior stimulation. Cells were analysed 5 minutes after stimulation with 30 μM TRAP-6.