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. 2016 May 6;6:25550. doi: 10.1038/srep25550

Figure 2. Deletion of the 3S motif is critical for ADAM17 mediated proteolysis.

Figure 2

(a) HEK293 cells were transfected with expression plasmids encoding either wildtype human IL-6R, human IL-6R∆ S353_V362 and human IL-6R∆ S359_S361. 48 h later, cell surface expression of the three IL-6R variants was determined via flow cytometry. (b) HEK293 cells were transfected with an expression plasmid encoding wildtype human IL-6R. 48 h later, cells were treated for 2 h with PMA (100 nM) or DMSO as negative control. Cells were pre-treated with GI or GW 30 min prior to PMA stimulation where indicated. Generation of the soluble IL-6R was determined via ELISA or sIL-6R was precipitated from the cell culture supernatant and visualized via Western blotting. Cells were lysed and IL-6R expression determined via Western blotting. β -actin served as loading control. (c) HEK293 cells were transfected with an expression plasmid encoding wildtype human IL-6R. 48 h later, cells were treated for 1 h with ionomycin (1 μ M) or DMSO as negative control. Cells were pre-treated with 3 μ M GI or 3 μ M GW 30 min prior to ionomycin stimulation where indicated. Generation of the soluble IL-6R was determined via ELISA. (d,e) The experiment was performed as described under panels (b,c), but cells were transfected with pcDNA3.1-IL-6R∆ S353_V362. (f,g) The experiment was performed as described under panels (b,c), but cells were transfected with pcDNA3.1-IL-6R∆ S359_S361. ELISA data are the mean (± S.D.) of three independent experiments, Western blots and flow cytometry data show one representative experiment out of three performed.