Figure 3. Substitution of the 3S motif only marginally influences proteolysis by ADAM17.
(a) HEK293 cells were transfected with expression plasmids encoding either wildtype human IL-6R, human IL-6R_AAA or human IL-6R_EKK. 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 human IL-6R_AAA. 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. (c) The experiment was performed as described under panel (b), but cells were transfected with pcDNA3.1-IL-6R_EKK. (d) HEK293 cells were transfected with an expression plasmid encoding human IL-6R_AAA. 48 hours 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. (e) The experiment was performed as described under panel (d), but cells were transfected with pcDNA3.1-IL-6R_EKK. ELISA data are the mean (± S.D.) of three independent experiments. Flow cytometry data show one representative experiment out of three performed.