Ba/F3 cells (ACC-300) were purchased from the Leibniz Institute DSMZ–German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). Murine Ba/F3-gp130 cells were obtained from Immunex (33). Murine Ba/F3–IL-6R cells were a gift from Garbers (University of Kiel, Germany). Ba/F3 cell lines were grown in Dulbecco’s modified Eagle’s medium (DMEM) high-glucose culture medium (Gibco, Life Technologies) supplemented with 10% fetal bovine serum (Gibco, Life Technologies), penicillin (60 mg/liter), and streptomycin (100 mg/liter) (Genaxxon bioscience GmbH) at 37°C with 5% CO2. Proliferation was maintained in Ba/F3 cells by adding 0.2% conditioned medium of WEHI-3B cells [IL-3 (10 ng/ml)] (DSMZ ACC-26). Ba/F3-gp130 cells or variants thereof were maintained in the presence of HIL-6, a fusion protein of IL-6 and the sIL-6R, which mimics IL-6 trans-signaling (14). Either recombinant protein (10 ng/ml) or conditioned cell culture medium from a stable CHO-K1 clone secreting HIL-6 (10 ng/ml) was used. Expression and purification of human HIL-6 and human IL-6 were performed as described previously (14, 34). pSTAT3 (Tyr705) (D3A7), STAT3 (124H6), and myc (71D10) antibodies were obtained from Cell Signaling Technology. The anti–hIL-6R mAb 4 to 11 was described previously (16). Anti–IL-6 biotin mAb was obtained from ImmunoTools. Allophycocyanin (APC) anti-human IgG Fc (HP6017) was obtained from BioLegend. Rabbit anti-human IgG Fc–specific and peroxidase-conjugated secondary mAbs were obtained from Pierce (Thermo Fisher Scientific). APC-streptavidin was purchased from BD Biosciences. APC (115-136-146)–conjugated goat anti-mouse IgG polyclonal antibody was obtained from dianova. Alexa Fluor 488 goat anti-rabbit IgG was purchased from Life Technologies (A11070). Anti-CD130 (gp130) antibody B-R3 (ab34315) was purchased from Abcam. InSolution JAK Inhibitor I (pan-JAK inhibitor P6) was obtained from Merck. The metalloprotease inhibitor GW280264X (GW, selective for both ADAM10 and ADAM17) was a gift from GlaxoSmithKline (35). Inhibitor stock solutions were prepared in dimethyl sulfoxide. Recombinant sgp130Fc, IL-6, and VHH6 were produced and purified as described previously (14, 17). Rabbit anti-human IgG Fc HRP was obtained from Thermo Fisher Scientific.

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