Socs3

Protein from the IFN- and/or tocilizumab-treated cells was extracted and applied to SDS-PAGE. DR4 (Santa Cruz Biotechnology, Inc., TX) and SOCS3 (Immuno-Biological Laboratories Co., Japan) were used as primary antibodies. Anti β-actin antibody (SIGMA, MO) was used as an internal control. Protein bands were visualized using SuperSignal West Dura Extended Duration Substrate (Thermo Scientific, IL), and imaged with the ChemiDoc XRS plus system (BIO-RAD, CA). Individual bands were quantified with Image Lab 3.0 software (BIO-RAD, CA), and normalized against the control value.

For the isolation of cellular proteins, cells were lysed in RIPA lysis buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 0.5% NP-40, 15% Glycerol, supplemented with 10 µg/ml of each aprotinin, leupeptin and pepstatin as well as 0.8 µM Pefabloc (Roche, Mannheim, Germany), 1 mM NaF, and 1 mM Na₃VO₄). The protein concentration of the lysates was determined using Bradford Assay according to manufacturer´s instructions (Carl Roth, Karlsruhe, Germany). Proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Antigens were detected by incubation with specific primary antibodies (1:1,000) followed by incubation with DyLight-coupled secondary antibodies (1:10,000) (Licor, Lincoln, NE, USA). List of primary antibodies: (p)Y SHP2 (#3751), (p)Y STAT3 (#9145), STAT3 (#9139), (p)Y/T ERK1/2 (#4370), ERK1/2 (#4695) (Cell Signaling Technology); SOCS3 (#18391) (Immuno-Biological Laboratories, Fujioka, Japan); HSC70 (#7009) (Stress Marq, Victoria, Canada); SHP2 (#K0810) (Santa Cruz Biotechnology, Dallas, TEX, USA); Tubulin (#T5168) (Sigma-Aldrich Chemie, Munich, Germany). Detection was performed using an Odyssey gel documentation system (Licor). Analysis of Western blots was performed using Image Studio Lite (version 5.2, Licor).