Multifunctional gel imaging system

Total protein was extracted by sonication of liver homogenates for determinations of cytokines. Protein levels were determined by the Lowry method (Waterborg, 2009 ). Equal amounts of protein samples were separated in 10% and 12% polyacrylamide gels and transferred to PVDF membranes. Membranes were blocked with 5% skimmed milk for 3 h at 4°C. The membranes were incubated with one of the following primary antibodies: β-actin (sc-1615), TNFα (sc-12744). These antibodies were obtained from Santa Cruz Biotechnology Inc. (Dallas, TX, United States), and diluted to 1:2000. Antibody for IL-6 (ab6672) was obtained from Abcam (Cambridge, United Kingdom) and diluted to 1:4000. After three washes with TBS-T, the membranes were incubated at 4°C for 3 h with 1:2000 donkey anti-rabbit IgG-HRP (sc-2305) and m-IgGκ BP-HRP (sc-516102), obtained from Santa Cruz Biotechnology Inc. (Dallas, TX, United States). Finally, the images were obtained in a multifunctional gel imaging system (Bio-Rad Laboratories, Hercules, CA, United States). The densities of protein bands were quantified using the ImageJ software (Schneider et al., 2012 (link)).

Spleen proteins were extracted using RIPA lysis buffer (Solarbio, Beijing, China) and centrifuged at 12,000 g for 10 min at 4°C. Protein concentration was assessed using the BCA Protein Assay Kit (Biosharp, Beijing, China). Samples containing equal amounts of protein (80 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to 0.45 μm polyvinylidene difluoride membranes (Biosharp, Beijing, China). The non-specific binding sites on the membrane were then blocked with 5% fresh non-fat dry milk in Tris buffered saline TBS with Tween (10 mM Tris, 150 mM NaCl, pH7.4) with 0.1% Tween 20) for 2 h. Subsequently, they were incubated with primary antibodies (anti-IL-2, anti-STAT1, anti-SHP2, anti-JAK1, and anti-p-JAK1; 1:1000 dilution; Abcam, Cambridge, UK) and primary β-actin antibody (1:1,000 dilution; abmart, Shanghai, China) overnight at 4°C, respectively. After this, the membrane was washed followed by three 10 min washes in TBST, and then incubated with anti−mouse secondary antibody (1:2000 dilution; Cell Signaling Technology, Shanghai, China) for 2 h at room temperature followed by three 10 min washes in TTBS. Finally, the chemiluminescent signals were observed with a multifunctional gel imaging system (Bio-Rad, USA) and densitometry for immunoreactive bands was performed with National Institutes of Health software (Image J).

Cell proteins were lysed with RIPA buffer (50 mM Tris, pH 7.4; 150 mM NaCl; 1% NP-40; 0.5% sodium deoxycholate; 0.1% SDS; and the 1% protease inhibitor) for 30 min, 4 °C. The xenograft tumor tissue proteins were prepared in tissue protein extraction reagent (Thermo Fisher Scientific). The protein (40 μg/20 μL) was separated equal amounts of by SDS-PAGE and transferred to PVDF membrane. Then the membrane was blocked for 1 h using 5% skim milk at room temperature, and the primary antibodies were used and incubated at 4 °C overnight. After washing 5 times with PBST (10 min/time), the membranes were incubated with peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG (1:1000 diluted, Beyotime, Shanghai, China) for 60 min. The blot images were obtained from multifunctional gel imaging system (Bio-Rad, USA).