Coomassie r 250

IL-7 samples were separated in Novex 16% tricine gels (Invitrogen, cat. no. EC6695BOX) as recommended by the supplier. Proteins in gels were stained with the SilverQuest Silver Staining Kit (Invitrogen cat. no. LC6070) or transferred onto PVDF membranes using the Trans-Blot Turbo Transfer System with associated materials and protocols (Biorad, cat. no. 1704150). For Edman sequencing, proteins on the PVDF membrane were stained with Coomassie Brilliant Blue (Coomassie R-250, Thermo Scientific, cat. no. 20278) and analyzed with the Procise 491cLC protein sequencer (Applied Biosystems) or the PPSQ-51A protein sequencer (Shimadzu). For the analysis of signaling molecules, proteins in cell lysates were separated in 4-12% Tris-glycine gels under reducing conditions and transferred to PVDF membranes. Membranes were blocked for 1 h in 5% BSA with TBST buffer (150 mM NaCl, 0.1% Tween 20, 50 mM Tris, pH 7.5) and incubated overnight with anti-pSTAT3 (Tyr⁷⁰⁵) (Cell Signaling, cat. no. 9138) or anti-β-actin (Proteintech, cat. no. 20536-1-AP) antibodies. After washing, the blot was incubated with peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG for 1 h at room temperature. Finally, Western blot images were developed using the Vilber Lourmat Fusion system (Labtech International) and Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific).

Concentrated CM (100 μg) was resolved on a 10% Tris-Glycine gel supplemented with 0.1% gelatin (Invitrogen). Gels were renatured in a buffer (2.5% (v/v) Triton X-100), followed by incubation in a developing buffer (5 mM Tris-Base, 4 mM HCl, 20 mM NaCl, 0.5 mM CaCl₂) overnight at 37 °C and visualised with Coomassie R-250 (Thermo Fisher Scientific). Images of stained gels were captured under illumination using the UVP Imagestore 5000 (Ultra-Violet Products).