P stat3

The spleen was made into a single cell suspension for flow cytometry, as described earlier [45 (link),55 (link)]. Briefly, the spleen was collected immediately after sacrifice and placed in a 70 μm stainless steel strainer, which was kept on top of a 50 mL conical tube containing RMPI-1640 medium. The spleen was then dilacerated with the piston of a syringe to collect a single cell suspension. Immune cells (1 million cells) in the splenic cell suspension were immunostained with monoclonal antibodies against cell surface receptors, i.e., CD4 coupled to APC-Cy7/FITC/APC (Biolegend, San Diego, CA, USA; Santa Cruz Biotech, Dallas, TX, USA). Immune cells were then permeabilized/fixed and processed for intracellular staining. Immune cells were then immunostained with monoclonal antibodies against intracellular proteins such as IL-10, p-ITK, p-NFkB, Foxp3, p-STAT3 and IL-17A coupled to APC/PE/FITC (Biolegend, San Diego, CA, USA; Cell Signaling Tech, Danvers, MA, USA). Immune cells were then acquired (10,000 events) on a flow cytometer and analyzed with Cytomics FC500 software (Beckman Coulter, Brea, CA, USA), as described earlier [45 (link),55 (link)]. CD4+ T cells and their intracellular markers were analyzed in a lymphocytic gate on a forward and side scatter plot.

Cells were rinsed in ice-cold PBS then lyzed in NP40 lysis buffer (1% NP40, 50 mM Tris-HCl pH 8.0, 150 mM NaCl) plus protease inhibitor cocktail (Pierce), 5 mM sodium fluoride, 2 mM sodium orthovanadate and 0.2 mM PMSF incubating on ice for 15 min. Lysates were cleared by centrifugation at 20,000 g for 15 min at 4°C then protein concentrations determined using Coomassie Protein Assay Kit (Thermo Scientific, UK). For each sample, 30 μg of total protein were separated on 7% Bis-Tris polyacrylamide gels in SDS running buffer then blotted onto Protran 0.2 mM Nitrocellulose (GE Healthcare, UK). Membranes were probed with 1:1000 dilution of the appropriate primary antibody (mouse anti-gp130; Santa Cruz sc376280), rabbit anti-Clathrin (Biolegend, 813901), STAT3 (Cell Signaling Technologies, #9139), P-STAT3 (Y705, Biolegend, #651006) P-STAT1 (Y701, Cell Signaling Technologies, #8009) or 1:5000 dilution mouse anti-GAPDH (Cell Signaling Technologies, #2118), then 1:5000 dilution of donkey anti-rabbit-HRP (Stratech, 711-035-152-JIR) or donkey anti-mouse-HRP (Stratech, 715-035-150-JIR) as the secondary antibody. Immobilon Western Chemiluminescent HRP substrate (Millipore, UK) was used for visualization.

Gp130 mutants were designed to substitute the Tyr residues previously reported as required for the phosphorylation of STAT1 and STAT3 (namely Y757, Y815, Y905 and Y915). These gp130 mutants were single mutants-Y757F, Y815F, Y905F and Y915F-, double mutants in which the two more distal Tyr residues were replaced by Phe (2F-Y905F+Y915F), triple mutants (3F-Y815F-Y905F+Y915F) and finally a gp130 mutant in which all the Tyr residues were replaced by Phe (4F-Y757F-Y815F-Y905F+Y915F). HeLa gp130 KO cells were transfected ON with these different constructs, and then used to analysed the requirement of the different Tyr residues to drive the phosphorylation of both STAT1 and STAT3 upon stimulation with different concentrations of HyIL6 for 15 min. Then, cells were fixed with PFA 2%, permeabilized with methanol 100% on ice for 30 min, stained for P-STAT1 (CellSignaling, 8009S) or P-STAT3 (BioLegend, 651007) and analyzed by flow cytometry (CytoFlex).