Anti pstat3 y705

STAT5 and STAT3 phosphorylation were measured from isolated PBMCs after a 15-min stimulation in the presence of exogenous IL-2 (10 U/ml and 320 U/ml) or IL-21 (10 ng/ml), respectively, in pre-warmed RPMI 1640. IL-2-stimulated cells were then fixed and permeabilized according to manufacturer’s protocol (Becton Dickinson) and stained with fluorescent-conjugated CD3 (Invitrogen), CD4, CD25, CD56 (BD Biosciences), and pSTAT5 (eBioscience) antibodies. Cells were analyzed by flow cytometry (NovoCyte model 3000 and NovoExpress software, Acea). IL-21 stimulated cells were stained with antibodies for CD4, CD8, and CD19 (BioLegend) for 20 min on ice and fixed with 4% formaldehyde (Thermo) for 10 min. After washing, cells were incubated in 90% methanol at − 20 °C overnight and stained with fluorescent conjugated anti-CD3 (BD), anti-pSTAT3 (Y705, BD), and anti-STAT3 (BD). Cells were analyzed using BD Fortessa flow cytometer and FloJo (v10.6) software.

At scarify, single-cell suspensions were isolated from dLNs, spleens, and CNS. Briefly, the brain and spinal cord were obtained, homogenized, and then incubated with collagenase D (2.5 mg/mL, Roche Diagnostics) and DNase I (1 mg/mL, MilliporeSigma) for 30 minutes. Mononuclear cells were enriched by gradient centrifugation at 670g for 30 minutes on a 37%/70% Percoll gradient without interruption. Before staining, cells were blocked with anti-CD16/CD32 antibodies. The following antibodies were used for flow cytometry: anti-CD3 (OKT3), anti-CD45 (30-F11), anti-MHCII (M5/114.15.2), anti-CD8 (53-6.7), anti-CD44 (IM7), anti-Foxp3(FJK-16S), anti–GM-CSF (MP1-22E9), anti-CD49d (R1-2), anti–IL-17A (eBio17B7), and anti–IFN-γ (XMG1.2) (eBioscience); anti-CD4 (GK1.5), anti-CD25 (PC61.5), anti-CD62L (MEL-14), anti–Ly-6G/Gr-1 (1A8-Ly6g),anti-F4/80 (BM8), anti-CCR2 (SA203G11), anti-CCR6 (29-2L17), and anti-CD29 (HMβ1-1) (BioLegend). In addition, anti–p-STAT3 (Y705) (catalog 557814) and p-STAT1 (Y701) (catalog 502069) antibodies were purchased from BD Biosciences. Phosphoflow cytometry analysis was performed using BD Phosflow buffers (554655 and 558050). Stained cells were analyzed by FACSCanto flow cytometer and were analyzed with FlowJo software (Tree Star).