Anti p stat5

Purified murine CD4⁺ T cells were left unstimulated or stimulated as indicated in the figure legends. For detection of Tyr⁶⁹⁴STAT5 and Tyr⁶⁴¹STAT6 phosphorylation, cells were preactivated under Th9 conditions in the presence of anti-mIFNγ (5 μg ml⁻¹) and rIFN-γ (5 ng ml⁻¹) for 2 days, then washed and rested in cytokine-free medium for 8 h. Preactivated cells were treated with rat IFN-γ (5 ng ml⁻¹) in combination with either rmIL-4 (20 ng ml⁻¹) or rhIL-2 (50 U ml⁻¹) for 20 or 60 min. Whole-cell lysates were prepared as described previously47 (link), 20 μg of total protein were loaded per lane and proteins were detected according to standard protocols. The following Abs were used: anti-β-actin (AC-15, Sigma-Aldrich), anti-IRF-1 (M-20, Santa Cruz), anti-IRF-4 (M-17, Santa Cruz), anti-STAT1 (#9172, Cell Signaling) anti-P-STAT1 (Tyr⁷⁰¹, D4A7, Cell Signaling), anti-STAT5 (C-17, Santa Cruz), anti-P-STAT5 (Tyr⁶⁹⁴, C11C5, Cell Signaling), anti-STAT6 (M-20, Santa Cruz), anti-P-STAT6 (Tyr⁶⁴¹, sc11762, Santa Cruz), anti-goat IgG-HRP (#sc-2020, Santa Cruz), anti-rabbit IgG-HRP (#sc-2004, Santa Cruz), and anti-mouse IgG-HRP (#sc-2055, Santa Cruz).

LepR-Ba/F3 cells were incubated in serum-free medium overnight and then treated with 100 ng/mL leptin (#Z02962-1; GenScript, Nanjing, China) or 500 ng/mL agonist-type antibody at 37 °C, 5% CO₂ for 30 min. The cells were collected and washed once with ice-cold PBS containing a Halt protease/phosphatase inhibitor cocktail (#78446; Thermo Scientific, Waltham, MA, USA), followed by 30 min of lysis with RIPA buffer containing a protease/phosphatase inhibitor cocktail on ice with shaking. The resultant supernatant was collected by centrifuging at 14,000× g rpm for 10 min. The amount of total STAT3 or STAT5 and phosphorylated STAT3 or STAT5 were determined by a western blot analysis of the cell lysates’ supernatant using anti-STAT3 antibody (#32500; Abcam, Cambridge, UK), anti-STAT5 antibody (#9363T; Cell Signaling Technology), anti-pSTAT3 (Tyr705) (#76315; Abcam), and anti-pSTAT5 (#4322P; Cell Signaling Technology, Danvers, MA, USA), respectively.

Splenocytes were incubated with recombinant mouse IL-7 (10 ng/ml) or IL-15 (20 ng/ml) (Peprotech, Rocky Hill, NJ) for 20 min. Cells were fixed immediately with 2% paraformaldehyde for 10 min at room temperature and permeabilized with cold methanol for 20 min. Surface staining was performed with anti-CD4, CD8, CD44 and CD62L Abs after washing. Phosphorylation of STAT5, ERK and S6K was detected with anti-p-STAT5, p-ERK and p-S6K Ab (Cell Signaling Technology, Danvers, MA) followed by Alexa647-conjugated anti-rabbit secondary Ab. Cells were analyzed by flow cytometry.

The levels of p-STAT3 and p-STAT5 were determined by cytometric analysis. In brief, PBMCs were isolated from pSS patients or healthy controls using density-gradient centrifugation. PBMCs were washed and fixed for 15 min at room temperature. Cells were stained with anti-CD4 (Clone L200, BD Biosciences), anti-p-STAT3 (Clone D3A7, Cell Signaling), and anti-p-STAT5 (Clone Y694, Cell Signaling) using BD Phosflow kit according to the manufacturer’s instructions. The median fluorescence intensities were analyzed for p-STAT3 and p-STAT5 levels.

House dust mites (HDM, ALK-Abello A/S, Denmar), Recombinant Human long-isoform TSLP (lTSLP) was obtained from R&D systems. Synthetic sfTSLP peptides (63aa: MFAMKTKAALAI WCPGYSETQINATQAMKKRRKRKVTTNKCLEQVSQLQGLWRRFNRPLLKQQ) were prepared by China Peptides (Shanghai, China). 3-PO (3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one) and BAY 87-2243 (Selleck, China). Rabbit anti-HIF-1α, anti-LDHA, anti-PHD and anti-VHL (proteintech, China), Rabbit anti-STAT5, anti-p-STAT5, anti-JAK1, anti-p-JAK1, anti-JAK2and anti-p-JAK2 (Cell Signaling Technology, USA), Rabbit anti-TSLPR and mouse anti-IL-7R (santa curz, USA). Rabbit anti-TSLP (Abcam, USA).

For western blot detection, crude proteins were extracted and the concentrations of proteins in individual cell lysate samples were determined using a Micro BCA protein assay kit (Thermo fisher). The cell lysates (30 µg/lane) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 10–12% gels, and transferred to polyvinylidene difluoride (PVDF) membranes. After being blocked with 5% fat-free dry milk in TBST, the membranes were probed with primary antibodies, including anti-c-Maf (diluted 1:500), anti-p-STAT3 (diluted 1:500), anti-STAT3 (diluted 1:1000), anti-p-STAT5 (diluted 1:500), anti-STAT5 (diluted 1:500), and anti-β-actin (diluted 1:1000), all of these antibodies were from Cell Signaling Technology. The bound antibodies were detected with horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz, CA, USA) and visualized by the enhanced chemiluminescent reagents (Thermo fisher). The levels of IL-17, IL-10, and TGF-β1 in the supernatants of cultured cells and IL-6, TNF-α, IFN-γ, and IL-17 in the serum of mice were measured by enzyme-linked immunosorbent assay (ELISA) using specific kits (eBioscience). For detection of TGF-β1, 100 μl of supernatants had been acidified with 20 μl 1N HCl for 10 min at room temperature and then neutralized with 20 μl 1N NaOH to activate latent TGFβ1 to the immunoreactive form.