M89 61

For analysis of surface markers CD71, CD98, and CD122, cells were stained in PBS containing 2% (wt/vol) FBS with antibodies from eBioscience or BD. Their expression level was presented as net mean fluorescence intensity (ΔMFI), which was determined by subtracting MFI of isotype control, or as fold relative to unstimulated, set as 1. Intracellular staining was used for NK cell transcription factor and phosphorylated proteins. NK cell transcription factors E4BP4, Eomes, and T-bet were stained with anti–mouse E4BP4 antibody (S2M-E19; eBioscience), anti-Eomes (Dan11mag; eBioscience), and anti–T-bet (eBio4B10; eBioscience) before and after overnight stimulation with IL-15–IL-15R complex according to the manufacturer’s instruction (eBioscience), similar to Foxp3 staining, respectively. For detection of phosphorylated signaling proteins, NK cells were fixed with Phosflow Lyse/Fix buffer, followed by permeabilization with Phosflow Perm buffer III (BD) and staining with antibodies to S6 phosphorylated at Serc235 and Serc236 (D57.2.2E; Cell Signaling Technology), Akt phosphorylated at Serc473 (M89-61; BD), and Thrc308 (J1-223.371; BD). Flow cytometry data were acquired on LSRII or LSR Fortessa (BD) and analyzed using FlowJo software (Tree Star). Net mean fluorescence intensity (ΔMFI) was calculated. Expression levels were presented as fold relative to unstimulated, set as 1.

CD3+ T cells were treated with CRP at the indicated concentrations, washed once with medium and stimulated with H₂O₂ for 30 min. Cells were stained by flow cytometry for cell surface and phosphoprotein makers (ZAP70 (pY319)/Syk (pY352) (17A/P-ZAP70, BD Biosciences), LCK (pY505) (4/LCK-Y505, BD Biosciences) and LAT (pY717) (I58-1169, BD Biosciences) (M89-61, BD Biosciences)) after fixation and permeabilization using a transcription factor phosphor-buffer set (BD Biosciences).