In terms of the underlying mechanism, we showed that GSNOR-dependent Akt denitrosylation was associated with increased Akt activity and Akt phosphorylation, in particular, at Ser473. The crosstalk between S-nitrosylation and other post-translational modifications such as phosphorylation, ubiquitination and acetylation has been extensively studied . immunization, NO produced by inducible nitric oxide synthase (iNOS) suppresses the survival of T cells to control the persistence of CD4+ and CD8+ T-cell immune memory . Moreover, accumulating evidence suggests a protective role of S-nitrosylation in LASS4 antibody various autoimmune diseases by modulating the differentiation of T helper (Th) cell subsets, including Th-1, ??2 and ??17 . These previous studies indicated the direct or indirect regulatory effects of S-nitrosylation on T-cell apoptosis, survival, differentiation and development, but the regulatory effects of S-nitrosylation on Hcy-induced primary T-cell activation, including cytokine secretion and proliferation, remain to be fully elucidated. Our previous work showed that HHcy promotes Akt phosphorylation in T cells to accelerate atherosclerosis . The phosphoinositide-3 kinase (PI3K)/Akt pathway is critical for regulating T-cell proliferation, metabolism, cytokine production and survival , , . Upon activation, na?ve T cells develop into Teff cells that enter the bloodstream and are recruited into atherosclerotic plaques, where they proliferate and produce proinflammatory cytokines . Recent reports have shown that Akt can be S-nitrosylated in muscle cells and esophageal squamous cells, leading to its inhibited kinase activity in diabetic models, post-burn injury, and squamous cell differentiation , , . Considering the crucial role of Akt-mediated T-cell activation in HHcy-accelerated atherosclerosis, whether and how S-nitrosylation of Akt regulates Hcy-induced T-cell activation and the mechanism underlying the intracellular pathway remain to be determined. In this study, we demonstrate that HHcy upregulated the expression of GSNOR in T cells. As a result, GSNOR induced denitrosylation of Akt in Hcy-activated T cells and in PBMCs, PBMCs (1? 106 and 5? 106 cells) were collected for flow cytometry and RNA extraction, respectively, after isolation. To Asiatic acid quantify the proportion of IFN-+ T cells, 1??106 PBMCs were suspended in RPMI 1640 medium (Gibco, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (Gemini Bio-Products, Asiatic acid West Sacramento, CA, USA). PMA and ionomycin (Sigma-Aldrich, St. Louis, MO, USA) were added at 100?ng/mL and 1?g/mL, respectively, for 12?h, and a 1000??brefeldin A solution (420601, Biolegend, San Diego, CA, USA) was added per the instructions 4?h before harvest. CD3, SNO-Cys and IFN- were stained and analyzed by flow cytometry as described below. 2.3. Cell isolation and culture Splenic T cells were isolated from mice and purified by positive selection with magnetic microbeads against CD90.2 (Miltenyi Biotec, Bergisch Gladbach, Germany) following the manufacturer’s protocol. Purified T cells were cultured in RPMI 1640 medium (Gibco, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (Gemini Bio-Products, West Sacramento, CA, USA) in cell culture plates containing plate-bound anti-CD3 antibody (1?g/mL, BD Pharmagen, Franklin Lakes, NJ, USA). Purified T cells were further treated with or without 100 mol/L Hcy for the indicated times. Under some conditions, T cells were pretreated with 50 mol/L GSNO (provided by Prof. C Chen) or S-nitrosylation inhibitors, 50 mol/L N-acetyl-L-cysteine (NAc, Sigma Aldrich) or 25 mol/L dithiothreitol (DTT) for 30?min, and then incubated together with or without 100 mol/L Hcy for the indicated times. 2.4. Detection of protein S-nitrosylation with the irreversible biotinylation procedure (IBP) Protein S-nitrosylation was detected with the IBP, as previously described , which is an improved method based on the original biotin switch assay . 2.5. Quantitative S-nitrosylation proteomics and data analysis The iodoTMT labeling was conducted by using iodoacetyl tandem mass tag? (iodoTMT?) reagents (90103, Thermo Scientific, USA) as described by Qu (forward, CAGGAACCTGAAACTCCCCA; reverse, AGAAAGTCCACCACAGTTGC), (forward, TGGCTGTTTCTGGCTGTTAC; reverse, TTCGCCTTGCTGTTGCTGAAG), (forward, TATTTCAACTGGCTACGG; reverse, CTCAAGGGCTGATCTCAT), (forward, AAACGCTTCACTTCCAATG; reverse, CAATCCACAACTCGCTCC), (forward, AGCTCGGAGGATCACAAA; reverse, TCTGCTGAGACCCCTCTTG), (forward, CTGTTCCAGCGAGGGTCTA; reverse, CACAGCCAATAGCAGCACA), (forward, ACAGAAAGCATGATCCGCGAC; reverse, CCGATCACCCCGAAGTTCAGTA), (forward, CAGATGCAGTTAACGCCC; reverse, ATTCCTTCTTGGGGTCAGC), (forward, TGTCAAATTCATTCATGGCCT; reverse, ATCGATTTCTCCCCTGTGAA), (forward, GTGACGTTGACATCCGTAAAGA; reverse, GCCGGACTCATCGTACTCC). The primer sequences based on human genes are as Asiatic acid follows: (forward, ATGGCGAACGAGGTTATCAAG; reverse, CATGTCCCAAGATCACTGGAAAA). 2.13. Plasmid construction and transfection Mouse Akt-WT was amplified from T-cell cDNA with primers containing 5 gene was ligated into the pET-28a(+) expression plasmid for protein expression. The construct for Akt was provided by Synbio Tech (ID: CN5905-2, Suzhou, China). The Akt protein was.