Anti cd3ε okt3

The following antibodies were used: anti-Nck1, anti-ZAP70, anti-Lck, anti-phospho-Lck (Src416) and anti-phospho-ZAP70 (Y319, Cell Signaling Technology), anti-phospho-ζ (Y142, Sigma-Aldrich), anti-idiotypic TCR (C305, Millipore), anti-CD3ε (M20, Santa Cruz Biotechnology), anti-CD3ε (OKT3, eBioscience), anti-ζ antiserum 448 [4 (link)], anti-phospho-CD3ε (Y188) [30 (link)], anti-GST (Bethly), anti-GAPDH (Sigma) and secondary antibodies for immunoblotting (Perbio). Alexa Fluor 647-labeled anti-CD3ε (UCHT1, BioLegend) was used for flow cytometry. Recombinant His-tagged Nck was from Sigma and recombinant active Lck (aa61-aa509) was a generous gift from B.F. Lillemeier, Salk Institute for Biological Studies, San Diego.

Human CD4⁺ T cells purified from peripheral blood mononuclear cells (PBMCs) using EasySep^TM human CD4⁺ T cell enrichment kit (StemCell Technologies, Vancouver, Canada) were stimulated with plate-bound anti-CD3ε (OKT3, eBioscience, San Diego, CA) plus soluble anti-CD28 (CD28.2 eBioscience, San Diego, CA). Procedures with human subjects were approved by the Institutional Review Board of the University of California, Irvine and were conducted in conformity with the 1954 Declaration of Helsinki in its currently applicable version. Similarly, mouse T cells were stimulated with plate-bound anti-CD3ε (2C11, eBioscience, San Diego, CA) plus soluble anti-CD28 (37.51, eBioscience, San Diego, CA). Mgat5 wild-type and heterozygous mice were used for mouse experiments and approved by the Institutional Animal Care and Use Committee of the University of California, Irvine. Both human and mouse cells were cultured in RPMI 1640 medium, supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 ug/ml streptomycin, and 50 μM β-mercaptoethanol. GlcNAc was obtained from Wellesley Therapeutics Inc. (Toronto, Canada) while all acetylated forms of GlcNAc were obtained from Santa Cruz Biotechnology (Dallas, TX). GlcNAc and its acetylated forms were >95% pure.