Anti slp76

CD4⁺ T cells, Teffs or Tregs (10⁵ cells per well) were incubated for 30 min at room temperature with BT-061, OKT-3, RPA-T4, SK3, QS4120, B-A1, MT441, MT310, EDU-2 or OKT-4 (1 μg ml⁻¹). The mAbs were cross-linked for 5, 10, 30 or 60 min at 37 °C either by anti-human IgG (20 μg ml⁻¹) (Life Technologies) or anti-mouse IgG (amIgG) (10 μg ml⁻¹) (Thermo Scientific, Waltham, MA, USA). For intracellular staining, cells were treated with fixation buffer, permeabilized with perm buffer III and stained with anti-Zap-70 (pY319)/Syk (pY352), anti-PLC-γ2 (pY759), anti-ERK1/2 (pT202/pY204), anti-NF-κB p65 (pS529), anti-PKCα (pT497), anti-p38 MAPK (pT180/pY182), anti-Btk (pY551)/Itk (pY511), anti-MEK1 (pS298), anti-Pyk2 (pY402), anti-SLP-76 (pY128), anti-Lck (pY505), anti-Akt (pS473), anti-LAT (pY226), anti-JNK (pT183/pY185), anti-SHP-2 (pY542) or anti-IKKγ (pS376) according to the manufacturer's instructions (all BD Phosflow, San Jose, CA, USA) before being analyzed on a FACS Canto II. Fold induction was calculated by dividing the mean fluorescence intensity of the measured value by the mean fluorescence intensity of the untreated control.