Phospho lat tyr171

Phorbol 12-myristate 13-acetate (PMA) and ionomycin (were purchased from Sigma-Aldrich (St. Louis, MO, USA) and were prepared in DMSO. Anti-LAT, anti-Lck, anti-PLC-γ, and phospho-Erk antibodies were from Santa Cruz Biotechnology (Heidelberg, Germany); antibodies binding ZAP-70, phospho-ZAP-70-Tyr319, β-actin, phospho-PLC-γ1-Tyr783, and phospho-LAT-Tyr171 were from Cell Signaling Technology (Danvers, MA, USA); anti-Grb2 mAb was from Abcam (Cambridge, MA, USA); and anti-Erk mAb was from BioLegend (San Diego, CA, USA). Stimulations were performed with the anti-human CD3 OKT3 monoclonal antibody (eBioscience, Thermo Fisher Scientific). Staining to assess CD3 expression on the membrane was performed with OKT3 antibody conjugated to APC (BioLegend, San Diego, CA, USA).

The anti-Fas (IgM) antibody was from Merck-Millipore; anti-LAT LAT-01 mAb was from EXBIO (Praha, Czech Republic); anti-LAT 11B.12, anti-PLC- γ, anti-PTP1B, and anti-caspase-3 monoclonal antibodies were from Santa Cruz Biotechnology (Heidelberg, Germany); antibodies binding phospho-Erk, β-actin, phospho-PLC-γ1-Tyr783 and phospho-LAT-Tyr171 were from Cell Signaling Technology; anti-6His-HRP antibody and anti-phospho-LAT-Tyr132 were from Abcam (Cambridge, United Kingdom). The protein synthesis inhibitor cycloheximide was purchased from Merck-Millipore. Stimulations were performed with the anti-human CD3 OKT3 monoclonal antibody (eBioscience).