Anti lat

Anti-LAT, anti-PLC-γ1, and anti-Erk antibodies were from Santa Cruz Biotechnology (Heidelberg, Germany). The anti-NTAL NAP-07 monoclonal antibody was from EXBIO (Prague, Czech Republic). Antibodies binding phospho-Erk, phospho-PLC-γ1-Tyr783, ZAP70, phospho-ZAP70-Tyr319, phospho-MEK-Ser221, and anti-MEK were from Cell Signaling Technology. Anti-β-actin, anti-CD3 (OKT3), and anti-CD4 monoclonal antibodies were provided by Biolegend (San Diego, CA, USA).

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-mouse PLSCR1 monoclonal antibody 1A8 has been described elsewhere [27 (link)] and was a generous gift of Dr. P.J. Sims (University of Rochester, Rochester, NY). The anti-Syk polyclonal antibody has been described [30 (link)]. Anti-Lyn, anti-Fyn, anti-LAT and anti-ERK antibodies were from Santa-Cruz Biotech (Santa-Cruz, CA). Anti-Akt and anti-PLCγ1 were from Cell Signaling Technology (Danvers, MA). Anti-DNP mouse monoclonal IgE clone DNP48 [31 (link)] was a kind gift of Dr. R.P. Siraganian (NIDCR, NIH, Bethesda, MD). Anti-actin and horseradish peroxidase-labeled secondary antibodies were from Sigma-Aldrich (St Louis, MO).