Phospho lat tyr191

The anti-Fas (IgM) antibody, anti-phospho-LAT-Tyr226, and rabbit polyclonal anti-human LAT were from Merck-Millipore; anti-PLC-γ1 mAb, anti-Lck, and anti-Erk were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); antibodies binding phospho-Erk, phospho-PLC-γ1-Tyr783, phospho-ZAP70-Tyr319, phospho-MEK-Ser221, phospho-LAT-Tyr191, and anti-MEK were from Cell Signaling Technology; anti-6His-HRP, anti-Grb2, and anti-phospho-LAT-Tyr132 antibodies were from Abcam (Cambridge, MA, USA); and anti-CD69-APC (allophycocyanin) and anti-β-actin monoclonal antibodies were from Biolegend. The protein synthesis inhibitor cicloheximide was purchased from Merck-Millipore. Stimulations were performed with anti-human CD3 (OKT3; eBioscience) or anti-human CD28 (CD28.2; BD Pharmingen) monoclonal antibodies.

Antibodies and reagents were purchased from the following sources: antibodies against ALDH2 (sc-48838) and Kit (sc-13508) were from Santa Cruz Biotechnology (Dallas, TX, USA) and used at a dilution of 1:1000 for Western blotting. Anti-phospho-Syk (Tyr525/526) (#2711), -phospho-LAT (Tyr191) (#3584), -phospho-PLCγ1 (Y783) (#2821), -phospho-Stat4 (Tyr693) (#5267), -phospho-Akt (Thr308) (#9275), -phospho-SAPK/Jnk (Thr183/Tyr185) (#9251), and -phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (#9101) antibodies were from Cell Signaling Technology (Beverly, MA, USA) and diluted to 1:2000 for Western blotting. Anti-β-actin (#A5316), used as a loading control at 1:5000 dilution, was purchased from Sigma-Aldrich (St Louis, MO, USA). Antibodies against Syk (sc-929), Shp-1 (sc-287), and Kit (sc-48838) for immunoprecipitations were from Santa Cruz Biotechnology. Cell culture reagents were from GIBCO/Invitrogen (Carlsbad, CA, USA). Dinitrophenyl (DNP)-specific monoclonal IgE and DNP-human serum albumin (BSA) were from Sigma (St. Louis, MO, USA).