Anti plcγ

Proteins in cell lysates were resolved by electrophoresis on 6–10% polyacrylamide gels and subsequently transferred to PVDF membranes (Millipore). The following antibodies were used: anti-EGFP (Clontech), anti-β-actin (Santa Cruz), anti-GST (Sigma), anti-UPF1 (Cell Signaling), anti-UPF2 (Santa Cruz Biotechnology), anti-calnexin (Santa Cruz Biotechnology), anti-REQ (in-house manufactured anti-rabbit peptide antibodies raised against the peptide Ac-LGEFPVSNSRARC-NH₂, Mimotopes) and anti-PLCγ (Santa Cruz Biotechnology). Antibodies were diluted 1:200–1:1500 in 5% non-fat dry milk in 0.1% Tween 20/TBS and then incubated overnight at 4°C with rocking. Immunoreactive bands were then visualized using ECL reagents (Thermo).

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 reagent Trizol, Lipofectamine 2000 transfection reagent, miR-519b mimic, and control miRNA were purchased from Life Technologies (Carlsbad, CA). Recombinant human TGF-β1 and the TGF-β1 ELISA kit were bought from PeproTech (Rocky Hill, NJ, USA). The antibodies against phospho PLCγ and phospho PKCα were the products from the Cell Signaling (Danvers, MA, USA). U73122, Gö6976, and GF109203X were bought from Enzo Biochem, Inc. (Enzo, New York, NY). Anti-PLCγ, anti-PKCα, anti-HO-1, anti-mouse and anti-rabbit conjugated horseradish peroxidase (HRP) antibodies were the products from Santa Cruz (Santa Cruz, CA, USA). The small interfering RNAs (siRNAs) against PLCγ, PKCα as well as the control siRNAs were the products from the Dharmacon Research (Lafayette, CO, USA). The pertinent chemicals not mentioned above were purchased from the Sigma-Aldrich (St. Louis, MO, 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).