Anti cd3ζ hrp

Cell pellets were resuspended in lysis buffer (50 mM Tris-HCl, pH 7.5, containing 150 mM NaCl, 100 mM NaF, 1% Triton X-100, 1 mM Na₃VO₄, 1 mM dithiothreitol and protease inhibitors (cOmplete Mini, Roche Diagnostics, Mannheim, Germany), incubated 20 min on ice, centrifuged (15 min, 4 °C, 13,000× g), and supernatants were collected. The Coomassie dye-binding assay (Bio-Rad Laboratories, Hercules, CA, USA) was used to determine cellular protein concentrations. Equal total protein amounts were separated by SDS-PAGE and transferred to nitrocellulose membranes (GE Healthcare Life Science, Solingen, Germany). Membranes were blocked in Tris-buffered saline containing 5% (w/v) milk powder and incubated with antibodies (anti-CD3ζ-HRP (1:1000, Santa Cruz Biotechnology, CA, USA) and anti-GAPDH-HRP (1:10,000, GeneTex/BIOZOL, Eching, Germany). Membranes were washed, incubated with SuperSignal^® West Pico chemiluminescent substrate (ThermoScientific, Rockford, IL, USA), and images were captured with the Fusion imaging system (Molecular Devices, Biberach, Germany).

Total protein concentrations of cell lysates were determined with the Coomassie dye-binding assay (Bio-Rad Laboratories, Hercules, CA, USA). For Western blot analysis, membranes were incubated with anti-CD3ζ-HRP (1:1000, Santa Cruz Biotechnology, CA, USA) in 5% milk. After detection, membranes were stripped and reprobed with anti-GAPDH-HRP (1:10,000, GeneTex/BIOZOL, Eching, Germany) in 5% milk.