Mouse anti human cd3ζ

To confirm the expression of EGFRvIII CAR protein, T cells (3×10⁶) were lysed in 100μl lysis buffer. After centrifugation, cell lysates were run on 12% SDS-PAGE under reducing conditions and transferred to PVDF membrane (Millipore) for immunoblot analysis. Membranes were incubated with mouse anti-human CD3ζ (Santa Cruz). Moreover, EGFRvIII expression in target cell was also detected. The samples were immuno-blotted with mouse anti-human EGFRvIII (Novus). Horseradish peroxidase conjugated anti-mouse IgG (Santa Cruz) was used as the secondary antibodies. Blots were visualized using the ECL (Millipore).

T cells (5×10⁶ per effector condition) were lysed with Laemmli buffer, and protein concentrations were quantified utilizing the Pierce BCA Protein Assay Kit (ThermoScientific, Waltham, MA). Membranes were incubated with 0.2 μg/mL mouse anti-human CD3ζ (Santa Cruz Biotechnology, SC-166275, Dallas, TX) and 0.04 μg/mL rabbit anti-human glyceraldehyde-3-phosphate dehydrogenase (Santa Cruz Biotechnology, SC-25778, Dallas, TX). Membranes were then washed and subsequently incubated with 0.05 μg/mL of corresponding secondary antibodies, goat anti-rabbit 680LT (LI-COR, 926-68021, Lincoln, NE), and goat anti-mouse 800CW (LI-COR, 32210, Lincoln, NE) in 5% blocking solution for 1 hour. After washing and drying, the membrane blots were developed utilizing the LI-COR Odyssey imaging system with both the 700 and 800 channels (Supplemental Figure 3).