Irdye 800cw goat anti mouse igg2b

10⁶ cells/sample were lysed in NP-40 lysis buffer. Cell debris was pelleted, supernatant collected and heated with reducing protein loading buffer (Nacalai-Tesque). To detect phosphorylation after stimulation, 10⁵ APC-CHO cells were seeded per well in a 24-well plate one day before stimulation. 10⁶ CAR-T or TCR-T-cells were added into each well and cocultured for the designated duration. Cells were collected and prepared as above. To detect FYN association with CAR, 10⁷ CAR-Jcam cells and 2 × 10⁶ CHO-L2 cells were mixed for 5min in 1mL cRPMI. Stimulation was stopped by adding 10mL pre-cooled PBS. Cells were collected and lysed in 200μL NP-40 lysis buffer. Lysate was incubated overnight with protein G Dynabeads (Invitrogen, 10003D) with anti-Myc. IP samples were washed in lysis buffer and heated with reducing protein loading buffer. All samples were loaded in a 4-12% Bis-Tris gradient gel (NuPAGE, Invitrogen) and transferred to a PVDF membrane (Immobilon-FL Transfer Membrane, Millipore). The membrane was blocked using blocking buffer (Odyssey, LI-COR). The membrane was probed with different primary antibodies, followed after washing by secondary antibodies: IRDye 800CW Goat anti-Mouse IgG2b (Cat# 926–32,352, LI-COR) and IRDye 680LT Goat anti-Rabbit (Cat#926-68021). Visualization and quantification of the blot was by the LI-COR Odyssey infrared imaging system.