For Jurkat cell lines, 2 × 106 cells were mixed with 250 pmol of scrambled or VAV1 siRNA in Ingenio electroporated buffer (Mirus) and electroporated using the H-10 program of an Amaxa device (Lonza). The same program was used for the electroporation of CD226 plasmids. Before electroporation, primary CD4+ T cells were briefly activated using plate-bound anti-CD3 (4 μg/ml; UCHT-1) and soluble anti-CD28 (1 μg/ml; CD28.2) in IL-2–containing medium (30 U/ml). After 48 hours of activation, cells were harvested and allowed to rest for 24 hours in complete medium. Cells (4 × 106) were then mixed with 250 pmol of scramble or VAV1 siRNA in Ingenio electroporated buffer (Mirus) and electroporated using the T-23 program of an Amaxa device (Lonza). Maximum VAV1 knockdown was achieved 72 hours after electroporation. Scramble siRNA (5′-AUUGUAUGCGAUCGCAGACdTdT-3′ and 5′-GUCUGCGAUCGCAUACAAUdTdT-3′) and VAV1 siRNA (5′-CGUCGAGGUCAAGCACAUUdTdT-3′ and 5′-AAUGUGCUUGACCUCGACGdTdT-3′) were purchased from Sigma-Aldrich.

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