Round-bottomed, 96-well plates were coated overnight with anti-CD3 antibody (10 μg/ml; 145-2C11, BioLegend, RRID: AB_2632707) in PBS at 4°C. Clone 4 CTLs or TILs were resuspended in complete medium at a concentration of 1.5 × 106 cells/ml and incubated at 37°C. CTLs or TILs (100 μl) were added to the well together with 100 μl of complete medium. Cells were centrifuged in the plate for 30 s at 250g to ensure uniform contact with the anti-CD3–coated plastic. The plate was immediately incubated at 37°C for 1, 2, or 5 min. After incubation, the cells were lysed with 100 μl of ice-cold radioimmunoprecipitation assay buffer (Cell Signaling Technology). Phos-tag reagent (Wako) was added to 15% SDS–polyacrylamide gel electrophoresis gels according to the manufacturer’s protocols. Standard ECL protocols were used for immunodetection. The antibodies used were anti-cofilin (D3F9, Cell Signaling Technology, RRID: AB_10622000), anti–glyceraldehyde-3-phosphate dehydrogenase (14C10, Cell Signaling Technology, RRID: AB_10693448), and horseradish peroxidase–conjugated anti-IgG (Cell Signaling Technology).

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