Phospho thr202 tyr204 clone d13.14.4e

For T cell activation, 5C.C7 T cells were preincubated with 1 μM jasplakinolide or DMSO for 15 minutes at 37°C. Following treatment, T cells were mixed with polystyrene beads coated with 1 μg/mL of MCC-I-E^k and 1 μg/mL ICAM-1 and incubated at 37°C. At various time points, cells were collected and lysed in cold lysis buffer containing 50 mM TrisHCl, 0.15 M NaCl, 1 mM EDTA, 1% NP-40, 0.25% sodium deoxycholate, phosphatase inhibitors (1 mM NaF and 0.1 mM Na₃VO₄), and protease inhibitors (cOmplete mini cocktail, EDTA-free, Roche). Activation of PI3K and MAP kinase signaling was assessed by immunoblot for pAkt (Phospho-Akt (Ser473) Ab; Cell Signaling Technology) and pErk1/2 (Phospho-Thr202/ Tyr204; clone D13.14.4E; Cell Signaling Technology), respectively.

In total, 0.2–1 × 10⁶ CTLs were lysed using cold cell lysis buffer containing 50 mM Tris-HCl, 0.15 M NaCl, 1 mM EDTA, 1% NP-40, and 0.25% sodium deoxycholate. Suppression of talin 1 was confirmed using an anti-talin 1 antibody (clone 8D4, Abcam). Talin head domain GFP fusion was detected using a polyclonal antibody against GFP (Invitrogen). Actin (clone AC-15, Sigma) or GAPDH (clone D16H11, Cell Signaling Technology) served as loading controls. For signaling assays, serum and IL-2 starved OT-1 CTLs were incubated with streptavidin polystyrene beads (Spherotech) coated with H-2K^b-OVA and ICAM-1 at a 1:1 ratio for various times at 37 °C and immediately lysed in 2× cold lysis buffer containing phosphatase inhibitors (1 mM NaF and 0.1 mM Na₃VO₄) and protease inhibitors (cOmplete mini cocktail, EDTA-free, Roche). Activation of PI3K and MAP kinase signaling was assessed by immunoblot for pAkt (Phospho-Akt (Ser473) Ab; Cell Signaling Technology) and pErk1/2 (Phospho-Thr202/ Tyr204; clone D13.14.4E; Cell Signaling Technology). Additional information about antibodies may be found in Supplementary Table 1. Uncropped images of blots are provided in Supplementary Fig. 12.