Figure 2

TRAF3 association with Csk in T cells. (a–d) T cells were stimulated via CD3/CD28 for the indicated times. Fab fragments targeting the stimulatory antibodies were added to prevent association of the stimulatory antibodies with the magnetic protein G beads. Samples were also precleared to remove any unbound stimulatory antibody. TRAF3 immunoprecipitation was then performed as described in Methods from whole cell lysates of WT primary mouse splenic T cells (a) or Hut28.11 T cells (b and d). (c) Immunoprecipitation of Csk from Hut28.11 whole cell lysates. (e) Cellular fractionation was performed as in Methods on HuT 28.11 T cells. TRAF3 was immunoprecipitated from soluble and insoluble cell lysate fractions, isolated as in Methods. (a–e) Analysis by Western blot to detect the indicated proteins. C = Control samples, in which cells were stimulated for 5′ and no immunoprecipitation Ab was added, to detect any residual stimulatory Ab association with the protein G beads. (f) HEK 293T cells transfected with TRAF3 and Csk constructs (depicted in Fig. 4a) were lysed, and Csk was immunoprecipitated. Total input prior to immunoprecipitation is shown at right. Full-length blots are presented in Suppl. Fig. S5. Data shown are representative of 3–6 independent experiments.