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The EMBO Journal (2004) 23, 2577–2585, doi:10.1038/sj.emboj.7600268 Published online 10 June 2004 Figure 4 Negative feedback loop in T-cell activation through MAPK-catalyzed threonine phosphorylation of LAT Satoshi Matsuda, Yoshihiro Miwa, Yasuko Hirata, Akiko Minowa, Junko Tanaka, Eisuke Nishida and Shigeo Koyasu

Figure 4 Effects of LAT phosphorylation at T155 on LAT function. (A) The cells were assayed for TCR-induced Ca2+ influx. [Ca2+]i levels were measured on a flow cytometer (right), and calculated as described in Materials and methods (left). (B) J.CaM2 cells stably expressing Myc-tagged LAT were assayed for TCR-induced Ca2+ influx (left). LAT expression levels were confirmed by immunoblot with the anti-LAT antibody and an anti-ZAP70 mAb (Transduction Labs.) (right). It should be noted that wild-type LAT is more expressed compared to T155A mutants. (C) The cells were stimulated with (+) or without (-) 2Ad2A2 for 2 min. ZAP70 and LAT were immunoprecipitated, and subjected to immunoblot with 4G10. (D) Raft fractions were prepared as described in Materials and methods. Existence of LAT was detected with the anti-LAT antibody.

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