Stromal contact selectively blocks late-phase Rap1 activation.

(A) K562 cells cultured with or without TPA and with or without HESS-5 stromal cell contact for the indicated durations were analyzed for Rap1 and Ras activation as well as for Akt activation. (B) Graph showing relative Rap1 activation was derived by scanning densitometry of the Rap1 signals in the immunoblot in panel A. The results shown are representative of experiments performed 3 times. Rap1 and Ras activation were assayed by pulldown using GST-RalGDS(RBD) followed by immunoblot detection. Akt activation was detected by probing immunoblots of whole cell lysates with antiphospho-Akt (Ser473).