Fig 1: Upstream signaling mechanisms induced by secreted eIF5A in cardiac myocytes.(a) Secreted re-eIF5A (10 μg/ml) activated initiator caspases, including caspase-8 (IMG-5703; IMGENEX), caspase-10 (ab25045; Abcam), caspase-2 (ab2251; Abcam), and caspase-9 (#9508; Cell Signaling), in cultured cardiac myocytes. The anti-actin antibody was sc-1616 from Santa Cruz. (b) Secreted re-eIF5A (10 μg/ml) rapidly increased the phosphorylation of Shc at Tyr 239/240 (#2434; Cell Signaling) (lower panels), but not FADD at Ser 191 (sc-33399; Santa Cruz) (upper panels), in cultured cardiac myocytes. The control antibodies were anti-Shc (sc-967; Santa Cruz) and anti-FADD (ab24533; Abcam). (c) Secreted re-eIF5A (10 μg/ml) increased the phosphorylation of Jak 1 at Tyr 1022/1023 (sc-16773; Santa Cruz) and Tyk 2 at Tyr 1054/1055 (sc-11763), but not Jak 2 at Tyr 1007/1008 (sc-21870). The control antibodies were anti-Jak 1 (sc-295), anti-Tyk 2 (sc-169), and anti-Jak 2 (sc-34479). (d,e) The effects of Jak inhibitor I (Santa Cruz; 1 μM, pretreated for 24 h) on the induction of apoptosis in cardiac myocytes by secreted re-eIF5A (10 μg/ml, 72 h) as determined by TUNEL staining (brown) and cardiac myosin immunostaining (blue), as shown in Fig. 3a. *P < 0.0001 vs. secreted re-eIF5A alone (mean ± s.e.m., n = 4 each) (Dunnett's multiple comparison test) (e). (f) SPR analysis of the interaction between secreted re-eIF5A and cardiac myocyte membrane proteins. The surface of the sensor chip was coupled with 10μg/ml of secreted re-eIF5A in 10 mM acetate buffer (pH 5.5) at a flow rate of 5 μl/min. Then, cardiac myocyte membrane proteins (100 μg/ml in HBS-EP buffer) were analyzed for their interaction with the surface at a flow rate of 10 μl/min for 4 min. The anti-eIF5A mAbs (YSP5-45-36 and YSPN2-74-18, 1 μg/ml for each) and BSA (100 μg/ml) were used as positive and negative controls, respectively. All experiments were performed at least in triplicate. The results shown are from one representative experiment.