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. 2004 Jun 21;101(26):9855–9860. doi: 10.1073/pnas.0403172101

Fig. 5.

Fig. 5.

EPO+IGF-I treatment promotes association of XIAP with activated caspase-3 and decreases NMDA-induced caspase activity in cerebrocortical neurons. (a) Treatment with EPO somewhat increased the degree of association of XIAP and cleaved caspase-3 in cells exposed to NMDA. IGF-I treatment alone did not alter XIAP expression, but also resulted in an increase in cleaved capsase-3 coimmunoprecipitated with XIAP. However, EPO+IGF-I exposure led to a large, significant increase in the amount of XIAP associated with active caspase-3. (Upper) Blots representing immunoprecipitates with anti-active caspase-3 Ab that were probed with XIAP Ab. (Lower) Blots from immunoprecipitates of the same lysates made with XIAP Ab and probed with anti-active caspase-3 Ab (a-casp. 3), which recognizes both the p17 and p20 subunits. The blots in a and b were repeated in three separate experiments with similar results. (b) EPO+IGF-I, but neither cytokine alone, reduced caspase-3 activity for at least 9 h after NMDA insult (*, P < 0.05). Caspase activity in cell lysates was assessed 0, 1, 3, 6, and 9 h after drug exposure and is shown as relative DEVDase activity, expressed as nanomoles of free 7-amino-4-trifluoromethylcoumarin (AFC) per microgram of protein. NMDA, EPO, and IGF-I were all added concurrently. (c) Transduction with dnAkt largely prevented the inhibitory effect of EPO+IGF-I on NMDA-induced caspase-3 activity. Caspase activity was assessed 6 h after drug exposure in this experiment (*, P < 0.05; **, P < 0.01). (d) NMDA exposure resulted in cleavage of caspase-3 in the absence of significant changes in XIAP at 6 h. Controls manifest no significant increase in caspase-3 cleavage or XIAP expression.