Fig. 3.

EPO+IGF-I treatment induces prolonged Akt phosphorylation. (a) Rat cerebrocortical cultures were treated for 1-180 min with EPO (10 units/ml), IGF-I (100 ng/ml), or both EPO+IGF-I. (Left) Whole-cell lysates were subjected to immunoblot analysis with anti-phospho-Akt Ab (pAkt, each lane loaded with 15 μg of total protein). The blots were then stripped and reprobed with an anti-total Akt Ab. (Right) Densitometry revealed a significant increase in phospho-Akt after incubation with EPO+IGF-I (^*, P < 0.05 EPO+IGF-I vs. IGF-I alone, or IGF-I vs. EPO). (b) Cerebrocortical cultures were incubated with EPO (10 units/ml), IGF-I (100 ng/ml), or both for 1-6 h. (Left) Whole-cell lysates were subjected to immunoblot analysis as described above (except each lane was loaded with 30 μg of total protein). (Right) Densitometry of blots. The level of phospho-Akt was corrected for total Akt in each lane (control lane served as the baseline). Because more protein was loaded in b than in a, the baseline was higher in b, creating somewhat different apparent fold-increases at the same time point between the two blots (e.g., at 1 h). Each experiment was repeated at least three times with similar results.