Effects of G-CSF and GM-CSF on expression of cIAP1 and cIAP2 mRNA and proteins in neutrophils.

(A) Neutrophils were cultivated in the presence or absence of G-CSF (50 ng/mL) or GM-CSF (5 ng/mL). After cultivation for 0.5 to 3 hours, the expression of cIAP1 and cIAP2 mRNA was analyzed by the real-time PCR. As an internal control, 18S rRNA was used. The ratios of cIAP1 to 18S rRNA and cIAP2 to 18S rRNA were calculated, and the ratio at hour 0 is expressed as 1. The data are expressed as means ± standard errors of the mean of 5 experiments. *Significantly increased as compared with control cells (P < .01). (B) Neutrophils were cultivated in the presence or absence of G-CSF (50 ng/mL) or GM-CSF (5 ng/mL). After cultivation for 1 to 4 hours, the expression of cIAP2 and p38 proteins was analyzed by immunoblotting using antibodies against each protein. The cell lysates equivalent to 2.5 × 10⁶ cells were loaded onto each lane. As a control, p38 was used. The ratio of cIAP2 to p38 band intensity was calculated, and the ratio at hour 0 is expressed as 1 (lower panel). The results shown are representative of 3 independent experiments. Lym indicates lymphocytes (2.5 × 10⁶ cells). (C) The expression of cIAP1, cIAP2, and p38 proteins in freshly isolated neutrophils (Neut), monocytes (Mono), and lymphocytes (Lym) was analyzed by immunoblotting using antibodies against each protein. The cell lysates from an equal number of cells (2.7 × 10⁶) were loaded onto each lane. The results shown are representative of 3 independent experiments.