Phosphorylation of PKB/AKT and ERK1/2 in response to fMLP and GM-CSF is decreased in MDS patients.

Neutrophils were isolated from whole blood from MDS patients and healthy controls as described. Cells were stimulated with 1 μM fMLP with or without priming with 5 ng/mL GM-CSF or with 5 ng/mL GM-CSF alone for the indicated time. Stimulation of the neutrophils was stopped by the boiling of samples in 1 × Laemmli buffer. Proteins were separated by SDS-PAGE, and PKB/Akt activation was detected by Western blotting, using antibodies against phosphorylated PKB/Akt (A-C, upper panels). The same blots were reprobed with an antibody against phosphorylated ERK1/2 (A-C, middle panels). ERK1/2 is shown in the lower panels and represents equal protein loading. The experiment was performed for 5 MDS patients and healthy controls. One patient was tested twice independently, with identical results. Results of 3 representative experiments are shown (A-C). (D) Phosphorylation of PKB/Akt and ERK1/2 was quantified by densitometry of the films. Level of phosphorylation of PKB/Akt and ERK1/2 in neutrophil lysates from healthy donors was expressed as a percentage of the phosphorylation observed in cell lysates from MDS patients, run on the same gel. Significant differences are indicated with asterisks (P < .05).