Effect of kinase inhibitors on G-CSF-induced activation of Akt or ERK1/2. (A) Inhibition of downstream substrates of Akt, mTOR, and GSK3 by a specific Akt inhibitor A838450. Ba/F3GR cells were pretreated for 1 hour with the indicated concentrations of Akt inhibitor A838450 or DMSO (the diluent control), then stimulated with or without 100 ng/mL G-CSF. Lysates were prepared, and immunoblotting was performed using anti-phospho-mTOR S2448 or anti-phospho-GSK3 S21/9 antibodies. Blots were stripped and reprobed with anti-mTOR or anti-GSK3 antibody to demonstrate comparable protein loading. (B) Akt inhibition. Ba/F3GR cells were pretreated for 1 hour with the indicated concentrations of Akt inhibitor A838450 or DMSO (the diluent control), then left unstimulated or stimulated with 100 ng/mL G-CSF. Lysates were prepared, and immunoblotting was performed using anti-phospho-AktS473 and anti-phospho-ERK1/2 T202/Y204 antibody. Blot was stripped and reprobed with anti-Akt or antiactin antibody to demonstrate comparable protein loading. (C) ERK1/2 inhibition. Ba/F3GR cells were pretreated with the indicated concentrations of MEK inhibitor or DMSO (diluent control) for 1 hour and then stimulated with 100 ng/mL G-CSF. Lysates were prepared, and immunoblotting was performed using anti-phospho-ERK1/2 T202/Y204 antibody or anti-phospho-Akt S473. Blot was stripped and reprobed with antiactin antibody to demonstrate comparable protein loading.