American Society of Hematology

Fig. 6.

$Fig. 6. GM-CSF increases the cytosolic accumulation and phosphorylation of Bad in neutrophils. / Human neutrophils were incubated for 30 minutes in the presence or absence of GM-CSF (10 ng/mL) with or without LY294002 (10 μM). Neutrophil lysates were prepared, and total Bad and phospho Ser-112 and Ser-136 Bad were quantified by Western blotting of cytosolic and membrane fractions, as detailed in the “Materials and methods” section. Cytosolic immunoprecipitates and blots were performed using cell lysates prepared with hypotonic lysis buffer containing 0.05% NP-40 and centrifuged at 22 000g (20 minutes, 4°C); detergent was omitted from the initial lysis buffer used to prepare the membrane fractions that were subsequently pelleted at 104 000g (30 minutes, 4°C) and resuspended in buffer containing 0.1% Triton-X100. (A) Representative immunoblots (with corresponding densitometry) demonstrating that GM-CSF increased the amount of total immunoreactive Bad within the cytosolic compartment of neutrophils and caused a reciprocal loss in membrane associated Bad. Both effects were inhibited by LY294002. (B, C) Phosphorylation status of cytosolic Bad was assessed using phosphospecific antibodies to Ser112 and Ser136. Representative immunoblots and densitometry analyses indicate that GM-CSF substantially increased the phosphorylation of cytosolic Bad at Ser112 and Ser136 through a PI3-kinase–dependent mechanism. Identical data were obtained in 3 further independent experiments.$

GM-CSF increases the cytosolic accumulation and phosphorylation of Bad in neutrophils.

Human neutrophils were incubated for 30 minutes in the presence or absence of GM-CSF (10 ng/mL) with or without LY294002 (10 μM). Neutrophil lysates were prepared, and total Bad and phospho Ser-112 and Ser-136 Bad were quantified by Western blotting of cytosolic and membrane fractions, as detailed in the “Materials and methods” section. Cytosolic immunoprecipitates and blots were performed using cell lysates prepared with hypotonic lysis buffer containing 0.05% NP-40 and centrifuged at 22 000g (20 minutes, 4°C); detergent was omitted from the initial lysis buffer used to prepare the membrane fractions that were subsequently pelleted at 104 000g (30 minutes, 4°C) and resuspended in buffer containing 0.1% Triton-X100. (A) Representative immunoblots (with corresponding densitometry) demonstrating that GM-CSF increased the amount of total immunoreactive Bad within the cytosolic compartment of neutrophils and caused a reciprocal loss in membrane associated Bad. Both effects were inhibited by LY294002. (B, C) Phosphorylation status of cytosolic Bad was assessed using phosphospecific antibodies to Ser112 and Ser136. Representative immunoblots and densitometry analyses indicate that GM-CSF substantially increased the phosphorylation of cytosolic Bad at Ser112 and Ser136 through a PI3-kinase–dependent mechanism. Identical data were obtained in 3 further independent experiments.

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