Upregulation of the B subunit depends on new protein synthesis. Neutrophils were cultivated for 2 hours in the presence (G-CSF ) or absence (Con) of G-CSF. Where noted, cycloheximide and actinomycin D (CHX/ActD) were added 30 minutes before the addition of G-CSF and included throughout the incubation. Cytosol and membrane fractions were prepared as described in the legend to Fig 1, and blots were probed with anti-B2 antibody (B) or with E-11 antibody against subunit E (E).