G-CSF inhibits the degradation of XIAP in neutrophils. Neutrophils were incubated in the absence or presence of G-CSF, cycloheximide (CHX), G-CSF + CHX (G + CHX), the calpain inhibitor CI3 (20 μM), the proteasome inhibitor MG132 (50 μM), the caspase inhibitor z-VAD (20 μM) or the Ca²⁺chelator BAPTA (5 μM) for 16 hours. After incubation, whole-cell lysates were prepared, run on SDS-PAGE gel and analyzed by Western blot for XIAP expression (A,B). indicates full-length XIAP (57 kDa) and its cleavage products (∼30 kDa and ∼20 kDa). The antibody is directed against a region at the C-terminal site of the protein, including the BIR3 domain. The blot is representative of 3 independent experiments. For panel B, the blot was reprobed to detect caspase-3. Survival was determined by annexin V staining on the flow cytometer (C). Data are expressed as a percentage (± SEM) of annexin V negative cells and represent 4 different experiments performed in duplicate. Significance was determined relative to the control (20 hours, no inhibitors) for all samples except G-CSF + CHX, which was compared with G-CSF, by paired, 1-tailed Student t test. *P < .05, **P < .001, and n.s. (not significant), P > .05.