Deletion of FAK in BMM and neutrophil cells results in reduced proliferation, survival, and activation of antiapoptotic proteins. (A) Cre-mediated Fak deletion in BMMs: after 1 month of poly (I):(C) injection, BM cells from WT and Fak^−/− mice were cultured for 7 days, and DNA was extracted. Lanes 1 and 2 represent WT Fak bands, and lanes 3 and 4 represent FAK deleted bands. (B) Western blot analysis demonstrating the expression of Fak in WT and Fak^−/− cells. Equal amount of cell lysates were subjected to Western blot analysis. Blot was probed with an antibody specific to FAK (raised against amino acids 903-1052). The same blot was reprobed for β-actin to show equal loading. (C) Cells were subjected to proliferation assay in the absence of growth factors as well as in the presence of indicated concentrations of M-CSF. After 48 hours of culture, cells were pulsed with [³H] thymidine for 6 hours. Concentrations of M-CSF are shown on the x-axis, and mean thymidine incorporation (in cpm) are shown on the y-axis. Shown are results from 1 of 3 independent experiments performed in quadruplicate; *P < .05. (D) Expression of F4/80 on WT and Fak^−/− cells. Cells were stained with PE-conjugated anti-F4/80 antibody and subjected to flow cytometric analysis. Solid histograms indicate the level of F4/80 expression on the surface of WT and Fak^−/− cells, while open histograms indicate the level of expression using an isotype control antibody. (E) BM cells grown in G-CSF were subjected to proliferation assay in the absence of growth factors and in the presence of indicated concentrations of various cytokines. After 48 hours of culture, cells were pulsed with [³H] thymidine for 6 hours. Concentrations of various cytokines are shown on the x-axis, and mean thymidine incorporation (in cpm) is shown on the y-axis. Bar graph shows data from 1 experiment performed in replicates of 4 (*P < .05). Similar findings were observed in 3 additional independent experiments. (F) BM cells grown in G-CSF derived from WT and FAK-deficient mice were starved of growth factors for indicated times and subjected to flow cytometric analysis after staining with Annexin V and 7-AAD. The percentages of early and late apoptotic cells at each time point are indicated. Shown is a representative dot blot from 1 of 3 independent experiments. (G) WT and Fak-deficient cells grown in the presence of IL-3 and G-CSF were starved of growth factors and stimulated for 5 minutes with G-CSF. Upper panel shows the activation of AKT as assessed by Western blot analysis using an anti–phospho AKT antibody. Bottom panel shows total AKT levels in each lane. (H) Actively growing neutrophils derived from WT and Fak^−/− mice were subjected to Western blot analysis using an anti–caspase 3 (upper panel), anti–Bcl-x_L (middle panel), and anti–β-actin (bottom panel) antibody. Similar results were observed in 2 additional independent experiments.