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rhIL-5 primed eosinophils (5 × 10⁵) were incubated in complete medium at 37°C with mAb 2C4 or IgG1 control for various time points. Next, 2x Laemmli buffer (Bio-Rad, Hercules, CA) was used to isolate total protein from cell lysates, which were then electrophoresed through mini-PROTEAN TGX precast gels and transferred to Immun-Blot low fluorescence PVDF membranes using the Trans-Blot SD semi-dry transfer cell (Bio-Rad), according to the manufacturer’s guidelines. After blocking with Odyssey Blocking Buffer (TBS) (LI-COR, Lincoln, NE), membranes were incubated overnight with various monoclonal or polyclonal antibodies (1:500 – 1:1000 in 5% BSA in TBS-T): phospho-Akt, Akt, phospho-p38, p38, phospho-JNK/SAPK, JNK/SAPK (Cell Signaling). Specific binding of these antibodies was detected with infrared dye (IRDye) 680RD or 800RD-conjugated secondary antibodies (LI-COR) using the Odyssey Imaging System (LI-COR).

Membrane and tissue homogenates were prepared as previously described [4 (link), 39 (link)]. Briefly, cells were incubated in the presence or absence of SDF-1 and/or MAPK signaling inhibitors; e.g., the p38 (SB203580; 10 μM), for 24 h. Cells were solubilized in lysate buffer (Cell Signaling Technology, Beverly, MA), supplemented with protease inhibitor cocktail (Sigma, St. Louis, MO). The homogenates were centrifuged and protein concentrations in the supernatants were determined by the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA). Equal amounts of protein were loaded onto wells and separated by 12% SDS-PAGE and transferred to Immobilon-P transfer membranes (Millipore, Bedford, MA). The membranes were incubated in blocking buffer for 1 h and incubated with antibodies to caspase 3, BAX, P38, JNK/SAPK, phospho-P38, phospho-JNK/SAPK, GAPDH (Cell Signaling Technology, Beverly, MA), Phospho-MAPKAPK2 and MAPKAPK2 (Abcam, Cambridge, MA) overnight at 4 °C. The membranes were washed and incubated in secondary HRP conjugated antibodies, 1:2000 dilutions (Cell Signaling Technology Beverly, MA). Immunocomplexes were visualized with enhanced chemiluminescence and autoradiographs were analyzed by laser densitometry. Three independent experiments were performed; the groups represent the mean of three separate experiments.