Syntenin increases IL-5–mediated signaling. (A) TF-1 cells ectopically expressing syntenin and PDZ1+2 were starved overnight in 0.5% serum and stimulated for 15 minutes with IL-5 or GM-CSF. Cell lysates were prepared, proteins quantified, and subsequent Western blots assessed for phosphorylated ERK1/2 (pERK1/2), phosphorylated STAT5 (pSTAT5), and actin in the top panel. A representative example of 4 independent experiments is shown. The bottom panel shows a representative example of Western blots assessed for phosphorylated and total JAK2, and tubulin. (B) pERK1/2 and pSTAT5 levels of 4 independent experiments as outlined in panel A were quantified using ImageJ software. Mean integrated intensity ± SD is indicated. *P < .05; **P < .01. (C) EGFP or ectopic syntenin-expressing cells were starved overnight and stimulated with IL-5 for indicated time points. Protein lysates were quantified for protein amount, and Western blots were prepared in duplicate and analyzed for pERK1/2 and total ERK levels. A reference sample (R) was included to facilitate comparison between blots; a lower amount of reference was loaded on the blots that were probed for total ERK. (D) Cells ectopically expressing EGFP, PDZ1+2, and syntenin were stimulated with IL-5 for indicated time points, and protein lysates were prepared and quantified. Subsequent Western blots were incubated with antibody recognizing phosphorylated PKB (pPKB) substrates and tubulin as loading control. One of 3 representative experiments is shown. (E) Cells ectopically expressing EGFP, PDZ1+2, and syntenin were cultured for 3 days in the presence of IL-5 and an inhibitor (U0126) of MEK1/2, the upstream kinases of ERK1/2. Viable cell numbers are depicted (average ± SD), and were determined by flow cytometry in 3 independent experiments.