Decreased IL-5R functions upon knockdown of syntenin in TF-1 cells. (A) Scrambled control siRNa and syntenin directed siRNA were electroporated into TF-1 cells using an Amaxa system. TF-1 cells recovered after 24 hours in the presence of IL-3 (cells were ∼ 90% trypan blue negative), and were starved for 4 hours before cells were stimulated with IL-5 for 15 minutes. Cells were lysed in Laemmli buffer, proteins content was quantified, and Western blots were stained for syntenin and tubulin as loading control. (B) pERK1/2 levels (± SD) were assessed by quantifying Western blot from 3 independent experiments as described in panel A using ImageJ software. (C) TF-1 cells were transduced with bicistronic constructs that express nonfunctional shRNA (control) or syntenin-targeting shRNA in combination with EGFP. EGFP cells were sorted beyond 95% purity and syntenin levels were assessed by Western blot. (D) Control and syntenin-targeting shRNA-expressing TF-1 cells were cultured for 3 days in the presence of IL-5 or GM-CSF. Viable cell numbers were determined by flow cytometry. Results depicted are from 2 experiments with 4 samples per group per experiment (± SD). *P < .05.