Figure 3.
FBXL19 mediates the IL-33-induced degradation of ST2L via the ubiquitin-proteasome system. (a,b) Immunoblot analysis of ST2L and β-actin in lysates of MLE12 cells given no pretreatment (top) or pretreated with leupeptin (100 μM; middle) or MG-132 (20 μM; bottom) and then treated with various concentrations (below lanes) of IL-33 (a), and densitometry analysis of band intensity in a (b). (c) Immunoassay of lysates of MLE12 cells treated for 0–2 h with IL-33 (10 ng/ml), assessed by immunoprecipitation with anti-ubiquitin and immunoblot analysis of precipitates (top) and input lysates (bottom) with anti-ST2 or β-actin. (d) Immunoprecipitation (as in c) and immunoblot analysis of ST2L, V5-tagged proteins and β-actin in lysates of MLE12 cells transfected for 72 h with plasmid encoding control or FBXL19-specific shRNA alone or with plasmid encoding FBXL19-V5, then left untreated or treated for 2 h with IL-33 (10 ng/ml). (e,f) Immunoblot analysis of ST2L, V5-tagged proteins and β-actin in lysates of MLE12 cells transfected as in d (Ctrl, control plasmid), then treated for 0–2 h with IL-33 (10 ng/ml; e), and densitometry analysis of band intensity in e (f). Data are from one experiment representative of three.