The Human IL-22 Receptor Is Regulated through the Action of the Novel E3 Ligase Subunit FBXW12, Which Functions as an Epithelial Growth Suppressor

Figure 3

FBXW12 depletion with shRNA increases IL-22R protein levels and activity. (a) HeLa cells transfected with FBXW12-Myc (2 μg) were transiently transfected with shRNA clones 1–6 (Origene) designed to target FBXW12 (2 μg), with reduction in immunoreactive FBXW12. (b) Control or FBXW12 shRNA (clone #4, 2 μg) was cotransfected into HeLa cells with FBXW9, W20, and W22 V5-tagged plasmids with immunoblot for V5. (c) Endogenous IL-22R immunoblot of cell lysates from HeLa cells transfected with control or FBXW12-targeting shRNA (clone #4) in the setting of PBS or IL-22 treatment (60 ng/mL for 60 min). In (d), IL-22R densitometry is shown from 3 shRNA experiments ( by ANOVA for FBXW12 shRNA compared to each other group). (e) Cells transfected with indicated plasmids were treated with IL-22 (60 ng/mL) for the indicated times and lysates probed for activated (Tyr705 phosphorylated) STAT3 with densitometry graphed on the right.

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