Regulation of Interleukin-10 Receptor Ubiquitination and Stability by Beta-TrCP-Containing Ubiquitin E3 Ligase
Figure 6
Inhibition of βTrCP-mediated IL-10R1 degradation leads to increased cellular responsiveness to IL-10.
(A and B): 293T cells stably expressing IL-10R1 were transfected with shCon or shBTR1/2 (A); 293T cells were transiently transfected with WT or 3SA IL-10R1 (B). Cells were treated with 5 ng/ml of IL-10 for indicated times and the levels of pStat3, Stat3, IL-10R1-Flag were determined by IB. The bands representing the mature form of IL-10R1 were denoted by arrow heads in the Flag panels. In (A), the levels of another βTrCP substrate, Cdc25A, were used to show the effectiveness of βTrCP knockdown. (C) Raw264.7 cells were transfected with pcDNA, WT or Ser320, 24, 67A (3SA) mIL-10R1, together with the M67-luciferase reporter construct and the pCMV-renilla luciferase control. Cells were treated with IL-10 (black bars) or without (open bars) for 6 h and samples were subjected to dual-luciferase activity assay. Cells were transfected in triplicates. Average Stat3 reporter activities represented by the relative ratios of firefly over renilla luciferase activities (fLuc/rLuc) were shown on the graph, with error bars showing standard deviations. Comparison was made between IL-10-treated WT versus 3SA groups and the p value was shown. Data shown are a representative from two independent experiments.