Figure 3.

E3 ligase RNF216 (ring finger protein 216) targets TLR8 for ubiquitination and degradation. (A) RNF216 associates with TLR8. HEK293 cells were transfected with an empty vector or TLR8 and subjected to His pulldown, and IB was performed for V5-TLR8, RNF216, or β-actin. (B) HEK293 cells were transfected with TLR8 (1 μg) or cotransfected with increasing amounts of RNF216, and IB was performed for V5-TLR8, RNF216, or β-actin 16 hours after transfection. IB is representative of three independent experiments. (C) Densitometric quantification of three independent experiments. The data represent mean ± SEM (n = 3). Adjusted P = 0.059 by Dunnett multiple comparisons test, one-way ANOVA compared with 0. (D) HEK293 cells were transfected with TLR8 (1 μg) or cotransfected with increasing amounts of RNF219, and IB was performed for V5-TLR8, RNF216, or β-actin 16 hours after transfection. (E) THP-1 cells were transfected with Dicer-substrate siRNA (dsiRNA) against RNF216 or a scrambled dsiRNA (scr), and IB was performed for RNF216, TLR8, or β-actin 72 hours after transfection. (F) THP-1 cells were transfected with dsiRNA against RNF216 or a scrambled dsiRNA (scr) and treated with CHX (40 μg/ml) for the indicated times, followed by IB for TLR8 or β-actin 72 hours after transfection.