Figure 7.

Parkin inhibits TRAF3 function in E3 ligase–independent manner. A–C, overexpression of Parkin (WT) or Parkin (C431S) showed similar inhibition in SeV-induced activation of the IFN-β, NF-κB and ISRE promoters. HEK293 cells were co-transfected with the indicated expression plasmids and luciferase reporter constructs driven by promoter of gene encoding IFN-β (A), NF-κB (B), or ISRE (C). pRSV/LacZ was used as an internal control. 24 h after transfection, the cells were infected with SeV for 24 h and then lysed for luciferase assays. D, overexpression of Parkin (WT) or Parkin (C431S) showed no different inhibition in SeV-induced transcription of the IFNB1 gene. HEK293 cells were transfected with the indicated expression plasmids. 24 h after transfection, the cells were infected with SeV for 6 h, and cell lysates were analyzed by qRT–PCR to examine IFNB1 transcript levels. E, overexpression of Parkin (WT) or Parkin (C431S) showed similar enhancement of TRAF3 degradation in a dose-dependent manner with co-expression of TBK1. HEK293 cells were transfected with the indicated expression plasmids. The cell lysates were analyzed by immunoblotting with the indicated antibodies. F, overexpression of Parkin (WT) or Parkin (C431S) showed similar increased Lys⁴⁸-linked ubiquitination of exogenous TRAF3. HEK293 cells were transfected with Flag–TRAF3 and HA–Ub–Lys⁴⁸ together with Parkin–Myc or Parkin (C431S)–Myc plasmids. 24 h after transfection, the cells were infected with SeV for 24 h and treated with MG132 (final concentration, 25 μm) for 4 h. The cell lysates were immunoprecipitated with anti-Flag beads and analyzed by immunoblotting with the indicated antibodies. Bottom panel, expression of exogenous proteins in WCL. The data in A–D are from a representative experiment of at least three independent experiments (means ± S.D. of duplicate assays in A-C; triplicate assays in D). Two-tailed Student's t test was used to determine statistical significance. *, p < 0.05; **, p < 0.01; ***, p < 0.001, versus control groups. S. E, short exposure; L. E, long exposure; Rel. Luc. Act, relative luciferase activity.