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. 2018 Jun 14;293(31):11996–12010. doi: 10.1074/jbc.RA117.001201

Figure 5.

Figure 5.

Parkin affects TRAF3 stability. A, overexpression of Parkin reduced the abundance of exogenous TRAF3 protein. HEK293 cells were transfected with Flag–TRAF3 and EGFP–Flag together with empty vector or Parkin–Myc plasmids. EGFP–Flag was used to determine transfection efficiency. 24 h after transfection, the cell lysates were analyzed by immunoblotting with the indicated antibodies. B and C, Parkin overexpression enhanced TRAF3 degradation in a dose-dependent manner with co-expression of IKKi or TBK1. HEK293 cells were co-transfected with TRAF3 and IKKi (B) or TBK1 (C) together with empty vector or increased dose of Parkin–Myc plasmids. 24 h after transfection, the cell lysates were analyzed by immunoblotting with the indicated antibodies. D, overexpression of Parkin shortened the half-life of endogenous TRAF3 in HEK293 cells. HEK293 cells were transfected with the indicated expression plasmids. 24 h after transfection, the cells were treated with CHX (final concentration, 50 μg/ml) for the indicated times. The cell lysates were analyzed by immunoblotting with the indicated antibodies. Densitometry analysis to quantify TRAF3 expression is shown in the right panel. E, Parkin deficiency enhanced TRAF3 protein stability in primary Parkin−/− MEFs. Primary Parkin+/+ and Parkin−/− MEFs were infected with SeV for 12 h. The cell lysates were analyzed by immunoblotting with the indicated antibodies. F and G, TRAF3 protein was more stable in Parkin−/− immortalized MEFs. Immortalized Parkin+/+ and Parkin−/− MEFs were infected with SeV (F) or VSVΔM51-GFP at MOI of 0.5 (G) for 6 h. The cell lysates were analyzed by immunoblotting with the indicated antibodies. H, the treatment of MG132, but not CQ, affected the stability of TRAF3 protein in Parkin+/+ immortalized MEFs. Immortalized Parkin+/+ and Parkin−/− MEFs were left untreated or treated with MG132 (final concentration, 25 μm) or CQ (final concentration, 25 μg/ml) for 10 h. The cell lysates were analyzed by immunoblotting with the indicated antibodies. I, autophagy inhibitor 3-MA failed to affect the stability of TRAF3 protein in Parkin+/+ immortalized MEFs. Immortalized Parkin+/+ and Parkin−/− MEFs were left untreated or treated with MG132 (final concentration, 25 μm) or 3-MA (final concentration, 5 mm) for 9 h. The cell lysates were analyzed by immunoblotting with the indicated antibodies. Numbers below lanes (top) indicate densitometry of the protein presented relative to GAPDH expression in the same lane.