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. 2013 Jan 7;8(1):e53717. doi: 10.1371/journal.pone.0053717

Figure 3. RNF11 interacts with TBK1/IKKi and blocks their Lys63-linked polyubiquitination.

Figure 3

(A) 293T cells were transfected with 1 µg of RNF11-GFP, Flag-IKKi and Flag-TBK1. Co-IPs were conducted using anti-Flag for IP followed by immunoblotting with anti-GFP and anti-Flag. Immunoblotting was performed with lysates using anti-GFP, anti-Flag and anti-Actin. (B) 293T cells were transfected with poly(I:C) (20 μg) and co-IPs were performed with anti-RNF11 or isotype control IgG followed by immunoblotting with anti-IKKi (top panel) or anti-TBK1 (lower panel). Immunoblots were also performed with lysates using anti-IKKi, anti-RNF11, anti-TBK1 and anti-Actin. (C) 293T cells were transfected with empty vector, Flag-RNF11 or RNF11-GFP (1 µg) and HA-Ub-Lys63-only (500 ng). Cells were transfected again 24 h later with poly(I:C) (20 μg) and co-IPs were conducted the next day using anti-TBK1 (left panel) or anti-IKKi (right panel) followed by immunoblotting with anti-HA, anti-TBK1 (left panel) and anti-IKKi (right panel). Immunoblotting was performed with lysates with anti-Flag, anti-GFP and anti-Actin.