Figure 7. SUMO-2/3-Ubiquitin chains drive an efficient IκBα degradation by the 26S proteasome.

(A) (B) In vitro ubiquitylation, SUMOylation or mixed assays using IκBα WT (A) or S³⁵ IκBα WT (B) as substrates. Suboptimal conditions of conjugation were used in this assay (see materials and methods). (A) Western blot detection with the indicated antibodies. (B) Detection of radio-labelled material. (C) In vitro ubiquitylation, SUMOylation or mixed assays using S³⁵ IκBα WT as substrate in the presence (+) or absence (-) of 26S proteasome. Saturating conditions of conjugation were used in this assay (see materials and methods). Different Ubiquitin: SUMO-2/SUMO-3 molar ratios were tested as follows: lane 1 = 4∶0/0, lane 2 = 3∶0.5/0.5, lane 3 = 2∶1/1, lane 4 = 1∶1.5/1.5, lane 5∶0:2/2. Detection of radio labelled material. (D) In vitro ubiquitylation, SUMOylation or mixed assays using S³⁵ IκBα WT as substrate in the presence (+) or absence (−) of 26S proteasome. Replicated reactions using saturating conditions and the following ubiquitin: SUMO-2/SUMO-3 ratios: 4∶0/0 for lanes 1 and 2, 0∶2/2 for lanes 3 and 4 and 2∶1/1 for lanes 5 and 6. Phosphorimager quantification of modified forms of S³⁵ IκBα WT in the presence or absence of 26S (n = 5). Standard deviation is indicated in the histograms. (E). Seventy-two hours after transfection with control or Ubc9 siRNA (100 nM), HeLa cells were pre-treated with MG132, stimulated with TNFα and lysed in a buffer containing TUBE-hHR23A. TUBE-captured material was submitted to IκBα immunoprecipitation. After IκBα-IP, extracts were eluted with glycine 200 mM pH2.5, equilibrated at pH 7.5 and submitted to an in vitro proteasome-mediated degradation assay at the indicated times.