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. Author manuscript; available in PMC: 2014 Mar 15.
Published in final edited form as: ACS Chem Biol. 2012 Dec 11;8(3):578–587. doi: 10.1021/cb300568r

Figure 2. TCH-013 causes the accumulation of ubiquitinylated proteins and inhibits the degradation of IκBα.

Figure 2

(A) Representative photos showing fluorescent confocal images of HeLa cells treated with either vehicle, or 1 μM TCH-013 for two hours and subsequently stained for the presence of ubiquitinylated proteins (red) and counterstained for DNA with DAPI (blue). Fluorescent intensity of the ubiquitinylated proteins obtained from each treatment were measured using Olympus FV1000 software (n=6, p=0.0291). (B) (top panel) PRMI-8226 cells were left untreated or treated with either vehicle, 1 μM bortezomib or 10 μM TCH-013 for thirty minutes and then stimulated with 10 ng/mL TNF-α. Whole cell extracts were probed for the presence of unmodified IκBα using a monoclonal antibody against the N-terminal of IκBα (L35A5). (middle panel) The same whole cell extracts were probed for the presence of unmodified and modified IκBα using a polyclonal antibody against the C-terminal of IκBα (C-21). The presence of actin was used as a loading control on the same blot. (C) HeLa cells were left untreated or treated with either vehicle, 1 μM MG-132 or 10 μM TCH-013 for thirty minutes and stimulated with 10 ng/mL TNF-α. (input) Whole cell extracts from the samples were probed for the presence of ubiquitin using a monoclonal antibody directed toward ubiquitin (P4D1) that detetects ubiquitin, polyubiquitin and ubiquitinylated proteins. The presence of actin was used as a loading control. (bottom panel) IκBα was immunoprecipitated from the whole cell extracts using a polyclonal antibody directed toward the C-terminal of IκBα (C-21). The IκBα immunoprecipitates were probed for the presence of ubiquitinylated proteins. (D) Representative photos showing fluorescent confocal images of HeLa cells treated with either vehicle or 1 μM TCH-013 and either stimulated with 10 ng/mL TNF-α or left untreated. The cells were fixed and subsequently stained for the presence of IκBα (red) and counterstained for DNA with DAPI (blue). (n=6, p=0.0001).