Figure 4. IκBα modified with hybrid SUMO-Ubiquitin chains is captured using Ubiquitin-traps.

(A) HEK293 cells were pre-treated with MG132 and stimulated with TNFα for 20 min. Cells were lysed in a buffer containing TUBE-hHR23A or GST used as a control. GST-captured material was eluted and submitted to IκBα immunoprecipitation. (B) Cells were treated as in (A) and lysed in a buffer containing TUBE-hHR23A. Captured material was eluted and submitted to IκBα or control immunoprecipitations. (C) Cells were treated with MG132 and stimulated with TNFα for the indicated time. Cells were lysed in a buffer containing TUBE-hHR23A. Captured material was eluted and submitted to IκBα immunoprecipitation. (D) Cells were treated as in (C) and lysed in a buffer containing TUBE-hHR23A. Captured material was eluted and submitted to IκBα, ubiquitin or SUMO2/3 immunoprecipitations.