Figure 6. SUMO-2 and Ubiquitin promote efficient chain extension on IκBα.

(A) Strategy used to make the different fusions proteins. (B) HEK293 cells were co-transfected with His6-ubiquitin and IκBα fusion-proteins as indicated. Cells were pre-treated with MG132 and stimulated 20 min with TNFα. His₆-ubiquitylated proteins were purified using denaturing conditions and Ni²⁺ chromatography. EV: Empty Vector. (C) HEK293 cells were co-transfected with His6-SUMO-2 and IκBα fusions protein as indicated. Cells were pre-treated with MG132 and stimulated 20 min with TNFα as in A. His₆-sumoylated proteins were purified using Ni²⁺ chromatography procedure. (D) In vitro SUMOylation assay using IκBα WT or fusion proteins as substrates. (E) HEK293 cells were transfected as indicated, pre-treated with MG132 and stimulated 20 min with TNFα. Cells were lysed in a buffer containing 3.5 µM of TUBE hHR23A. TUBE-captured material was eluted and submitted to IκBα immunoprecipitation. EV: Empty Vector.