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. 2016 Dec 8;6:38553. doi: 10.1038/srep38553

Figure 5. GSK-3β/IKKγ interaction modulates the NF-κB pathway.

Figure 5

(a) HEK293 cells were transfected with wild-type NEMO or with the S8, 17, 31A mutant of NEMO. The lysates (CL) were assayed for protein expression before immunoprecipitation as indicated. (b) Lysates from TNFα-stimulated (10 ng/ml) cells were immunoprecipitated (IP) with anti-GFP, followed by immunoblotting (IB) with anti-NEMO, anti-IKKα, anti-IKKβ and anti-GSK-3β. Part of the immunoprecipitation was examined by native gel electrophoresis (upper band) to detect the dimerization of NEMO. (c) HEK293 cells were transfected with wild-type NEMO or with the S8, 17, 31A mutant of NEMO. The lysates (CL) were assayed for protein expression as indicated. The lysates were immunoprecipitated (IP) with anti-IKBα followed by immunoblotting (IB) with anti-ubiquitin. (d) After 2 h pretreatment with SB216763 (1 μM), cells were stimulated with TNFα (10 ng/ml) for 1 h. Lysates were immunoprecipitated (IP) with anti-NEMO and were followed by immunoblotting (IB) with anti-NEMO, anti-IKKβ, and anti-GSK-3β. (e) After 2 h pretreatment with SB216763 (1 μM), cells were stimulated with TNFα (10 ng/ml) for 1 h. Lysates were immunoprecipitated (IP) with anti-IĸBα and were followed by immunoblotting (IB) with anti-Ubiquitin and IĸBα.