Figure 4
GSK-3β-mediated phosphorylation induces NEMO stabilization.
(a) The lysates from cells transfected with NEMO constructs were immunoblotted (IB) with the antibodies indicated. β-Actin was used as a loading control. Quantitative analysis (lower panel) of NEMO. (Normalized to β-actin, the ratio at control cells was set as 1.) (b) Cells were lysed 48 h after transfection and immunoblotted (IB) with anti-NEMO. β-Actin was used as a loading control. Quantitative analysis (lower panel) of NEMO. (Normalized to β-actin, the ratio at control cells was set as 1.) (c,d) The lysates from cells transfected with NEMO constructs were immunoblotted (IB) with the antibodies indicated. Quantitative analysis (lower panel) of NEMO. (Normalized to Vinculin, the ratio at control cells was set as 1.). Vinculin was used as a loading control. (e, f) NEMO protein turnover was evaluated by cycloheximide half-life experiments. Immunoblot analysis of whole-cell lysates of HEK293 cells transfected with vectors encoding GFP-tagged wild-type or mutant NEMO and treated with cycloheximide (CHX) (50 μM) for 0–24 h. (g) HEK293 lysates of cells transfected with wild-type NEMO or with the S8A, S17A, S31A mutant of NEMO in combination with various HA-tagged ubiquitin species were subjected to anti-GFP immune-precipitation followed by anti-ubiquitin, anti-P47 and anti-NEMO immunoblotting (IB). Quantitative analysis (lower panel) of P47. (Normalized to NEMO, the ratio at control cells was set as 1.) (h) HEK293 cells were transfected with wild-type NEMO or with the S8, 17, 31A mutant of NEMO. After 48 h of incubation, lysates from TNFα-stimulated (10 ng/ml) cells were immunoprecipitated (IP) with anti-GFP, followed by immunoblotting (IB) with anti-TRAF6 and anti-RIP1. Quantitative analysis (lower panel) of RIP1 and TRAF6. (Normalized to immunoprecipitated NEMO, the ratio at control cells was set as 1).
