Effect of L353A mutation in the ULD of IKKβ on structural stability or degradation of IKKβ.

(A) Effect of different HA-IKKβ mutations on IKKβ-induced NF-κB activation in terms of NF-κB luciferase activity. HEK293 cells were transfected with NF-κB reporter and HA-IKKβ mutants. After 18 h, luciferase activities were measured. (B) Effect of HA-IKKβ mutants on IKKβ autophosphorylation. HEK293 cells were transfected with HA-IKKβ mutants and phosphorylation of IKKβ was detected by western blot analysis. (C) Effect of HA-IKKβ on IKKβ-induced IκBα phosphorylation. HEK293 cells were transfected with HA-IKKβ mutants together with HA-IκBαRR, and phosphorylation of IκBα was detected by western blot analysis. (D) Role of L353A mutation in HA-IKKβ translocation into detergent insoluble or insoluble fractions of HEK293 cells. Cells were transfected with wild type or mutated HA-IKKβ(L353A), and expression levels were investigated in total cell lysates, detergent soluble and insoluble fractions. (E) Effect of L353A mutation on IKKβ degradation in different fractions. HEK293 cells were transfected with wild type or mutated HA-KKβ(L353A) mutant. After incubation with 10 μg/ml CHX, expression levels were investigated in total cell lysates, detergent soluble and insoluble fractions. (F) Autophagy mediates degradation of L353A mutant. HEK293 cells transfected with HA-IKKβ or HA- KKβ(L353A) mutants were incubated with CHX in the presence or absence of MG132 and chloroquine. * P < 0.05, ** P < 0.01.