Fig. 5

CRYAB interacts with IKKβ and IKKα and inhibits IKKβ activity. a CRYAB inhibited IKKβ-driven p65 phosphorylation, IκBα phosphorylation, and degradation. HCT116 and SW480 cells were transfected with control vector PRK7, Flag-IKKβ, or Flag-CRYAB plasmids, respectively. After 24 h of culture, the cells were lysed and immunoblotted with anti-FLAG, p65, p-p65, IκBα, p-IκBα, and p-IKKβ, respectively. b CRYAB inhibits the IKKβ activity. HT29 cell was treated with TNF-α (10 μg/ml) or control for 6 h in the absence or presence of pretreatment of TAT-CRYAB recombined protein (5 μg/ml) for 12 h. Specific IKKβ activity was then measured using a peptide substrate and a small molecular IKKβ inhibitor; *p < 0.05, **p < 0.01. c, d Co-immunoprecipitation of IKKβ and CRYAB. HCT116 cells were transfected to express HA-CRYAB and Flag-IKKβ. Cell lysates were immunoprecipitated with anti-HA beads and blotted with anti-FLAG antibody or immunoprecipitated with anti-FLAG beads and blotted with anti-HA antibody. e, f Co-immunoprecipitation of IKKα and CRYAB. Cell lysates were immunoprecipitated with anti-HA beads and blotted with anti-FLAG antibody or immunoprecipitated with anti-FLAG beads and blotted with anti-HA antibody. g, h CRYAB inhibits the interaction between IKKα and IKKβ. HCT116 cells were co-transfected with plasmids expressing Flag-IKKβ, HA-IKKα, and HA-CRYAB. Co-immunoprecipitation of expressed Flag-IKKβ and HA-IKKα was significantly inhibited compared with the cells without HA-CRYAB and vice versa. All data are shown from three independent experiments