Anti IKK E, phospho Thr501  

IFI44 decreases the kinase activity of IKKβ and IKKε. Human 293T cells were silenced for IFI44, or for FKBP5, and were transfected with plasmids expressing His-IKKε (A) or MYC-IKKβ (B), together with IFI44-HA, and FKBP5-FLAG expression plasmids. At 24 hpt, IKKε (A) and IKKβ (B) complexes were purified with anti-His and anti-MYC antibodies, respectively, and these complexes were assayed in kinase assays using IRF-3 (for the IKKε complexes shown in panel A) and IkBα (for the IKKβ complexes shown in panel B) as substrates. The levels of phosphorylated and unphosphorylated forms of IRF-3 (panel A, bottom blot) and IkBα (panel B, third and fourth blots) were analyzed by Western blotting using specific antibodies. Levels of IKKε were analyzed using an anti-His-specific antibody (A, first blot) and anti-pIKKε (A, second blot), and levels of IKKβ were analyzed using an anti-MYC-specific antibody (B, first blot) and anti-pIKKβ (B, second blot). Western blots were quantified by densitometry using ImageJ software (v1.46). Protein expression levels in cells expressing IKKε (A) and IKKβ (B) alone were assigned a value of 100% for comparisons with the levels of expression in cells expressing the different combinations of IKKε/IFI44/FKBP5 (A) or IKKβ/IFI44/FKBP5 (B) (numbers are indicated below each plot). pIRF-3 and IRF-3 levels (observed in the same bottom blot in panel A) and pIkBα and IkBα (third and bottom blot in panel B) are represented with numbers below each blot. Levels of pIRF-3 and pIkBα normalized to the levels of IKKε and IKKβ are represented in the bottom graphs in panels A and B, respectively. Molecular weight markers are indicated (in kilodaltons) on the right. Figure provided by CiteAb. Source: MBio, PMID: 31455651.