Fig. 3: Functional analyzes on the YDDΦxΦ docking interaction.

a In vitro kinase activity experiments using purified full-length 6xHis-IκBα proteins and endogenous IKK immunoprecipitated from HEK293T cells. Aliquots of the reactions were taken at the indicated time points and immunoblotted using anti-His (IκBα), anti-phospho-Ser32/Ser36 IκBα (pS-IκBα) and anti-IKKβ (IKKβ) antibodies. Samples were run on two separate 10% SDS-PAGE gels for detection of total IκBα and pS-IκBα. (Left panel) Representative Western blot images. (Right panel) Quantification of pS-IκBα levels normalized to IκBα at 30 min (100%). The data (mean +/− SD) are obtained from three independent kinase activity experiments. The indicated P-values (p) are obtained from a two-tailed unpaired t-test, n = 3 biological triplicates. Black: differences between IκBα YD/SS and IκBα wt; red: differences between IκBα C308L/F and IκBα YD/SS. b In cellulo degradation of retro-transduced IκBα proteins in MEF cells KO for IκBα, IκBβ, IκBε. Cells were stimulated with TNFα (20 ng/ml) and collected at the indicated time points. Cellular extracts were immunoblotted using anti-IκBα (IκBα) and phospho-specific p65 (P-p65) antibodies. IκBα SS/AA: IκBα S32A/S36A. (Upper panel) Western blot images. Each IκBα mutant was migrated together with wt IκBα on the same gel (see Source Data file). (Lower panel). Quantification of IκBα levels normalized first to tubulin and, then, to the IκBα values at time 0 (100%). The data (mean +/− SD) are obtained from three measurements, with bars reporting on the quantification error. Similar IκBα degradation profiles were obtained in a second independent experiment. c Detection of IκBα tyrosine phosphorylation in HEK293T cells. Cells were transfected with the indicated HA-tagged IκBα constructs, incubated, or not, with pervanadate (1 mM) for 30 min and stimulated, or not, with TNFα (10 ng/ml) for 15 minutes before collection. Cleared lysates were immunoprecipitated with anti-HA beads, and immunoblotted with anti-phospho-Tyr (pY-IκBα and pY-IκBαΔ73) and anti-HA (IκBα and IκBαΔ73) antibodies. The results were reproduced in a second independent experiment. d ITC data on the IKKα interaction with synthetic phospho-Tyr305 IκBα pep (pY-IκBα pep). e Competition MBP-pulldown experiment using 6xHis-IKKα(10-667) EE, MBP-IκBα pep coupled to amylose resin and an excess of synthetic IκBα pep or pY-IκBα pep peptides. IKKα : (pY-)IκBα pep stoichiometric ratios: 1:100 ( + ), 1:200 ( + + ); 1:500 (+++). Samples were migrated on a 10% SDS-PAGE gel and stained by Coomassie. These results were reproduced in a second independent pulldown experiment. Source data for this figure are provided as a Source Data file.