Fig. 7: The interaction of KRT32 with NEMO promotes NEMO degradation via K48-linked polyubiquitination modification and also inhibits the formation of the IKK complex.

KRT32 expression level does not affect the transcription of IKBKG (the gene encoding NEMO) in Ker-CT cells. The mRNA level of NEMO in Ker-CT cells overexpressing KRT32 detected using RNA-seq (A) and in KRT32 knockdown cells detected by RT-qPCR analysis (B). Data are means ± SEM of three independent experiments in (A) and (B), and P value was calculated using a two-sided unpaired Student’s t test. C, D Immunoblotting of NEMO in Ker-CT cells with overexpressing KRT32 and knockdown with and without TNF treatment. E The impact of KRT32 wildtype and mutations overexpression on NEMO protein level in Ker-CT cells analyzed by Western blot. F Cycloheximide (CHX) chase assay to analyze the protein stability of NEMO in Ker-CT cells overexpressing KRT32. Cells were treated with 50 µg/ml CHX for indicated time. G The levels of NEMO were measured by western blotting with 5 μM MG132 in Ker-CT cells overexpressing KRT32 wildtype or negative control for 12 h. H Co-immunoprecipitation of HA-KRT32 and Flag-NEMO in HEK 293T cells, followed by detection of ubiquitin levels of NEMO by Western blotting. I Co-transfection of Flag-NEMO with HA-Ub-WT, HA-Ub-K48 or K48R (containing lysine at residue K48 or lysine to arginine mutation at residue K48) along with Myc-KRT32 in HEK 293 T cells, followed by immunoprecipitation and Western blotting analysis. J Immunoprecipitation of lysates with NEMO antibody from Ker-CT cells overexpressing KRT32 wildtype or mutations, and then detect the Ub-K48 modification of NEMO. PLA analysis in Ker-CT cells overexpression KRT32 wildtype and mutations to assess the interaction of Ub with NEMO (K) and IKKα with NEMO (M). Scale bars represent 10 μm. L, N Quantification of the number of PLA foci per cell detected in (K) and (M), separately. Each dot on the graph corresponds to a specific analyzed cell. Red bars represent the mean ± SEM from the indicated number (N) of cells. The number of cells analyzed per group varies as follows: N = 315, 302, 302, 310, 302, 315, 313, 311 in (L) and N = 243, 211, 214, 220, 217, 214, 205, 208 in (N), with each group consisting of three biological replicates. P value was calculated using a two-sided unpaired Student’s t test. O GST-NEMO (120-419aa) fusion protein was incubated with excess E. coli extracts containing His-KRT32 (wildtype or six mutants), His-IKKα, and His-IKKβ. GST complex was pulled down with glutathione-Sepharose beads, and the protein complexes were analyzed by western blotting. P Statistical analysis was performed on the binding ability of IKKα/β and NEMO with wildtype KRT32 and its mutations addition from tree-independent experiments of (O). Data shown as means ± SEM of three independent experiments in (P). P value was calculated using a two-sided unpaired Student’s t test. Q Immunoblotting assay of phosphorylated IKKα/β in Ker-CT cells overexpressing KRT32 wildtype and mutations. One representative experiment from two independent experiments with similar results is shown in (C–G) and (J, Q). One representative experiment from three independent experiments with similar results is shown in (O). Source data are provided as Source Data file.