Fig. 6: MAP3K13-232 directly binds to the kinase domain of IKKα.

A Proteins interacting with MAP3K13-232aa were identified by immunoprecipitation followed by mass spectrometry (IP-MS). A total of 2305 proteins were pulled down by anti-flag antibody from lysates of cells transfected with circMAP3K13 or empty vector using an anti-FLAG antibody. Of these, 435 proteins were unique to the circMAP3K13 group. Differentially abundant proteins (fold change >2) were subjected to KEGG pathway enrichment analysis. B Fifty-three proteins with a fold change >13, including the IKKα, with fold changes >13 were enriched in cancer-related pathways; several enriched pathways identified in the enrichment analysis are shown. C The interaction between MAP3K13-232aa and IKKα was modeled using Alphafold3. IKKα is shown in green, and the purple structure is MAP3K13-232aa. Chemical interactions were visualized using PyMOL. Yellow dotted lines represent hydrogen bonds, red dotted lines indicate hydrophobic interactions, and blue dotted lines denote salt bridges. D Co-immunoprecipitation experiments were performed to detect the interaction between MAP3K13-232aa and IKKα. E Schematic of the five GST-tagged IKKα recombinant protein constructs used in the GST pull-down assay: GST-WT (full-length recombinant GST-IKKα); GST-IKKα-ΔNEMO (lacking the NEMO-binding region); GST-IKKα-ΔKinase (kinase domain of GST-IKKα was deleted); GST- IKKα-Kinase (only the kinase domain); GST-IKKα-NEMO (only included the NEMO-binding region of GST-IKKα and lacked the kinase domain, lacking the kinase and leucine zipper regions). F Five “GST-tagged constructs and a His-tagged MAP3K13-232aa construct were expressed in E. coli BL21(DE3). Purified proteins were incubated to form immunocomplexes, followed by western blotting using anti-GST and anti-His antibodies. G AGS cells were transfected with ATG-mutation, circMAP3K13, wild-type circMAP3K13, empty vector (PCDH), linear MAP3K13-232aa, or kinase-dead MAP3K13-232aa. Whole-cell lysates were collected and subjected to immunoprecipitation for kinases enrichment. In vitro kinase assay was performed. Upper panel: IP FLAG; middle and lower panels: IP IKKα. Substrates used were IκB for IKKα and MBP for MAP3K13-232aa. Unless otherwise specified, statistical analysis was performed using a two-tailed Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001.