Fig. 4: MIB1 interacts with FTO and mediates FTO protein degradation.

a Dual omics-based screening workflow was employed. For RNA-seq and Mass spectrometry analysis, n = 3 biological independent experiments. b The ten proteins associated with FTO were identified using mass spectrometry. n = 3 biologically independent experiments. c–f Immunoprecipitation (IP) and western blot analysis were conducted to validate the interaction between FTO and MIB1, HERC2, and HUWE1 proteins in U87MG cells. n = 3 biological independent experiments. g, h Schematic diagrams of the MIB1 and FTO fragments used in the immunoprecipitation experiments shown in i, j. i, j The IP assay was used to map the interaction domain between MIB1 and FTO proteins. n = 3 biological independent experiments. k RT-qPCR was utilized to detect the effect of MIB1 transfection on FTO mRNA levels. n = 3 biological independent experiments. l Western blot analysis demonstrated the effect of MIB1 transfection on FTO expression. 293 T cells were co-transfected with Flag-tagged FTO and increasing doses of HA-tagged MIB1 for 48 h. n = 3 biological independent experiments. m The effect of MIB1 transfection on the polyubiquitination modification of FTO protein was analyzed in U87MG cells. n = 3 biological independent experiments. n, o The molecular structure of the FTO protein was generated using SWISS-MODEL (https://swissmodel.expasy.org/). The predicted ubiquitination sites were labeled using UbPred (http://www.ubred.org/). p, q IP and western blot analysis were performed to identify the ubiquitination site of FTO mediated by MIB1. U87MG cells were co-transfected with HA-Ubiquitin and indicated FTO mutants. n = 3 biological independent experiments. Statistical significance was determined using two-tailed Student’s t-test in k. Data represent the mean ± SD. Source data are provided in the Source Data file.