Fig. 4: Parkin promotes ubiquitination and degradation of ITF2.

A A list of the ITF2-interacting E3 proteins from the BioGRID database. B ITF2 interacts with Parkin. HEK293T cells were transfected with either F/S-ITF2B or Myc-PRKN vector or both. Protein interactions were analyzed by immunoprecipitation-immunoblotting. n = 3, biologically independent experiment. C In vitro binding analysis. Purified Myc-ITF2B and F/S-Parkin proteins were mixed in a test tube, and pulled-down proteins were immunoblotted. n = 2, biologically independent experiment. D The N-terminal of ITF2 interacts with Parkin. Each Myc-tagged ITF2B fragment was co-expressed with F/S-PRKN in HEK293T cells. After being immuno-precipitated with α-Myc, proteins were analyzed using α-Flag. n = 2, biologically independent experiment. E Parkin binds to the N-terminal of ITF2 between Lys residues (171–175) based on the AlphaFold2 (AF2) prediction result. F Plasmids encoding WT F/S-ITF2B or F/S-ITF2B-ΔK (171–175) mutant were co-transfected with HA-Ub into HEK293T cells in the absence or presence of Myc-PRKN. Cell lysates were immunoprecipitated with α-Flag antibodies and immunoblotted. n = 3, biologically independent experiment. G Competitive interaction between p65 and Parkin for binding with ITF2B. pcITF2 was co-transfected into HEK293T cells with Myc-PRKN and increasing (1, 3 μg) Flag-p65 amounts (left). pcITF2 (0.5 μg), Flag-p65 (0.5 μg) and Myc-PRKN (1, 3 μg) were co-transfected (right panel). The protein interactions were analyzed by immunoprecipitation-immunoblotting. n = 3, biologically independent experiment. H The effects of Flag-p65 overexpression and PRKN knockdown on ITF2 ubiquitination. Each Flag-tagged p65 fragment was co-expressed with pcITF2 in HEK293T cells with or without siRNA targeting PRKN. n = 3, biologically independent experiment. In all immunoblot analyses, the data are representative of three independent experiments. Source data are provided in the Source Data file.