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. 2024 Jan 15;15:532. doi: 10.1038/s41467-024-44843-w

Fig. 1. DUSP22 induces UBR2 degradation.

Fig. 1

a The identified peptide sequences of endogenous UBR2 protein by mass spectrometry-based analyses using Myc-tagged immunocomplexes from the lysate of Myc-DUSP22-transfected HEK293T cells. b-d Immunoblotting of Flag-tagged UBR2, Myc-tagged DUSP22, and tubulin proteins in HEK293T cells co-transfected with different amounts of Flag-UBR2 plus either Myc-DUSP22 (b and c) or Myc-DUSP22 (C88S) (d) plasmids. Tubulin immunoblotting was performed by reprobing the Flag (UBR2) immunoblot membrane (b and d). Arrow, intact UBR2 protein; asterisk, degraded UBR2 protein. e Schematic diagram of the murine DUSP22 wild-type (WT) alleles and the targeted DUSP22 mutant alleles. P1 and P2, the primers for PCR. f Characterization of DUSP22-knockout mice. PCR analyses of wild-type and DUSP22 mutant alleles in the genomic DNA from mouse tails. The PCR product of the upper band (300 bp) denotes the wild-type allele, and the lower band (145 bp) denotes the DUSP22 mutant allele. g Immunoblotting analysis of UBR2 protein levels in multiple tissues of wild-type and DUSP22 knockout mice. h, i DUSP22-induced UBR2 proteasomal degradation. Flag-UBR2 and Myc-DUSP22 plasmids were co-transfected into HEK293T cells. The transfected cells were treated with MG132 (25 µM), Z-VAD-FMK (50 µM), or chloroquine (50 µM) for the indicated time points and then subjected to immunoblotting analysis. Anti-tubulin immunoblotting was performed by reprobing the anti-Myc (DUSP22) immunoblot membrane (h). Vinculin immunoblotting was performed by reprobing the anti-Flag (UBR2) immunoblot membrane (i). j, k UBR2 interacted with DUSP22 or DUSP22 (C88S) mutant proteins. Coimmunoprecipitation and immunoblotting analyses of UBR2, DUSP22, and DUSP22 (C88S) mutant proteins in HEK293T cells co-transfected with Flag-UBR2 plasmid plus either Myc-DUSP22 or Myc-DUSP22 (C88S) mutant plasmid. l Proximity ligation assays (PLA) showed in vivo UBR2-DUSP22 interaction in Jurkat T cells. Red fluorescence represents interactions ( < 40 nm) of Flag-UBR2 and Myc-DUSP22 proteins. Images were captured by confocal microscope (Leica TCS SP5 II). Original magnification, 400X. Cell nucleus was stained with DAPI. Scale bar, 10 μm. m In vitro binding assays of purified Flag-tagged UBR2 and recombinant GST-tagged DUSP22 proteins.