FIGURE 4.

TRIM21 downregulates ZHX3 expression in UCB cell ubiquitin‐mediated degradation. A, Silver staining results showed the protein bands that bind to ZHX3, and mass spectrometry was used to identify these proteins. The band corresponding to TRIM21 is indicated by an arrow. B, CoIP results show the relationship between TRIM21 and ZHX3 in T24 cells with stable ZHX3 overexpression. C, Confocal staining revealed the colocalization of TRIM21 and ZHX3 in T24 cells. D, Knockdown and overexpression of TRIM21 resulted in increased or decreased ZHX3 protein levels, although TRIM21 protein levels had no obvious change after knockdown of ZHX3. E, Silencing TRIM21 by siRNA in T24 cells resulted in no obvious change in ZHX3 mRNA levels. Similarly, there was no obvious change in TRIM21 mRNA levels in ZHX3‐silenced shRNA in T24 cells. Error bars indicate the means ± SD of 3 independent experiments. ns: negative significance. F, The T24‐Vector and T24‐TRIM21 cells were cultured and treated with 50 µg/mL CHX. Cells were harvested at 0, 4, 8, or 12 h, and the levels of ZHX3 protein were detected by western blotting. G, In T24‐Flag‐ZHX3 cells, si‐NC, or si‐TRIM21 cotransfected with or without HA‐ubiquitin plasmids. Before harvest, cells were cultured with 10 µM MG132 for 8 h. Cell lysates were immunoprecipitated with Flag antibody and immunoblotted as indicated. H, In T24‐Flag‐ZHX3 cells, Vector, or Myc‐TRIM21 plasmids cotransfected with or without HA‐ubiquitin plasmids. Before harvest, cells were cultured with 10 µM MG132 for 8 h. Cell lysates were immunoprecipitated with Flag antibody and immunoblotted as indicated