Fig. 6: CRL4B complex catalyzes polyubiquitination of p53 at K164 to promote its degradation.
From: CUL4B protects kidneys from acute injury by restraining p53/PAI-1 signaling
A The level of Trp53 mRNA in kidneys from the indicated mice. N = 5. B The level of TP53 mRNA in the indicated HK2 cells. N = 5. Data are presented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. ns: no significance. C Western blots showing the level of p53 protein in HK2 cells with CUL4B stably knocked down (shCUL4B) or control HK2 cells (NS) treated with 20 μg/ml cisplatin (20 μg/ml) or vehicle control (Con). D Western blots showing the level of p53 protein in the cisplatin-treated HK2 cells with or without CUL4B knocked down at different time points after treating with CHX and the quantification of the protein levels. E The co-immunoprecipitation (IP) assay showing p53 interacts with CUL4B and DDB1 in HK2 cells treated with cisplatin and MG132. Ubiquitination assay showing ubiquitination of p53 in HK2 cells stably knocking down CUL4B (F) or overexpressing CUL4B (G). Cells were transfected with HA-Ub plasmid and treated with MG132. The lysates were immunoprecipitated with anti-p53 antibody. H Western blots showing the level of wild type or K164R mutant p53 in HEK293T cells at different time points after treating with CHX and the quantification of the protein levels. Cells were transfected with 5×Myc: p53-WT or 5×Myc: p53-K164R. I Ubiquitination assay showing the levels of polyubiquitination on wild type or K164R mutant p53 in CUL4B-overexpressing HEK293T cells. Cells were transfected with the indicated plasmids and treated with MG132. The lysates were immunoprecipitated with anti-Myc antibody. J Schematic showing the molecular mechanism underlying CUL4B regulation of AKI.
