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. 2019 Mar 15;10(4):254. doi: 10.1038/s41419-019-1488-2

Fig. 8. RBCK1 associates with p53 and decreases p53 stability.

Fig. 8

a RBCK1 promotes proteasome-mediated p53 degradation. After 24 h of transfection, cells were treated with 10 μM MG132/vehicle for the indicated time. Cell lysis was prepared for Western blot analysis. The following Image is a histogram of the relative expression of p53 protein. b RBCK1 decreases p53 half-life in RCC cells. Caki-1 cells were transfected with siControl and siRBCK1. After 24 h, cells were treated with 100 μM cycloheximide/vehicle for indicated times. Cell lysates were prepared for Western blot analysis. Image J was used to quantify the p53 protein level. For p53 density, each cycloheximide treated time point was normalized to its zero time point for each group. c RBCK1 UBL and/or RBR domain decreases p53 stability. HEK293 cells were transfected with vector or different RBCK1 deletion mutant plasmids. After 24 h, cell lysis was prepared for Western blot analysis. d HEK293 cells were transfected with 2 μg EGFP-p53 plasmid, 0.5 μg HA-Ub plasmid and 0.5 μg flag-RBCK1/ flag -domain/flag -vector plasmids. The cell extracts were immunoprecipitated with p53 antibody. The poly-ubiquitinated p53 was detected by HA antibody