dp53m preferentially forms a ternary complex with p53-R175H and CRBN for degradation. (IMAGE)

Science China Press

dp53m preferentially forms a ternary complex with p53-R175H and CRBN for degradation.

Caption

Molecular dynamics simulations calculate the structure of p53m and its binding interfaces with p53-R175H and CRBN (a), and PAGE electrophoresis confirms the successful connection between p53m-DA and CRBNL(b). Microscale thermophoresis measures the affinity of dp53m in p53-WT and p53-R175H (c). Streptavidin pull-down assays show that excess dp53m competitively binds p53-R175H (d). Fluorescence complementation experiments indicate that dp53m, CRBN, and p53-R175H form a ternary complex (e). AlphaLISA (f) and NanoBRET (g) experiments demonstrate that dp53m and CRBN can form a ternary complex with p53-R175H, but not p53-WT. Immunoblotting (h) and quantitative analysis (i) show that dp53m degrades p53-R175H in H1299 cells, with no effect on p53-WT. Immunoblotting (j) and quantitative analysis (k) show the impact of dp53m treatment on H1299 cells over specified time intervals. qPCR analysis shows the level of p53 mRNA in H1299-R175H cells 24 hours after transfection with dp53m (l). PAGE electrophoresis indicates the serum stability of dp53m (m, n).

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