Arsenic Trioxide Reactivates Proteasome-Dependent Degradation of Mutant p53 Protein in Cancer Cells in Part via Enhanced Expression of Pirh2 E3 Ligase
Figure 3
Ectopic expression of Pirh2 promotes arsenic-induced degradation of mutant p53 protein.
(A–B) Western blots were prepared with extracts from HaCaT (A) and MIA PaCa-2 (B) cells, which were transfected with pcDNA3 or pcDNA3-FLAG-Pirh2 for 24 h, and then treated with 5 or 7.5 µM ATO for 6 h. The blots were then probed with antibodies against FLAG tag, p53, and actin, respectively. (C) Schematic presentation of the Pirh2 protein along with locations of the Zn Finger and RING domains, two substitution mutations C145S and C148S in the RING domain (Pirh2-DN), and deletion of the RING domain in Pirh2 (Pirh2-ΔRING). (D–E) Ectopic expression of Pirh2-DN or Pirh2-ΔRING has little if any effect on the level of mutant p53. Western blots were prepared with extracts from HaCaT (D) and MIA PaCa-2 (E) cells, which were transfected with pcDNA3, pcDNA3-FLAG-Pirh2-DN, pcDNA3-FLAG-Pirh2-ΔRING, or pcDNA3-FLAG-Pirh2 for 24 h. The blots were then probed with antibodies against FLAG tagged Pirh2, p53, and actin, respectively.