Figure 5. Hoxa2 inhibits RCHY1-dependent ubiquitination of p53 and stabilizes p53.

(A) Ubiquitination assays for p53. HEK293T cells were co-transfected with indicated plasmids and treated with MG132. Cells were lysed and Ni-NTA beads were used to pull down 6His-ubiquitin-conjugated proteins. Proteins were separated by SDS–PAGE and western blotting was performed using an anti-p53 antibody to detect ubiquitinated forms of p53. Lysate samples were loaded on a SDS-PAGE to verify protein levels prior to purification (Input; beta-actin was used as a protein load control). Lane numbering under the gels identifies cell samples. I: input sample; P: Ni-NTA purified sample. (B) p53 protein stabilization by Hoxa2. PA1 cells were transfected with indicated plasmids and treated with MG132, or DMSO as control. Cells were lysed, proteins were separated by SDS-PAGE and western blot detection was performed with indicated antibodies.