Investigating p53-HDM2 Protein Interactions

A375 cells seeded in 6-well plates were co-transfected with pcDNA-Flag-p53 (Addgene) and pCMV-myc3-hDM2 (Addgene) for 6 h using TurboFect transfection reagent following the instruction from manufacturer. Cells were then cultured in Dulbecco's Modified Eagle's Medium (DMEM) complete medium overnight. Serial dilutions of organometallic compounds in low FBS buffer were placed into the wells, and incubated with cells for additional 6 h. Protein samples were collected, and the concentration in the supernatant was determined with the bicinchoninic acid (BCA) method. The organometallic compounds of p53/hDM2 in the protein samples were pulled down using anti-Flag magnetic beads to capture the FLAG fusion proteins (Sigma) as previously described [50 , 51 (link)]. The protein-binding beads were washed three time with TBS buffer (50 mM Tris HCl, 150 mM NaCl, pH 7.4) to remove non-specifically bound proteins, and subsequently subjected to the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by detection with anti-Flag (1:1000, Sigma-Aldrich) and anti-myc antibodies (1:1000, Beyotime).