Ser 215 phosphorylated p53 has been shown to possess paid down DNA binding activity. However, the crosstalk between the Aurora A pathways and p53 remains unclear. In this study, we demonstrate that Aurora A mediated phosphorylation of p53 takes place at CX-4945 yet another site to Ser 215 and Ser 315. A combination of immobilized metal affinity chromatography and phosphoserine certain chemical changes were used to enrich for putative phosphorylated peptides. Following mass spectrometric analyses of these chemically modified proteins generated the identification of a book phosphorylated Ser106 on p53. This phosphorylation was then verified in vitro and in vivo. Eventually, this story phosphorylation was shown to prevent the interaction between p53 and MDM2 as well as being able to prolong the half life of p53. GST p53 WT encodes glutathione S transferase fused to human wild type p53. Likewise, GST p53 S106A, GST S215A/S315A, and GST S106A/215A/S315A encode the GST merged p53s with mutations at the websites. Mammalian indicated pFlag CMV2 p53 and pFlag CMV2 Aurora A were supplied by Prof. Fung Fang Wang and Prof. Chromoblastomycosis Chi Ying F. Huang, respectively. All mutants of p53 and Aurora A for transfection in to H1299 cells were created by way of a mutagenesis set. The cDNA fragment of p53 was cloned in to the pGEX 4T2 vector and developed from the cDNA library by PCR. Mutant constructs of p53 were prepared by mutagenesis kit using pGEX 4T2 p53 because the template. All constructs were expressed in Escherichia coli BL21 in line with the manufacturers protocol to obtain fairly pure fusion protein. Recombinant p53 was purified from 300 ml of bacterial lysate using GSH beans. Recombinant wild type or mutated p53 protein was natural product library pre incubated with human Aurora A kinase in kinase buffer on ice for 10 min and then incubated with cold ATP at 30 C for 3 h or ATP at 30 C for 30 min. The reaction was stopped and then analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Harvested cells were lysed using radioimmune precipitation assay load, 150 mM NaCl, 0. Fortnight Nonidet P 40, 0. Twenty five percent salt deoxycholate, 5 mM EDTA, and 1 mM EGTA in the current presence of general protease inhibitor. Complete cell lysate was analyzed by SDS PAGE according to Laemmlis process. Similarly, Phos tag SDS PAGE utilizing an 8% polyacrylamide gel containing 50 uM Phos tag acrylamide and 100 uM MnCl2 was also completed based on the manufacturers guidelines. For the following Western blot analysis, the gels from either technique were transferred to PVDF membrane. The ensuing membranes were incubated firstwith blocking solution for 1 h and then with primary antibody for over night at 4 C. The extra horseradish peroxidase conjugated antibody was then included with the filters for 1 h at room temperature.