Fig. 6. TAD mutations affect the state of phosphorylation of mutant p53.

Western blot analysis of equal amounts of extracts from a H1299 cells expressing p53-R273H, -L22Q/W23S/R273H, and vector control; b H1299 cells expressing p53-R175H, -L22Q/W23S/R175H, and vector control; and c murine lung cells expressing p53-R172H, -L22Q/W23S/R172H, and p53–/– control were developed with antibodies against different phosphorylation sites on p53. The data presented show that phosphorylation at amino acid position 20 went down significantly. d Transactivation assays of mutant p53 and its derivatives by Ala substitution mutations at positions of phosphorylation using a mutant p53 inducible gene promoter. Transient transcriptional assays were carried out in H1299 cells after transfection of NF-κB2-luc and indicated expression plasmid or empty vector. Cells were harvested after 24 h of transfection. Firefly luciferase readings were plotted as relative light units (RLU). Data are presented as the mean ± SEM (n = 3, performed twice). A two-sided Student’s t-test was performed. Ser20Ala substitution reduced the transactivation ability the greatest. Substitution of S20 to Glu or Asp did not regain transactivation ability either. e Western blot analysis showing level of different p53 mutants after transfection. *p-value < 0.05, **p-value < 0.01, and ***p-value < 0.001. NS no statistically significant difference from control.