The effects of Ser215 and Ser269 mutation on p53 function; phosphomimetic substitution or double alanine substitution of p53 at Ser215 or Ser269 inactivates p53. The effect of serine 215 and serine 269 mutation on expression of the downstream target genes of p53 was examined by immunoblotting lysates derived from H1299 cells transfected with pcDNA vector containing p53 S215A, S215D, S269A, S269D, S215A/S269A, S215A/S269D, S215D/S269A, or S215D/S269D. Lysates were blotted for reactivity to DO-1 (A), MDM2 (B) (2A10), and p21 (C).