Figure 4. Phosphorylation of p21 increases its stability.
A) Phospho mimics of p21 increase steady levels of p21 in HCT116 cells. HCT116 cells were transfected with equal amounts of one of the HA-tagged p21 plasmids (WT, T/D, S/D, T/A, S/A, DD or AA). Forty hours post transfection, cells were harvested and the steady level of the p21 proteins were evaluated by Western blot with anti-HA antibody. The membrane was reprobed with anti-actin antibody to verify equal loading. B) Stability assay of various p21 proteins. HCT116 cells were transfected with one of the HA-tagged p21 plasmids (DD, T/D, S/D, WT, S/A, T/A, or AA). Thirty-six hours after transfection, cells were treated with 25μg/ml cycloheximide (CHX) and harvested at indicated time points. The stabilities of p21 and actin proteins were analyzed by Western blot with anti-p21 and anti-actin antibodies.