FIG. 2.

14-3-3σ increased p53 stabilization. (A) Ectopic expression of 14-3-3σ increased the stabilization of p53. H1299 cells were transfected with equal amounts of p53 expression vector and increasing amounts of the Flag-tagged 14-3-3σ expression vector. Equal amounts of protein from cell lysates were immunoblotted with anti-p53, anti-Flag, and antiactin. Actin served as a loading control. (B) Immunofluorescence studies of p53 stability. R1B/L17 cells were transfected with either plasmid pCMV expressing p19^ARF, p53, or 14-3-3σ or with an empty cytomegalovirus vector to examine the immunostaining of endogenous p53 as detected by immunofluorescence. The cells that received the indicated plasmids were seeded at 2 × 10³ cells per chamber slide. The cells were fixed, and p53 was immunodetected with anti-p53 antibody (FL293; Santa Cruz), followed by indocarbocyanine-conjugated anti-rabbit immunoglobulin (B, D, F, and H). DAPI staining was used to show the localization of nuclei (A, C, E, and G). (C) p53 binding activity of 14-3-3σ and p19^ARF. R1B/L17 cells were transfected with the indicated expression vectors. Cell lysates were immunoprecipitated (i.p.) with anti-p53 antibody (DO-1) and immunoblotted (I.B.) with anti-Flag antibody (M2) to observe the association between p53 and 14-3-3σ or p19^ARF. Immunoprecipitation with normal rabbit serum (NRB) was used as a negative control. Equal amounts of protein from cell lysates were immunoblotted with anti-p53 to indicate the expression of p53. Actin served as a loading control.