FBP interacts with p53 and its regulatory proteins, and regulates their expression. (A) Effect of radiation-induced stress on the expression of p53, p21, BCCIP and TCTP in control and FBP-kd Huh 7 cells. Cells grown for 48 h were irradiated with 3-Gy of γ-ray. The irradiated cells were grown for 0, 3, 6, and 12 h and examined for the expression of p53, p21, BCCIP and TCTP by Western blotting. Lanes 1-4, show results from cells grown for 0, 3, 6 and 12 h post-irradiation (B) Fold change in the mRNA level of FBP1, p53, p21, BCCIP and TCTP in irradiated control and FBP-kd Huh7 cells. Cells grown for 48 h were irradiated with 3-Gy of γ-ray and grown for 0, 3, 6, and 12 h. Fold changes in mRNA level of FBP1, p53, p21, BCCIP, and TCTP were determined by real-time quantitative PCR. Upper and lower panels show fold change in mRNA level in irradiated control and FBP-kd cells, respectively. Lanes 1-4, show results from cells grown for 0, 3, 6 and 12 h post irradiation. (C, D) Co-immunoprecipitation of FBP1, p53 and BBCIP. IP was done with either (C) anti-FBP1 antibody or (D) anti-BCCIP antibody, and the IP complexes were captured on protein A/G PLUS-agarose beads, resolved on 8% polyacrylamide SDS-PAGE gel and Western blotted for p53, BCCIP and TCTP and FBP1. (E) FBP, p53 and BCCIP form ternary complex in-vitro. We incubated 500 ng of each of the recombinant FBP, p53 and BCCIP at 4oC for 30 min. The complex was then subjected to FBP-IP, p53-IP or BCCIP-IP. The IP complex was resolved by SDS-PAGE and Western blotted for FBP, p53 and BCCIP. Lanes 1-3, show ternary complex of FBP1-p53-BCCIP captured by immunoprecipitation of FBP, p53 and BCCIP. Lane 4-6 show the formation of binary complexes between FBP1-p53, p53-BCCIP and FBP1-BCCIP, respectively.