Fig. 2

PCBP1 interacts with p27 mRNA 3'-UTR mainly through its KH1 domain. (a) Luciferase activities induced by the full-length p27 3'-UTR (construct a) and its serial deletions (construct b-m) in PCBP1 over-expressing cells or the control cells. Top panel shows the schematic diagram of human p27 mRNA. Full-length p27 mRNA 3'-UTR fragment and its deleted mutants were fused downstream of luciferase coding region in pGL3 plasmid. A2780 cells were transiently co-transfected with the indicated constructs with pRL-TK plasmid for 24-48 h and lyzed for enzyme activity measurement. Relative luciferase activity (Firefly/Renilla) was normalized from three independent experiments (mean±SD). (b) Immunoblot of p27 protein expression affected by the indicated PCBP1 mutations. A2780 cells transiently transfected with the pEGFP vector (Lane 1) or plasmids coding the wild-type PCBP1 (Lane 2) or its mutations (Lane 3-9) as indicated. The protein band intensity was scanned and measured by Image J software. Relative p27 level against GAPDH is further normalized against GFP and shown at the bottom. Results are representative of at least three independent experiments. (c) Effect of PCBP1 KH1 domain to Luciferase activity. A2780 cells were triply co-transfected with luciferase reporter containing full-length p27 3'-UTR (construct a) and plasmids encoding GFP-PCBP1 KH1 mutant, GFP-PCBP1, or GFP with pRL-TK, respectively. 48h after transfection, the enzyme activates were measured as in (a) Meanwhile, the reporter plasmid without PCBP1-binding p27 3'-UTR region (construct k), as a negative control, was similarly used for the above triple transfection. Relative luciferase activity (Firefly/Renilla) is presented from three independent experiments (mean±SD). * and ** represent p<0.05 and 0.01, respectively. ns indicates no difference. (d) RT-PCR detection of p27 mRNA bound with wild-type PCBP1 and the KH1 mutant. The experiments were conducted as in Fig. 1. The input or bound p27 mRNA was quantified based on band intensity and shown under each lane