Figure 3
From: PRAS40 promotes NF-κB transcriptional activity through association with p65
PRAS40 regulates NF-κB transcriptional activity. (a) Dual Luciferase Reporter assay of NF-κB transcriptional activity. ConA-Luc and 3κB-Luc was the Firefly plasmid for the control and reporter, respectively. Renilla was transfected for the endogenous control for the experiment. The ratio of Firefly to Renilla is presented on the y axis. The activity of 3κB-Luc in the cells expressing GST-PRAS40 was significantly enhanced relative to cells transfected with the GST control and pcDNA (P<0.001). Two PRAS40-targeted shRNAs significantly decreased the relative activity of 3κB-Luc compared with control shRNA (P<0.01). There were no significant differences of relative activity between the groups of ConA-Luc. NS=P>0.05, *P<0.05. (b) In order to determine the transfection efficiency, the cell lysates were analyzed by western blotting. HSP90 was used as a loading control. (c) Confirmation of PRAS40 knocking down in melanoma A375 and lung cancer A549 cells. (d) Knocking down of PRAS40 suppressed NF-κB p65 DNA-binding activity in both A375 and A549 cells. Data are expressed as mean±s.d. *P<0.05, **P<0.01. (e, f) Knocking down of PRAS40 downregulated A20 and COX-2 mRNA levels in A375 (e) and A549 (f) cells. Data are expressed as mean±s.d. *P<0.05, **P<0.01.
