Figure 6
From: PRAS40 promotes NF-κB transcriptional activity through association with p65
p65 180-200 peptide disrupts the PRAS40–P65 interaction and regulates NF-κB transcriptional activity. (a) The Venus-tagged p65 180-200 peptide could disrupt the PRAS40–P65 association (lane 4 vs lane 3). There is no significant difference between the cells co-transfected with GST PRAS40 DNA and 0.75 or 1 μg of Venus-P65 (lane 2 vs lane 3). (b) HA-tagged p65 180-200 could disrupt the PRAS40 association with exogenously expressed or endogenous p65 (lane 1 vs lane 3; lane 2 vs lane 5). The remaining blots served as negative controls. The blot of the Ex-/En-P65 shows the exogenous and endogenous P65 expression, respectively. (c) In all, 46.3% of NF-κB transcriptional activity in the 3κB-Luc group cells with PRAS40 overexpression was reduced by the 180-200 fragment of p65 (P<0.001). The mutants of PRAS40 increased the NF-κB transcription activity comparing to the pcDNA control group. There are no significant differences of relative activity between the groups of ConA-Luc. NS=P>0.05, *P<0.01. (d) The p65 180-200 peptide significantly reduced the basal NF-κB transcription activity in cells with endogenous PRAS40 expression. *P<0.01. (e) The 3κB-Luc group cells with Venus and Venus-P65 180-200 used in the above experiment (d) were utilized to validate the transfection efficiency. Flag was used as the referral control of transfection because flag tag was included in the Venus tag vector in this study.
