Fig. 5

SELENBP1 transcriptionally stimulates p21 expression in a p53-independent manner. a Total RNA was isolated from indicated stable transfectants, and then subjected to RT-PCR assay for determining p21 mRNA levels. The mRNA levels of GAPDH were used as a loading control. b The potential binding sites of multiple transcription factors in the p21 promoter are shown in upper panel, and full-length (2.4 Kb), 1.8 Kb, 1.3 Kb and 200 bp of p21 promoter-driven luciferase reporters are schematically shown in lower panel. c Stable UMUC3 (Vector) and UMUC3 (HA-SELENBP1) cells were transiently co-transfected with pRL-TK and p21 promoter-driven luciferase reporters as described in “b”. Following 36 h of transient transfection, luciferase reporter activity was determined and normalized to that of corresponding UMUC3-Vector. The asterisk (*) indicates a significant difference in comparison to that of corresponding control transfectants (p < 0.05). d Cell lysates of HCT116 WT and HCT116 p53^−/− were subjected to immunoblotting assay for identification of p53 expression (left panel). Following 48 h of transient transfection with HA-SELENBP1 or empty vector, HCT116 p53^−/− cells were extracted and then subjected to immunoblotting assay with anti-SELENBP1, anti-p21 or anti-GAPDH antibodies (right panel). Densitometric quantification of p21 (relative to GAPDH) is shown