Fig. 1.

SIAH1 mRNA is under PUM and NANOS repression. a Protein extracts from HEK293FT cells transfected with plasmids expressing PUM1 or PUM2 were resolved by SDS-PAGE followed by western blot with the indicated antibodies. β-ACTIN was used as loading control. All values (quantification of the bands) were significantly higher than the negative control (cells transfected with empty vector) (P < 0.01, P < 0.005). The image on the left is representative of three independent experiments, while the middle panel is a statistical assessment of those three experiments. Western blot analysis showing overexpression of PUM1 and PUM2 compared to endogenous PUM1 and PUM2 protein level is on the right. The lower panel represents analysis of SIAH1 mRNA level upon PUM siRNA silencing and overexpression. b Schematic diagram of luciferase reporter construct carrying the 3′UTR of SIAH1 mRNA. c Luciferase reporter repression under overexpression of single PUM or NANOS proteins or PUM/NANOS combination. All values (RLU%) were significantly lower than the negative control (luciferase reporter construct only) (P < 0.001). Luminescence values (RLU relative luciferase units) are expressed as % of the RLU of samples transfected with reporter construct only, which was set to 100%. Renilla luciferase values were normalized using firefly luciferase measurements. d Control western blot showing effect of single (P1 or P2) or double (P1/P2) siRNA downregulation of PUM protein expression compared to β-ACTIN. e Effects of PUM1 and PUM2 siRNA silencing on luciferase reporter repression mediated by NANOS1, NANOS2, and NANOS3. All values (RLU%) were significantly changed (P < 0.001) compared to the negative control (luciferase reporter construct only), C transfected with control siRNA. Each group of experiments in a, c, and e was performed three times, and on each occasion, they were performed in triplicate. Error bars denote standard deviation (n = 9)