Figure 3.

The putative Pumilio cap-binding motif is not required for repression. (A) Repression activity was measured for three amounts of transiently transfected wild type or cap-binding mutant (W873G) Pum N-terminus via the tethered function dual luciferase assay using the Nluc 2× MS2 pA reporter. Repression activity was calculated relative to the MS2-EGFP negative control at the lowest transfected amount (100 ng). Empty expression vector, pIZ, was used to balance the total mass of transfected plasmids in samples with 100 and 500 ng of MS2 effector plasmid. Mean log₂ fold change and 95% credible intervals for three experimental replicates with four technical replicates each are reported in the graph. Data and statistics are reported in Supplementary Table S1. For significance calling, a ‘*’ denotes a posterior probability >0.95 that the difference relative to the negative control is in the indicated direction. The ‘**' indicates a posterior probability of >0.95 that the indicated difference is at least 1.3-fold. An ‘x’ marks a posterior probability >0.95 that the indicated difference is no more than 1.3-fold in either direction. (B) Western blot of the V5-epitope tagged MS2 fusion effector proteins used in panel A from a representative experimental replicate. Equivalent mass of protein from each sample was probed with either anti-V5 antibody or anti-IntS1 as a loading control. (C) Repression activity was measured for three amounts of transfected wild type or cap-binding mutant (W873G) full length Pum via dual luciferase assay using the Nluc 3× PRE pA reporter. The fold change values were calculated relative to the equivalent amount of transfected RNA-binding defective mutant Pum (mut R7) negative control. V5-tagged EGFP plasmid served to balance the total mass of transfected plasmids in samples with 50 and 500 ng of Pum effector plasmid. Mean log₂ fold change and 95% credible intervals for three experimental replicates with four technical replicates each are reported in the graph. Data and statistics are reported in Supplementary Table S1. (D) Western blot of the V5-epitope tagged Pum effector and EGFP balancer proteins used in panel C from a representative experimental replicate. Equivalent mass of protein from each sample was probed with anti-V5 antibody and, to assess equal loading of lanes, anti-tubulin antibody.