Figure 5.

CNOT components are involved in Pum RD mediated repression. (A) The efficiency of RNAi-mediated depletion of Not1 mRNA after 3 days of dsRNA treatment was measured using RT-qPCR. Fold changes were calculated relative to non-targeting control (NTC). Mean log₂ fold change and 95% credible intervals are reported in the graph for one representative experiment with three technical replicates of each measurement. Data and statistics are reported in Supplementary Table S1. (B) Western blot with anti-Not1 antibody confirms depletion of endogenous Not1 protein from a representative experiment. Equivalent mass of cellular extract was loaded for each sample. Anti-tubulin western blot serves as a loading control. (C) Tethered function dual luciferase assays measured the effect of Not1 depletion on the repression activity of Pum N-terminus and RDs. Non-targeting control (NTC) serves as negative control for comparison. Activity of each effector was determined relative to the corresponding negative control effector MS2-EGFP within each RNAi condition. Mean log₂ fold change and 95% credible intervals are reported in the graph for three experimental replicates with four technical replicates each. 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. (D) The efficiency of Not2 mRNA depletion after 3 days of dsRNA treatment was measured using RT-qPCR. Fold changes were calculated relative to non-targeting control (NTC). Mean log₂ fold change and 95% credible intervals are reported in the graph for one representative experiment with three technical replicates of each measurement. Data and statistics are reported in Supplementary Table S1. (E) Western blot confirms RNAi-mediated depletion of endogenous Not2 from a representative experiment using an anti-Not2 antibody. Equivalent mass of cellular extract was loaded for each sample. Anti-actin western blot serves as a loading control. The * designates a protein that cross-reacts with the Not2 antibody. (F) Tethered function dual luciferase assays measured the effect of Not2 depletion on the repression activity of Pum N-terminus and RDs. Non-targeting control (NTC) serves as negative control for comparison. Activity of each effector was determined relative to the corresponding negative control effector MS2-EGFP within each RNAi condition. Mean log₂ fold change and 95% credible intervals are reported in the graph for three experimental replicates with four technical replicates each. Data and statistics are reported in Supplementary Table S1. (G) The efficiency of Not3 mRNA depletion after 3 days of dsRNA treatment was measured using RT-qPCR. Fold changes were calculated relative to non-targeting control (NTC). Mean log₂ fold change and 95% credible intervals are reported in the graph for one representative experiment with three technical replicates of each measurement. Data and statistics are reported in Supplementary Table S1. (H) Western blot confirms RNAi depletion of endogenous Not3 protein from a representative experiment using an anti-Not3 antibody. Equivalent mass of cellular extract was loaded for each sample. Anti-tubulin western blot serves as a loading control. (I) Tethered function assays measured the effect of Not3 depletion on the repression activity of Pum N-terminus and RDs. Non-targeting control (NTC) serves as negative control for comparison. Activity of each effector was determined relative to the corresponding negative control effecto MS2-EGFP within each RNAi condition. Mean log₂ fold change and 95% credible intervals are reported in the graph. Data and statistics are reported in Supplementary Table S1. (J) Western blot of the V5-epitope tagged MS2 fusion effector proteins used in panels C, F and I from a representative experiment. Equivalent mass of protein from each sample was probed with anti-V5 antibody and IntS1 loading control.