Repression of Raf mRNA and protein levels by Pum. (A) A western blot confirmed depletion of endogenous myc-tagged Pum by RNAi relative to NTC negative control in three biological replicate samples of DL1 V5-Raf, Pum-myc cells. (B) Increased expression of Raf mRNA and protein levels in Pum RNAi samples relative to NTC was measured by RT-qPCR and quantitative western blotting, respectively. Mean LFC values ±SEM are plotted relative to the NTC RNAi condition. RT-qPCR measurements were made from nine replicate samples. Quantitative western blot measurements were made in three independent experiments, each of which had three biological replicate samples with four technical replicate measurements per condition. Significance calling is as follows: (*) P < 0.05, (***) P < 0.001, using unpaired, two-tailed Student's t-test. Details of quantitation are described in the Materials and Methods section. (C) Representative western blots of V5-tagged endogenous Raf protein in three biological replicate samples from an RNAi experiment wherein the effect of Pum RNAi was compared to NTC control in DL1 V5-Raf, Pum-myc cells. The indicated amount of total cellular protein per lane, as measured by DC Lowry assay, for each replicate was analyzed in the western blots.
