Fig. 10
CSDE1 binds mRNAs involved in neurogenesis and post-transcriptionally regulates their steady-state levels. a RIP with CSDE1 antibody. Quantitative PCR analysis of the indicated genes is expressed as fold enrichment over RIP performed with FLAG control antibody. Graph (relative enrichment to FLAG antibody) represents the mean ± s.e.m. (n = 4 independent experiments). b Western blot analysis of H9 hESCs with antibody to SEMA4A. GAPDH is the loading control. c mRNA levels were determined after the indicated time of actinomycin D (ActD) treatment (5 μg ml−1) by qPCR. For each gene, the right graph corresponds to a representative experiment showing the percentage of mRNA relative to time = 0. In the left panel, mRNA degradation is shown as relative slope to non-targeting shRNA hESCs (mean ± s.e.m. of four independent experiments). d Polysome profiling experiments followed by qPCR analysis. Total and polysome fractions mRNA levels are expressed as relative values to total and polysome fractions of NT shRNA hESCs, respectively. Graph represents the mean ± s.e.m. (n = 4 independent experiments). No further changes in SEMA4A, EPHB3, CDH2, and FZD7 were observed in polysome fractions compared with total cell extracts. In a and c the statistical comparisons were made by Student’s t-test for unpaired samples. In d the statistical comparisons were made by Student’s t-test for paired samples (the mRNA polysome fraction was paired to its corresponding total mRNA in each independent experiment). P-value: *(P < 0.05), **(P < 0.01), ***(P < 0.001), **** (P < 0.0001)
