Role of CTCF Protein in Regulating FMR1 Locus Transcription
Figure 5
CTCF knock-down in WT and UFM fibroblasts.
(A) Western blotting analysis of CTCF, visualized with ECL kit. Protein extracts from untreated (UT) and siRNA-treated WT and UFM fibroblasts were probed with an anti-CTCF rabbit polyclonal antibody (top panel) and one against GAPDH (bottom panel). After CTCF depletion a major reduction of the corresponding protein is visible. (B) Relative quantification through RT-PCR of CTCF and FMR1 sense and antisense transcripts in those knock-down experiments in which CTCF depletion is followed by FMR1 transcript reduction both in WT (upper panel) and UFM (bottom panel) fibroblasts. FMR1 sense transcript is reduced to around 50% in both cell lines with a consistent (about 80%) reduction of CTCF mRNA levels. Depletion of FMR1-AS1 (80 and 60% in WT and UFM cells, respectively) is also observed and directly correlates with CTCF reduction in both cell lines. The two cell lines were also transfected with a scramble siRNA (scRNA) without any modifications of CTCF transcript levels. Histograms represent mean and standard deviation of 10 independent knock-down experiments for UFM and WT.