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Intronic PAH gene mutations cause a splicing defect by a novel mechanism involving U1snRNP binding downstream of the 5’ splice site

Fig 4

RNA oligonucleotide affinity studies.

A) Schematic representation of the exon 11-intron 11 junction, the predicted binding sites for splicing factors and the RNA oligonucleotides used; B) Western blot gels after pull-down experiments; the blots shown are representative results from three independent pull-down experiments; C) Coomassie stained gels; 15 μg of HeLa nuclear extract (NE input), corresponding to 1/50 of the total nuclear extract used as input per pull-down reaction, equal amounts of nuclear extract collected after the binding reaction (NE output), and 7.5 μl (1/6) of the eluates were loaded and separated on an SDS-PAGE gel, and stained with Coomassie; D) Quantification of the pull down experiments: the intensity of the signal from western blots was quantified and normalized to the signal obtained from the pull-down reaction with the WT sequence. Student t-test was used to evaluate the differences, * p<0.05. BL and NE indicate control lanes without RNA oligonucleotides or with nuclear extract alone, respectively.

Fig 4