Fig 1: Compartment-specific RBP capture by eRIC is highly specific and sensitive. (A, B) Inputs and 10% of RNase eluates from eRIC captures were separated by SDS-PAGE and the proteins were visualized by silver staining (A) and western blotting (B). 250 ng and 0.05% of inputs were loaded onto the gels for silver staining and western blotting, respectively. (B) The western blots were immunoblotted against the known RBPs TIA-1 and ELAVL1, or against the non-RNA-binding proteins Histone H4 and a-tubulin as negative controls. The observed upwards shift in gel mobility of TIA-1 and ELAVL1 between the inputs and eluates of the UV-crosslinked samples is likely due to the presence of small cross-linked RNA fragments in the eluates. (C–H) captured RNA species were analyzed by RT-qPCR. The relative abundance of mRNA (C, D), rRNA (E, F), gDNA (G), and pre-mRNA (H) in cytoplasmic (Cyto) and nuclear (Nuc) inputs and eluates, from untreated and arsenite-treated (+ars) cells were quantified. The data represent the mean with standard deviation from three biological replicates. For analysis of captured pre-mRNA and gDNA samples where reverse transcriptase (-RT) was omitted from the cDNA synthesis was included to evaluate the presence of a gDNA-derived template. Note that mRNA is effectively captured by eRIC while rRNA is strongly selected against compared to inputs. Also gDNA contamination is low as observed by minor amplification in –RT samples. Further, the pre-mRNA capture is restricted to the nuclear fractions and eRIC efficiently discriminates against gDNA capture as observed by only minor amplification in –RT eluates (note the log10 scale in the Y-axis). The horizontal lines marked (*) above the columns indicate the compared groups. Long vertical ticks indicate the control-samples and short ticks indicate P< 0.05 (unpaired two-tailed t-test).