Fig. 2: Capture of RBPs from HEK293 cells using HARD-AP.

a Schematic of the HARD-AP procedure and LC-MS/MS analysis. b The silver stained SDS-PAGE gel showing protein precipitated from HEK293 cell lysates under indicated conditions. This experiment was repeated once with similar results. c Western blot analysis for indicated proteins in precipitates obtained under indicated conditions. This experiment was repeated once with similar results. d Volcano plot of distributions of proteins captured by HARD beads compared to EGFP beads in UV-crosslinked samples (top, n = 3,457), proteins captured by HARD beads in samples without UV treatment compared to EGFP beads (middle, n = 3594), and proteins captured by HARD beads in UV-crosslinked samples compared to non-UV samples (bottom, n = 1719). The fold changes were calculated from means of the ion intensities of three independent biological samples. The significance (p) was determined using the two-tailed Student’s t-test and further adjusted using the Benjamini-Hochberg correction for multiple testing (p-adjust). e Left: Venn diagram comparing HARD-AP-derived RBPs using the EGFP-AP control sample (EGFP) and sample treated with RNase (RNase) as the negative control respectively; Right: Venn diagram comparing HARD-AP-derived RBPs in the UV-crosslinked samples and non-UV samples. f Venn diagram comparing RBPs isolated by HARD-AP to published RBPs. g Top GO terms over-represent in RBPs of HEK293 cells identified using HARD-AP. The GO enrichment analysis used the two-sided Fisher’s exact test with the p-value adjusted using the Bonferroni correction for multiple testing. h Matrix bubble plot showing the comparison of Integrator, RNA Exosome, Mediator, 26S proteasome, and Spliceosome complexes captured by indicated methods from HEK293 cells. Hit% is the percentage of subunits of each complex captured by indicated method. The number of subunits captured by indicated methods is labeled on each bubble. The color scale indicates the enrichment. i Scatter plot of distributions of hydrophobicities vs. isoelectric points (pIs) of RBPs identified by HARD-AP (red), polyA-based methods (blue), and non-polyA-based methods (green) in HEK293 cells. Color scales indicate densities. Density plots outside axes illustrate distributions. Source data for (b-c, d-h) are provided as a Source Data file.