Crosslinking immunoprecipitation of U2AF2 under HNRNPA1 modulation. (A) Western blot analysis of SRSF1, U2AF2, HNRNPA1, the T7 epitope, and EWSR1 in control (lane 1) and HNRNPA1 overexpression HEK293 cells. (B) Examples of iCLIP autoradiographs for U2AF2 either in control or following overexpression of HNRNPA1. Protein–RNA complex shifts are UV-, antibody-, and micrococcal-nuclease–sensitive. Bars denote the region of nitrocellulose blot excised for RNA isolation for iCLIP library preparation. Micrococcal nuclease treatment at 15 U (high) and 0.015 U (low). (C) CLIPper analysis of iCLIP RNA distribution for U2AF2 in control and HNRNPA1 overexpression conditions. (D) Top HOMER consensus binding motifs for U2AF2 in control and HNRNPA1 overexpression conditions in peaks that do not overlap repetitive elements. (E) Proportion of U2AF2 binding site peaks lost (green), gained (red), or unchanged (blue) in HNRNPA1 overexpression cells.
