(a) Coomassie staining of the recombinant four-protein mini-complexes containing PHF5A-WT or PHF5A-Y36C used for Scintillation Proximity Assays. (b) The competitive titration curves of non-radioactive splicing modulators to 3H-labelled pladienolide analogue (10 nM) binding to the WT four protein complex. (c) Overall surface view of modelled C36 overlaid onto WT (Y36 show in cyan stick) and zoom-in PHF5A surface view at Y36 and C36. Surface potential coloured in red: −8 kBT/e, blue: +8 kBT/e and white: 0 kBT/e was calculated by APBS. (d) Scintillation Proximity Assay of the 3H-labelled pladienolide analogue (10 and 1 nM) binding to protein complexes containing WT or Y36C PHF5A. Error bar indicates s.d., n=2. (e) Western blot analysis of PHF5A levels in parental and indicated PHF5A variants expressing HCT116 cells. GAPDH is shown as a loading control. (f) Unsupervised clustering heatmap of the IC50 shift between indicated PHF5A variant expressing cell lines as compared to WT cell lines. The shift is shown as fold changes and calculated from IC50 values extracted from dose–response curves in (g). Each row represents indicated PHF5A variant and each column corresponds to indicated compound. Colour key is shown on the top right corner. (g) Seventy-two hours growth inhibition profiling (CellTiter-Glo cellular viability assay) of parental and indicated PHF5A variant expressing HCT116 cells’ response to indicated compounds. Error bar indicates s.d., n=3.