Fig. 2

RPAP3-Cter interacts with RUVBL1/2 hexamers. a SILAC proteomic analysis of RPAP3-Cter. The graph depicts the proteins identified in anti-GFP immuno-precipitates of HeLa cells expressing GFP-RPAP3-Cter. Each dot is a protein and the color code is indicated below the graph. X-axis: protein abundance (Log10 of signal intensity); y-axis: enrichment over a control IP (Log2 of SILAC ratio). b NMR interaction analysis of RPAP3-Cter with recombinant RUVBL1/2 complex. The graph depicts 1D NMR METHYL-SOFAST-HMQC spectra in the methyl region of ¹³C-labeled RPAP3-Cter alone (top lane) or mixed with recombinant RUVBL1/2 complex (bottom lane). Intensity of the NMR signal (arbitrary units, Y-axis) is plotted against the ¹H chemical shift (in ppm, X-axis). c SPR binding assays of RPAP3-Cter with RUVBL1/2. The graph depicts the response upon injecting the RUVBL1/2 complex (t = 0 s), or upon washing (t = 300 s), on immobilized RPAP3-Cter. X-axis: time (s); Y-axis: response (arbitrary units). These data have been obtained with the same batch of RUVBL1/2 complex as in the control experiment (Fig. S2E, F). d Chromatographic analysis of the RUVBL complexes. The graph depicts the chromatograms of purified RUVBL1–RUVBL2 (dashed gray, left Y axis) or RUVBL1-RUVBL2-RPAP3-Cter (black line, right Y axis), on a Superose 6 16/70 XK. X-axis: elution volume; Y-axis: absorbance.  e Electrophoresis of the purified RUVBL1–RUVBL2–RPAP3–Cter complex. The gel shows the peak fraction of the complex eluted from the column (black line in d), with a purity estimated to ~95 %. Lane 1: Precision Plus Protein Unstained Standards (Biorad); Lane 2: denatured RUVBL1–RUVBL2–RPAP3–Cter complex. Black and white arrows: RUVBL1 and RUVBL2 (52 and 53 kDa, respectively); gray arrow: RPAP3-Cter (15 kDa). f Native mass spectrometry analysis of recombinant RUVBL complexes. The upper mass spectrum presents the purified RUVBL1/2 complex. The bottom mass spectrum presents the same complex after addition of RPAP3-Cter. Y-axis: signal intensity; X-axis: m/z. Insets: zoom over the 8000–9000 m/z region. Schematics depict the complex observed. Blue: RUVBL proteins; red: RPAP3-Cter