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. 2017 Jun 23;36(15):2263–2279. doi: 10.15252/embj.201695757

Figure 2. Two separate internal regions of PWWP2A confer nucleosome binding and H2A.Z‐specificity.

Figure 2

  1. Pull‐downs of GST or GST‐PWWP2A with mononucleosomes (input) derived from HeLaK cells. Precipitated recombinant GST proteins and histones are detected with Coomassie blue staining and H2A and H2A.Z with specific antibodies in immunoblots. * indicates right sizes of purified and precipitated GST and GST‐PWWP2A.
  2. Immunoblots of GST‐PWWP2A IPs with mononucleosomes derived from HeLaK cells stably expressing GFP‐H2A (H2A), GFP‐H2A.Z.1 (Z.1), or GFP‐H2A.Z.2 (Z.2).
  3. Representative competitive EMSA using recombinant H2A (bottom)‐ or H2A.Z (top)‐containing nucleosomes incubated with indicated increasing concentrations of GST‐PWWP2A. GST alone served as negative control. * indicates nucleosome; ** indicates nucleosome‐GST‐PWWP2A complex.
  4. Schematic representation of recombinant GST‐tagged PWWP2A and PWWP domain deletion (ΔPWWP) and PWWP domain only (PWWP)‐containing constructs (top) used in cell‐derived mono‐IPs followed by Coomassie staining and immunoblotting (bottom). * indicates respective GST proteins. Notice that both the PWWP domain alone as well as the PWWP‐deletion protein are able to interact with nucleosomes, indicating at least two independent nucleosome binding sites within PWWP2A.
  5. Schematic representation of recombinant GST‐PWWP2A deletions (top) used in cell‐derived mono‐IPs followed by Coomassie and immunoblotting (bottom). See also Appendix Fig S1B for protein purification.
  6. Schematic representation of recombinant GST‐PWWP2A internal deletions (top) used in cell‐derived mono‐IPs followed by Coomassie and immunoblotting (bottom). See also Appendix Fig S1C and D for protein purification and further IPs.
  7. IPs as described in (F) with mononucleosomes derived from HeLaK cells stably expressing GFP‐H2A and GFP‐H2A.Z isoforms detected with anti‐GFP antibody.

Source data are available online for this figure.