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. 2016 Oct 12;35(22):2447–2467. doi: 10.15252/embj.201694574

Figure 3. Heh2 directly binds Chm7 and Snf7.

Figure 3

  • A, B
    Schematics of the domain organization and secondary structure of Heh2 and Snf7 and truncations with amino acid numbers. LEM is LAP2‐emerin‐MAN1 (green), TM is transmembrane (black), MCHD is MAN1 C‐terminal homology domain (brown), and MIM is microtubule interacting motif (purple). The striped α‐helix 5 represents the autoinhibitory helix.
  • C
    GST, GST‐Snf7, and GST‐snf7OPEN were immobilized on GT‐resin and incubated with buffer (−) or recombinant heh2(1‐308)‐His6. Bound proteins separated by SDS–PAGE were visualized by Coomassie stain and by Western blot with anti‐His6 antibody and ECL detection (WB; top panel). Middle panel shows indicated cropped region of the gel where contrast has been increased. Numbers on the side of the gel show position of molecular weight (MW) markers.
  • D
    In vitro transcription translation (IVT) reactions generating radiolabeled (35S) truncations of Heh2 (inputs) were incubated with bead‐bound GST, GST‐snf7OPEN, or GST‐Snf7 before washing, elution, and detection of bound proteins by autoradiography (top panel) or Coomassie staining (bottom).
  • E
    As in (C) except with GST‐Chm7 constructs. Upper panel shows Western blot (WB) with anti‐His6 antibody. Asterisk (*) indicates that chm7‐NTD is also expressed with a His6 tag. Bottom panel is Coomassie stained.
  • F
    As in (E) except with MBP‐Snf7.