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. 2011 Aug 22;194(4):551–565. doi: 10.1083/jcb.201010090

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© 2011 Tao et al.

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Figure 5. — Set7 and MyoD interact in vitro and in vivo. (A) MyoD specifically interacted with GST-Set7 and GST–Set7-dn, but not with GST alone, in GST pull-down assays. Coomassie-stained proteins corresponding to relative amounts of GST and GST-Set7 proteins used in the pull-down assay are shown. (B) HEK293T cells were transfected with Flag-tagged Set7 and/or Myc-tagged MyoD as indicated. (top) Set7 proteins were immunoprecipitated by anti-Flag antibodies, and anti-Myc antibodies were used to detect the presence of MyoD proteins in the immunoprecipitates by Western blot analysis. (bottom) One fifteenth of cell extracts were directly immunoblotted to detect the presence of Set7 and MyoD proteins. (C) Cell extracts from C2C12 myocytes at indicated differentiation dates (0, 1, or 3 d [D0, D1, and D3]) were immunoprecipitated using anti-Set7 antibodies (or IgG in controls), and the presence of MyoD proteins in the immunoprecipitates were detected by anti-MyoD antibodies. The expression of Set7 and MyoD was demonstrated in Western blot analysis. β-Tubulin was used as a loading control. (D, top) Schematic diagram of Set7 mutants for MyoD interaction. The arrowhead in indicates the Set7 point mutation that created the Set7-dn. (bottom) Myc-tagged MyoD was detected in Flag-Set7 aa 1–212 and aa 1–343, but not in aa 212–343 immunoprecipitates. (E) Schematic diagram of MyoD mutants for the Set7 interaction. (F) Myc-tagged MyoD mutants were cotransfected with Flag-tagged Set7, and their interaction was detected in Flag-Set7 immunoprecipitates. IB, immunoblot; HLH, helix-loop-helix.