The mR3-3 DBD demonstrates a selective defect in H4 tail binding. A. Alignment of histone H3 and H4 tails is shown, with basic residues highlighted. Sequences are from H. sapiens (Hs), G. gallus (Gg), X. laevis (Xl), and S. cerevisiae (Sc). B. The ability of in vitro translated 35S-labeled c-Myb DBD to interact with GST-histone tail fusions (~25–27 kDa) was tested under three conditions: (1) NETN buffer; (2) GST binding buffer with low salt (50 mM KCl); (3) GST binding buffer with high salt (150 mM KCl). Similar amounts of wild-type (wt) and mR3-3 (mR3) 35S-labeled c-Myb DBD, shown with a vector only control (v/o), were used in each precipitation (different exposure than GST binding assays). Staining by Coomassie revealed a similar amount of GST-histone tails in each precipitation. A selective defect in the ability of mR3-3 to bind GST-H4 tail and be precipitated compared to wild-type was found under condition 2 (*). The broad bands directly above the GST-histone tails (conditions 2 and 3) reflects the use of nonfat milk and are likely the major casein proteins (~30–35 kDa). The Benchmark prestained protein ladder (Invitrogen) separates GST-H4 tail and GST-H3 tail (top to bottom are ~50, 40, 25, 20, and 15 kDa). C. Relative amount of the 35S-labeled c-Myb DBD bound to the GST-H4 tail was quantitated for two separate experiments using the ImageJ program http://rsb.info.nih.gov/ij/. The wild-type (wt) c-Myb DBD was normalized to 100% and the mR3-3 (mR3) was scaled accordingly.