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Fig. 4. Domains necessary for Gli3-Hoxd interaction. (A) Relevant Gli3 and Hoxd12 coding domains used in assays shown in B-D. All input lanes show 5-10% of the assay input (B D). (B) The N-terminal Gli3 domain interacts with the C-terminal homeodomain (HD) of Hoxd12 in normalized Gst pull-down assays. The HD-region in Hoxd12/Gst is essential for interaction with in vitro translated (IVT) full-length (FL; B, left panel) or truncated (TR; B, middle panel) Gli3. Hoxd12 mutated in DNA-binding function (mtHD) and N-terminal Gli3 lacking zinc fingers (N-ZnF) still interact (B, middle, right panels). (C) 5'Hox proteins interact with Gli3 preferentially over 3'Hox proteins. Hoxd13(HD)/Gst fusion also binds Gli3 TR (C, left panel). IVT tagged-Gli3 TR (precipitated with Anti-Xpress) also binds full-length IVT Hoxd11 (C, middle panel). Hoxd12 binds preferentially in assays challenged with IVT full-length Hoxa1 or Hoxb1 (C, right panel). (D) Hoxd12 and Gli3 co-immunoprecipitate (IP) from co-transfected cells. Full-length wild-type (wt) or mutant (mtHD) Hoxd12 binds co-transfected Gli3 TR, whereas homeodomain-deleted Hoxd12 ({Delta}HD) does not (D, left panel). A representative input is shown; all inputs were similar and Hoxd12 recovery in IPs were equivalent (not shown). Hoxd12 binds Gli3 TR preferentially over co-transfected Hoxb1 (D, right panel). (E) Gli3 and Hoxd12 co-localize in transfected cell nuclei, as revealed in optical sections with FITC and Alexa Red antibodies. There are no differences in localization compared with the controls of cells transfected singly and expressing either Gli3 TR or Hoxd12 (full length) alone (data not shown).