Fig. 3

Co-purification of TRβ cofactors.

(a) Affinity purification of TRβ from zebrafish embryos. Blastula-/Gastrula-stage embryos were heat induced for 20 min and recovered for 2 h at room temperature. After purification, the eluate was run on an 8% SDS PAGE and then silver stained. Protein bands (indicated as 1–6) were cut and in gel digested followed by mass spectrometry. Identified protein IDs are shown. M = protein marker. (b) Immunodetection of HIPK3. Western blotting using a HIPK3 antibody (lower panel) shows that a subfraction of the protein co-purifies with the bait protein. Presence of the TRβ protein in the same fractions, detected using Flag-M2 antibody, is shown above. (c) HEK293 cells were transfected with FLAG-HIPK3 and one of three different Gal expression constructs containing the FSH-tag: Gal, Gal-TRβ (aa 189–461) and TRβ_ΔAF2 (aa 189–451). After 48 h of transfection, the cells were harvested in IP buffer and Gal and Gal fusion proteins immunoprecipitated using Streptactin Sepharose. Western blotting using a FLAG M2 antibody shows HIPK3 in the Gal-TRβ and Gal-TRβ_ΔAF2 pull downs. (d) Transcriptional activity of TRβ proteins. 293 cells were transfected with a 2xUAS-Luciferase reporter construct (0.2 μg/well) and Gal or Gal-TRβ constructs (0.05 μg/well): Gal-TRβ (aa 189–461) and TRβ_ΔAF2 (aa 189–451) in the presence of 100 nM TRIAC. Fold activation was measured in the presence (0.05 or 0.1 μg/well) or absence of HIPK3. Luciferase values were normalized against β-Gal. (e) Effects of HIPK3 on Gal-TRβ induced repression. 293 cells were transfected with a 2xUAS-Luciferase reporter construct and either Gal, Gal-TRβ or GAL-TRβ_ΔAF2. Fold repression was measured in the presence or absence of HIPK3. Luciferase values were normalized against β-Gal. Transfections as under 3d. (f) The effect of HIPK3 was investigated in the absence or presence of the co-repressor N-CoR. Transfections were performed as in 3d using the Gal-TRβ_ΔAF2 (aa 189–451) construct. Fold repression of the TRβ mutant in the presence of HIPK3 and/or N-CoR (0.05 μg/well) are shown.