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. 2005 Dec 2;33(21):6805–6815. doi: 10.1093/nar/gki985

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© The Author 2005. Published by Oxford University Press. All rights reserved

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Interaction between Glis proteins and CtBP1 is specific for Glis2. (A) Mammalian two-hybrid analysis. CHO cells were transfected with (UAS)₅-LUC, phRL-SV40, pM-Glis1, pM-Glis2, pM-Glis3, pVP16, pVP16-CtBP1 and pVP16-CtBP2. Reporter activity was assayed 48 h after transfection and the relative LUC activity calculated and plotted. (B) Immuno-pulldown analysis. CV-1 cells were transfected with p3XFlag-CMV-Glis1, p3XFlag-CMV-Glis2, p3XFlag-CMV-Glis3, pCMV-myc-CtBP1, p3XFlag-CMV and pCMV-myc as indicated. Cell lysates were prepared and Flag-Glis2 protein complexes isolated using anti-Flag M2 affinity resin as described in Materials and Methods. Proteins in the cellular lysates and immunoprecipitated (IP) proteins were subsequently examined by Western blot (WB) analysis using anti-Flag M2 or anti-myc antibody. Asterisk indicates the position of the different Flag-Glis fusion proteins (Glis1, 84.3 kDa; Glis2, 55.8 kDa; Glis3, 83.8 kDa).