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

Figure 6.

Figure 6

(A) Effect of various deletions in Glis2 on its interaction with CtBP1. CV-1 cells were transiently transfected with pCMV-myc-CtBP1 and p3XFlag-CMV-7.1 containing various Glis2 deletion mutants. (B) At 48 h after transfection, protein lysates were prepared as described in Materials and Methods. One part was used in western blot (WB) analysis using anti-Flag M2 (upper panel) or anti-Myc antibodies (middle panel). The remaining was used in immuno-pulldown (IP) assay using anti-Flag M2 affinity resin. Bound proteins were then examined by western blot analysis using anti-Myc antibody (lower panel). Lane 1, Flag-Glis2(1–143); lane 2, Flag-Glis2(1–200); lane 3, Flag-Glis2(83–323); lane 4, Flag-Glis2(170–323); lane 5, Flag-Glis2(323–520); lane 6, Flag-Glis2(170–520); lane 7, Flag-Glis2(full-length). (C) Mammalian two-hybrid analysis. CHO cells were transfected with (UAS)5-LUC (0.1 µg), pM-Glis2 (FL), or pM-Glis2(1–325), pM-Glis2(170–323), pM-Glis2(164–520) deletion mutant constructs (0.1 µg), phRL-SV40 (5ng), and pVP16 or pVP16-CtBP1 (0.1 µg) as described in Materials and Methods. Reporter activity was determined and relative LUC activity plotted. Data are means ± SEM of triplicate samples.