Supplemental Material

This article contains the following supporting material:

• Supplemental Figure 1 - (A) Glis2 does not bind to δ-catenin. HEK293 cells were transiently transfected with FLAG-p120 or -δ-catenin, and HA-Glis2. Cell lysates were immunoprecipitated with anti-FLAG antibody followed by Western blotting using anti-FLAG and anti-HA antibodies. (B) The full-length and C-terminus of p120 induce cleavage of Glis2, but the N-terminus of p120 does not. FLAG-Glis2 was expressed with HA-p120 (full length), p120-N-terminus (amino acids 1-647: designated as N) or p120-C-terminus (amino acids 385-910: designated as C) in HEK293 cells. Cell lysates (same as used in Figure 1E) were analyzed by Western blotting with anti-FLAG antibody. (C) p120 is tyrosine phosphorylated by co-expression of Src. HEK293 cells were transiently transfected with HA-p120 and Src. Cell lysates were immunoprecipitated with anti-HA antibody, followed by Western blotting using anti-phosphotyrosine (PY) and anti-HA antibodies.
• Supplemental Figure 2 - (A) Co-expression of Glis2 ΔC and the N-terminus of p120 does not affect neuronal differentiation. HH stage 10-12 embryos were electroporated with Glis2 ΔC and p120-N-terminus (amino acids 1-647), and incubated until HH stage 22. Transfected cells were identified by co-expression of GFP. Expression of GFP and Lim1/2 were examined by confocal microscopy using anti-GFP and anti-Lim1/2 antibodies, respectively. (B) Neither Glis2 nor Glis2 ΔC affect the expression pattern of Pax6 and Nkx2.2 in chick neural tube. Staining of Pax6 and Nkx2.2, markers for different neuronal subtypes in the neural tube. HH stage 10-12 embryos were electroporated with Glis2 or Glis2 ΔC, and incubated until HH stage 22. Transfected cells were identified by co-expression of GFP. Expression of GFP, Pax6 and Nkx2.2 was examined by confocal microscopy using anti-GFP, anti-Pax6 and anti-Nkx2.2 antibodies, respectively.