Fig. 3: JNK activation by PGE2 protects Gli2 from ubiquitin-proteasomal degradation by phosphorylating Gli2 at Thr1546.

A Immunoprecipitation-western blot analysis of the interaction between endogenous Gli2 with JNK in 293T cells treated with PGE2, SP, and MG132 as indicated for 1 h. B Immunoprecipitation-western blot analysis of the interaction between exogenous Gli2 with JNK in 293T cells expressing Flag-tagged JNK1, andMyc-tagged Gli2 after exposure to PGE2, SP, and MG132 as indicated for 1 h. C Immunoprecipitation-western blot analysis of Thr/Ser phosphorylation of endogenous Gli2 in LS174T cells after exposure to PGE2, SP, and MG132 as indicated for 1 h. D Immunoprecipitation-western blot analysis of Thr/Ser phosphorylation of exogenous Gli2 in LS174T cells expressing HA-tagged Gli2 after exposure to PGE2, SP, and MG132 as indicated for 1 h. E The in vitro kinase assay was performed with recombinant JNK1 and indicated His-tagged Gli2 fragments, followed by immunoblotting with anti-phosphorylation-Thr/Ser antibody. F The in vitro kinase assay was performed with recombinant JNK1 and His-tagged Gli2-F3 fragments and His-tagged Gli2-F3 harboring the alanine substitution of theronine 1546 (Gli2-F3 T1546A), followed by immunoblotting with anti-phosphorylation-Thr/Ser antibody. G Immunoblot analysis of Thr1546 phosphorylation of Gli2 in LS174T cells expressing GFP, and Jnk1a1(apf) after exposure to PGE2, MG132 as indicated for 1 h using specific phosphor-Gli2Thr1546 antibody. H Immunoblot analysis of Myc-tagged Gli2 (Gli2-Myc WT), Myc-tagged Gli2 with the glutamine substitution of threonine 1546 (Gli2-Myc T1546E), and Myc-tagged Gli2 with the alanine substitution of threonine 1546 (Gli2-Myc T1546A) in LS174T cells treated with PGE2 as indicated for 1 h. I Immunoprecipitation-western blot analysis of the ubiquitination of Gli2 (detected with anti-HA) in LS174T cells expressing HA-tagged ubiquitin, Gli2-Myc WT, Gli2-Myc T1546E, and Gli2-Myc T1546A after exposure to PGE2, and MG132 as indicated for 1 h.