Figure 4

SPOP inhibits Hh/Gli signaling activity. (A) SPOP interacts with Gli2 protein in MKN28 cells by co-immunoprecipitation method. Goat anti-rabbit IgG is used as negative control. (B) Quantification of Gli1 and Gli2 mRNA in Myc-SPOP transfected MKN28 cells. (C) Increasing SPOP reduced Gli2 expression. AGS cells were transfected with different amount of Myc-SPOP for 48 h. Full length of Gli2 is detected in cell lysates by Western blotting. (D) SPOP promotes Gli2 degradation through proteasome pathway. MKN45 cells were treated with vehicle (DMSO) or miR-SPOP for 48 h, with or without 10 mM MG-132. In order to limit toxicity, MG-132 was added 4 h before cell harvest. Lysates were subjected to immunoblotting with indicated antibodies. (E) Dual luciferase reporter assay is performed in HEK293T cells. Myc-SPOP were transfected into the cells and lysed after 48 h incubation. The percentage of decrease in luciferase activity was calculated. (F) SPOP affects Gli2 abundance in cytoplasm. Immunofluorescent stainings of transfected Myc-SPOP and endogenous Gli2 were performed in MKN45 cells. MKN45 cells were transfected with Myc-SPOP for 48 h. Myc-SPOP was detected by incubating cells with mouse anti-Myc antibody and subsequently Alexa Fluor 594 goat anti-mouse antibody. Gli2 was detected by incubating cells with rabbit anti-Gli2 antibody and subsequently Alexa Fluor 488 donkey anti-rabbit antibody. Nucleus was identified by DAPI staining.