Figure 6.

SPOP mediating the ubiquitination and degradation of Gli2 and modulating apoptosis signaling. (a) The interaction between SPOP and Gli2 was detected by co-immunoprecipitation. Goat anti-rabbit IgG is used as negative control. (b) Ubiquitination assays of Gli2 were performed by co-transfection of FLAG-Gli2 and HA-Ub. After immunoprecipitation by FLAG antibodies, HA antibody was used to detect poly-ubiquitinated Gli2. (c) The expression levels of Hh/Gli2 pathway-related apoptotic proteins Bcl-2, cleaved Caspase-3 and cleaved PARP were detected with western blotting. (d) The mRNA levels of Gil2 were detected by qRT-PCR and the protein levels were quantified based on (b) lower panel. (e) Gli2 stability was detected by cycloheximide protein degradation rate experiments. (f) The representative photos of SPOP and Gli2 expression in CRC tissue microarray. The number of CRC patients is 118. The correlation between SPOP and Gli2 expression was examined by Spearman correlation test. The numbers are referring to the predicted protein sizes. The experiment was repeated three times. *P<0.05