Research Paper Volume 15, Issue 20 pp 11131—11151

Figure 6. Triptolide enhanced the protein degradation of Gli1 and Gli2 through the ubiquitin-proteasome-dependent pathway. (A) TPL (4 nM) was applied to SKOV3 and A2780 cells for 24 h, and the results were separately detected using immunofluorescent assays with anti-Gli1 and anti-Gli2 antibodies. Scale bar: 20 μm. (B, C) SKOV3 and A2780 cells were incubated with TPL (4 nM) for 48h and then MG132 (10 μM) for 4h before harvesting and were subjected to Western blot for Gli1 and Gli2. (D, E) Cells were treated with cycloheximide (20 μM) with or without TPL (4 nM) and detected the protein levels of Gli1 and Gli2 by Western blot assay. (F, G) HEK293T cells were cotransfected with the Myc -Ub, Gli (GFP-Gli1 or GFP-Gli2), or the GFP control vector plasmids, and then the cells were further cultivated for 36 h with TPL after being cultured for 24 h. During the last 4h of TPL treatment, MG132 (10 μM) was added to every group. The protein lysates were examined by immunoprecipitation with an anti-MYC antibody, and the ubiquitinated protein levels were detected by Western blot in an anti-GFP antibody. All results were derived from three independent repeated experiments and represented by mean ± SD, * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. control; ns, no significance.