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Fig. 5 | BMC Cancer

Fig. 5

From: Up-regulation of GLI1 in vincristine-resistant rhabdomyosarcoma and Ewing sarcoma

Fig. 5

MVP is a direct transcriptional target of GLI1. a Electrophoretic mobility shift assays were completed using Rh30 cell lysate and 5 different probes that span the MVP promoter region and include each of the GLI consensus binding sites. Shifted bands (indicated by arrows) were visualized by anti-digoxigenin antibody and chemiluminescence for MVP probe 5 (lane marked -). Cold competitor was used to show specificity of binding in the lanes marked +. The gel has been cropped and the full-length gel is presented in Supplementary Figure 5A. b Chromatin immunoprecipitation was performed using Ruch-2 (left panel) and Rh41 (right panel) cells. PCR-amplified DNA bands are indicated in each of the cell lines by the arrows. IgG was used as negative control and anti-RNA polymerase2 (alpha-RNAP2) was used as positive control for the ChIP. Alpha-GLI1 = anti-c-terminal-GLI1 antibody. MVP 4,5 = PCR primers spanned the region from MVP probe 4 through MVP probe 5, which were also used in 5A. The gels have been cropped and full-length gels are presented in Supplementary Figure 5B. c Cotransfection assays were completed by transfecting HeLa cells with 0–1000 ng pCMV-GLI1 (black bars) or pCMV-control (yellow bars) effector plasmids, 200 ng of pMVP407 reporter construct and 20 ng of Renilla control reporter DNA. The experiments were performed in triplicate and results are expressed as an average with standard deviation. * indicates statistically significantly increased reporter activity using a given amount of effector pCMV-GLI1 DNA compared with control effector DNA (p ≤ 0.05). Microsoft Exel and Adobe Photoshop were used to prepare Figure 5

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