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. 2024 Dec 18;10:498. doi: 10.1038/s41420-024-02260-4

Fig. 5. SMURFs modulation affects GLI1 ubiquitination and protein levels in MB cells.

Fig. 5

A, D Endogenous Co-IP assays on DAOY and ONS-76 cells. Cells were lysed and the lysate was immunoprecipitated with either a GLI1 antibody or a control IgG antibody. Antibodies anti-GLI1, anti-SMURF1 and anti-SMURF2 were used to detect the immunocomplexes. E, F Ubiquitination assays on DAOY and ONS-76 cells. Cells were transfected with either control vector, MYC-SMURF1 or MYC-SMURF2. 24 h after transfection, cells were lysed, and the ubiquitination status was evaluated following GLI1 immunoprecipitation. Proteins were detected using antibodies anti-GLI1, anti-SMURF1, anti-SMURF2, anti-Ubiquitin and anti-ACTIN (used as a normalizer) G–J Analysis of GLI1 protein levels following SMURFs silencing. DAOY and ONS-76 cells were stably transduced with the indicated shRNAs against SMURF1 or SMURF2. Cell lysates were analysed using SDS-PAGE. Proteins were detected using antibodies anti-GLI1, anti-SMURF1, anti-SMURF2 and anti-ACTIN (used as a normalizer). K, L Ubiquitination assays on DAOY and ONS-76 cells stably transduced with the indicated shRNAs against SMURF1 or SMURF2. The ubiquitination status was evaluated following GLI1 immunoprecipitation. Proteins were detected using antibodies anti-GLI1 and anti-Ubiquitin.