Fig. 4: IR treatment promotes ubiquitination and degradation of Survivin in Skp2-deficient OSCC cells.
From: E3 ligase Skp2-mediated stabilization of survivin contributes to radioresistance
A Skp2-deficient OSCC cells were treated with IR for 24 h and given 20 μM MG132 for 6 h, followed by IB assay. B, C (B) Immunoprecipitation (IP) was performed to determine the changes in the ubiquitination level of Survivin after co-treatment of Skp2-deficient OSCC cells with IR and MG132. C Immunoblotting experiments were performed to detect Survivin phosphorylation and total protein expression levels. D The protein expression level of Survivin was analyzed by IB assay after transfection of Flag-Survivin-WT/T34D into CAL27 cells for 48 h and IR treatment for 24 h. E The corresponding plasmids were transfected into OSCC cells for 48 h followed by IR treatment with CHX (20 μg/ml) for different incubation times. IB assay was performed on WCE to analyze the half-life of OSCC cells. Below, qualification for the above immunoblotting bands. F IP assay to determine the ubiquitination of Flag-Survivin-WT/T34D with IR treatment. G Skp2-deficient OSCC cells were transfected with Flag-Survivin-T34D for 48 h, treated with IR, and cultured for 24 h. WCE was subjected to analyze the protein levels of Survivin and Skp2 by IB. H–J MTS (H) assay, soft agar colony formation assay (I), and caspase 3 activity (J) were examined in Flag-Survivin-T34D transfected cells with IR treatment. ***p < 0.001.
