Fig. 6

CircSCAP downregulates SF3A3 protein level by promoting the ubiquitination-proteasome pathway. A-B NSCLC cells (A549 and H1650) with circSCAP overexpression and control vector were treated with cycloheximide (CHX; 20 μg/ml) for the indicated times followed by immunoblotting to determine the protein stability of SF3A3. C CircSCAP overexpression notably prolonged the half-life of SF3A3 in A549 and H1650 cells. D Higher level of circSCAP in A549 and H1650 led to decrease of SF3A3 protein level which was restored by MG132 treatment (10 μM) for 24 h, as shown by immunoblotting. E–F A549 cells with circSCAP overexpression (E) or knock-down (F) were treated with MG132 (10 μM) for 24 h, and then cell lysates were immunoprecipitated with SF3A3 antibody and the precipitated complexes were subjected to western blot with ubiquitin-HA antibody (upper); or the cell lysates were immunoprecipitated with ubiquitin-HA antibody and the precipitated complexes were subjected to western blot with SF3A3 antibody (lower). The experiments were aimed to detect the ubiquitination status of SF3A3 protein after overexpression or knock-down of circSCAP. G-H A549 cells with circSCAP overexpression (G) or knock-down (H) were treated with MG132 (10 μM) for 24 h, and then cytosolic and nuclear components of cell lysates were separated and subjected to the same immunoprecipitation and immunoblotting as in E–F. * means P < 0.05. ** means P < 0.01