Fig. 6.

CircGPRC5A stabilizes PPP1CA protein by inhibiting the binding between UBA1 and PPP1CA. A–D After silencing or overexpressing circGPRC5A in HT29 and HCT116 cells, respectively, transfected CRC cells were treated with CHX (100 µg/mL) at the indicated times, and PPP1CA protein was evaluated using western blotting. E–F After silencing or overexpressing circGPRC5A in HT29 and HCT116 cells, respectively, transfected CRC cells were treated with MG132 (20 µM) for 6 h and then PPP1CA was evaluated using western blotting. G–H An IP assay was used to detect the polyubiquitination level of PPP1CA after silencing or overexpressing circGPRC5A. The immunocomplexes were examined using western blotting with anti-Ub and anti-PPP1CA antibodies. I-J PPP1CA was immunoprecipitated with an anti-PPP1CA antibody, and IgG was used as the negative control. The immunocomplexes were analyzed using western blotting, and then the SDS-PAGE gel was stained with Coomassie Brilliant Blue. K Mass spectrometry analysis was used to analyze the products of immunocomplexes immunoprecipitated with anti-PPP1CA antibody. Ubiquitination related proteins are shown. L CircRNA inhibited the binding between UBA1 and PPP1CA. The immunocomplexes with an anti-PPP1CA antibody were examined by western blotting. M–N CircGPRC5A inhibited PPP1CA ubiquitin levels by inhibiting the binding between UBA1 and PPP1CA. Immunocomplexes with an anti-PPP1CA antibody were assessed using the indicated antibodies by western blotting