Fig. 5

PPP1CA protein may be stabilized by circGPRC5A-PPP1CA binding. A RNA pulldown using the MS2-tagging system is outlined in a flow chart. B Following transfection of the expression plasmids circGPRC5A, NC, and circGPRC5A-MS2 into CRC cells, the relative expression of circGPRC5A was assessed using qRT-PCR. C Following transfections of MS2-CP and circGPRC5A-MS2 into CRC cells, fluorescence and bright field images were obtained. D The qRT-PCR was used to measure the enrichment of circGPRC5A in a complex with MS2-CP-Flag E MS2 combined protein in various groups in the RNA pulldown assay was examined using western blotting. F-G CircGPRC5A and PPP1CA were compared using the RIP assay to assess their relationship. The qRT-PCR was used to identify circGPRC5A expressions in the immunoprecipitates. H Immunofluorescence-FISH was used to examine co-localizations of circGPRC5A and PPP1CA in HT29 and HCT116 cells. I Following the overexpression or silencing of circGPRC5A, the relative expression of PPP1CA mRNA was assessed using qRT-PCR. J–L PPP1CA protein levels were assessed using western blotting and immunofluorescence after circGPRC5A was silenced or overexpressed. Values from three independent experiments are presented as the mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001