Fig. 5

CircRNA circPPP1R12A encodes a small unchartered protein circPPP1R12A-73aa. a The 1138-nt circPPP1R12A was potentially translated into a 73-aa (~ 10 kDa) protein (circPPP1R12A-73aa). b To establish a detectable PPP1R12A-C expression vector, flag-labeled circPPP1R12A sequence was cloned into a CMV-induced expression vector (Lv-flag-circPPP1R12A). For the circPPP1R12A-73aa deleted circPPP1R12A expression vector, flag-labeled circPPP1R12A sequence with start codon mutant (ATG/ACG) was cloned into a CMV-induced expression vector (Lv-flag-circPPP1R12A-Mut). For the circPPP1R12A-73aa positive control, the flag-labeled circPPP1R12A-73aa sequence was cloned into a CMV-induced expression vector (Lv- circPPP1R12A-73aa). The schematic structure of these vectors was shown. c The expression level of circPPP1R12A was analyzed by real-time PCR. d The expression level of flag-indicated circPPP1R12A-73aa was detected by Western blotting analysis. e The pulldown cell lysates from indicated groups were separated by SDS-PAGE. Protein bands at 10 kDa were manually excised and submitted for identification by LC-MS/MS. f LC-MS/MS-identified sequences covered 76% of the aa sequences of circPPP1R12A-73aa. Unique aa sequences of spectrum (GRLRHVNCLSPGVQD) formed by circular junction of circPPP1R12A-73aa are shown. The data are represented as the means ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001