Fig. 4: NOVA1 promotes the circ-SNRK formation binding to flanking introns directly.
From: Negative feedback of SNRK to circ-SNRK regulates cardiac function post-myocardial infarction
A qRT-PCR analysis of circ-SNRK in NRCMs with Ad-SNRK or si-SNRK. B qRT-PCR analysis of circ-SNRK in NRCMs transfected with si-NOVA1, si-RED1 or si-DDX1. C The level of circ-SNRK in NRCMs with overexpressed NOVA1. D The schematic of pre-circ-SNRK. Primer 1, 2, 3 targeted the predicted four binding sites respectively, primer 3 covered 2 sites because they closed to each other. E RIP analysis of predicted 4 NOVA1-binding sites in introns of pre-circ-SNRK with NOVA1. (Upper) Co-immunoprecipitation (Co-IP) of endogenous NOVA1; (Lower) Agarose gel electrophoresis showed the PCR products (primer1, 2, 3) of RNAs pulled down by NOVA1. F Immunoblot analysis of proteins pulled down by biotin-labeled RNAs of predicted 4 NOVA1-binding sites with anti-NOVA1. G (Left) Confocal analysis of circ-SNRK (red) in HEK293 with upregulation of NOVA1 following be transfected with pCMV-wt-pre-circ-SNRK or pCMV-mut-pre-circ-SNRK. Scale bar = 10 um; (Right) Quantification of fluorescence intensity of circ-SNRK via ImageJ. H Native-PAGE analysis of total protein from NRCMs with anti-NOVA1. I Immunoblot of NOVA1 in hearts from different group or in NRCMs treated with hypoxia or not. A–C, G All data were expressed as mean ± SD, n = 3, two-tailed t-test, *p < 0.05, NS not significant.
