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. 2019 Jul 29;20(15):3705. doi: 10.3390/ijms20153705

Figure 4.

Figure 4

ESRP1 binds the cyclin A2 mRNA 3’UTR. (A) RPISeq was used to predict the likelihood of interactions between ESRP1 and the cyclin A2 mRNA, with any probabilities greater than 0.5 being deemed “positive”, suggesting a likely interaction. (B) RIP was performed using antibodies against FLAG in ESRP1-overexpressing HeLa cells, with ESRP1-bound transcripts being analyzed via qRT-PCR. (C) HEk293 cells in 24-well plates were co-transfected with full-length or deletion mutant vector (0.5 μg/well), along with ESRP1 vector (0.1 μg/well), and after 48 h cells were lysed and luciferase activity was assessed. (D) EMSA was performed using RNA transcript probes and FLAG -ESRP1. Arrowheads indicate the RNA-protein complexes. Competition experiments with a 50- and 150-fold excess of unlabeled RNA transcript probes were also performed. For normalization of luciferase activity, the signal (1 μg protein) for control cells was set to 1. This experiment was repeated three times (n = 3); error bars indicate standard error; * p < 0.05, ** p < 0.01.