Effect of MCP-M protein binding on reporter translation. (A) Luciferase signal from reporter genes without and with MS2 hairpins, as a function of MCP-M proteins with different RNA-binding affinities. NanoLuc luminescence was normalized to firefly luciferase signal and plotted relative to signal in the presence of the NB mutant (Hatfield et al. 2024). Protein-encoding plasmids were transfected at 5 or 15 ng of plasmid per well. Mean ± SEM of biological triplicates are shown. (*) P-value < 0.05; Student's t-test. (B) Relative reporter mRNA levels in the presence of nonbinding MCP-M (NB) or MCP-M with KD of 10 nM. Protein-encoding plasmids were transfected at 15 ng/well. Mean ± SEM of biological duplicates, two technical measurements each, are shown. (C) Relative inhibition of NanoLuc signal normalized to signal in the presence of the nonbinding MCP-M (NB), as a function of protein amount (based on protein-encoding plasmid transfected) and affinity. Complete data sets are presented in Supplemental Figures S1 and S2.
