Tmsb4x titrates efficiently miR-1a/miR-206 in differentiated C2C12 cells. (A) Mutagenesis strategy. A luciferase reporter and G418 resistance cassette was introduced 286 bp downstream from the Tmsb4x poly(A) signal, and the Tmsb4x 3′ UTR was replaced by a copy where the miR-1a/miR-206 seed match is either replaced by itself (“wt”) or by a hexamer that is not matched by any known murine miRNA seed (“mutant”). (B) Luciferase activity was assessed in each of the five wild-type and four mutant polyclonal cell lines after differentiation. Each cell line was analyzed as 12 technical replicates (replicates for the same cell line are represented by the same symbol and same color). Mixed-effect linear modeling (taking into account heteroscedasticity within each genotype) shows that genotype of the Tmsb4x miR-1a/miR-206 binding site has a significant effect on reporter activity (p = 0.0285).