Mutation of the predicted RFX-binding site in the spag6 promoter decreased expression of the NanoLuc luciferase to an average of 61% of wild-type activity. Three biological replicates were assayed, with 3–6 technical replicates per construct in each replicate. Y-axis shows normalized NanoLuc expression relative to a construct expressing NanoLuc under the strong EFL promoter, included as a positive control in all experiments. Individual values are plotted in gray and averages for each biological replicate are plotted in orange. The average across biological replicates is represented by a horizontal bar. p values are shown for a paired t test between Pspag6-wt and Pspag6-ΔTFBS reporters transfected into each genotype, using the mean value for each biological replicate (n = 3), as well as un unpaired t test for Pspag6-wt transfected into either cRFXaWT or cRFXaPTS-1 cells, again using the mean value for each biological replicate (n = 3). See alsoData S1.
