(A) Schematic and equation describing SPS-GBE induced turnover of NICD. In both wild type (left) and nuclei with SPS-GBE sites inserted (right), NICD is produced and enters the nucleus at a constant rate (). NCM complexes form on SPS, where NICD is phosphorylated by Cdk8 at a rate . Phosphorylated NICD degrades faster () than unphosphorylated NICD (). Subscripts denote phosphorylated, unphosphorylated, bound, and unbound NICD. (B) Simulations of NICD levels as a function of SPS number. The three curves correspond to simulations with indicated values of . NICD starts from a common level () and initially decreases linearly with SPS number, with a slope proportional to . (C) Wing notching penetrance and severity in flies with indicated genotypes. Proportional odds model was used to assess significance. (D–G”) Wing discs from flies containing 6xSPS-GFP (6S-GFP) and either GBE-lacZ (G-lacZ) or (G6S)1,2 or 3-lacZ stained with cut (magenta). (H) Quantified GFP levels in wing discs with increasing SPSs (0, 6, 12, 18 correspond to (G6S)1,2 or 3-lacZ) in either wild type (blue) or skd heterozygotes (red). Each dot represents the average GFP level in margin cells from a single wing disc. Error bars show means and S.E.M for each disc. Solid lines represent linear fit to mean GFP values of the first three points of wild-type (blue) and the four points of skd heterozygotes (red). Ratio of slopes is indicated. Effective number of endogenous SPS, , is estimated by extrapolating the y axis intersect of dashed lines. (I) Estimated phosphorylation rates by a single SPS, . Values of (color-bar) were estimated for a range of values of and . Dashed line represents lower limit of the physiological range of kinase activities. (J) Quantified number of gained/lost macrochaetae from indicated genotypes in N55e11/+ (blue bars) or H1/+ (red bars) background. Proportional odds model with Bonferroni adjustment. Data are mean ±95% confidence interval. (K) NICD level simulations as a function of time after Notch activation (at t = 0 min) in wild-type (blue), N heterozygotes (yellow) and SPS-GBE flies (red). In tissues with long duration Notch activation (black dash line), N heterozygotes and SPS-GBE sites similarly reduce NICD levels. In tissues with short duration Notch activation (red dash line), NICD levels are weakly affected by SPS-GBE compared to N heterozygotes (arrows).