Magnesium regulates tubulin equilibrium through β-CTT. Magnesium regulates tubulin equilibrium through β-CTT. (A) Polymerization rate plotted as a function of tubulin concentration. Crosses represent data from experiments conducted at 1 mM MgCl2 and dots are data points from Fig 1D from experiments conducted at 5 mM MgCl2. Black: tubulin and red: S-tubulin. Data points are mean ± 95% CI plotted for each concentration. Each data point represents at least 61 polymerization events from at least 24 microtubules, collected from at least three separate experiments. Linear regressions were fit to the data with at least three different concentrations and plotted as lines. (B) Depolymerization rate plotted as a function of tubulin concentration. Each data point represents at least 45 depolymerization events from at least 24 microtubules, collected from at least three separate experiments. Linear regressions were fit to the data with at least three different concentrations and plotted as lines. (C) Slow depolymerization rate (Fig 3B, labeled “S”) for tubulin and S-tubulin microtubules after washing out free tubulin. The MgCl2 concentration indicated is for the washout buffer. The 5 mM plots are data from Fig 3E, provided for comparison. Data for the 1 mM MgCl2 represent 45 microtubules for untreated tubulin (4.8–6.8 μM) and 158 microtubules for S-tubulin (7.8–12.0 μM), pooled from at least four different experiments for each. Microtubules without a detectable slow depolymerization rate and/or a rate calculated to be zero were excluded from this analysis. *P = 0.05 and **P « 0.001, significance determined by the Mann–Whitney U test. (D) Fast depolymerization rate (Fig 3B, labeled “F”) for tubulin and S-tubulin microtubules after washing out free tubulin. The 5-mM plots are data from Fig 3F, provided for comparison. Data for the 1 mM MgCl2 represent 52 microtubules for untreated tubulin (4.8–6.8 μM) and 260 microtubules for S-tubulin (7.8–12.0 μM), pooled from at least four different experiments for each. N = 179 tubulin and n = 92 S-tubulin microtubules, pooled from at least four different experiments for each. *P = 0.01 and **P « 0.001, significance determined by the Mann–Whitney U test. Colby P Fees, and Jeffrey K Moore LSA 2018;1:e201800054 © 2018 Fees and Moore