Extended Data Fig. 7: Purification, characterization, and activity assays of Nf1-23a.

a, Size-exclusion chromatography purification of Nf1-23a in the absence (blue line) and presence (red line) of EDTA. In both preparations, Nf1-23a purified as a single peak, but with a higher void peak (~9 mL) in the presence of EDTA in comparison to without. b, SDS-PAGE analysis of corresponding fractions from the size-exclusion chromatography. Nf1-23a is highly pure by SDS-PAGE. For gel source data see Supplementary Figure 1. c, Total reflection X-ray fluorescence (TXRF) spectrum of purified Nf1-23a identified the presence of Zn in native Nf1. Measurements on both sample and buffer were done in presence of a gallium internal standard at 2 mg L⁻¹, added to the samples (1:1, v/v) before measurements. Buffer subtracted difference spectrum normalized by internal standard. Kα X-ray emission lines for different metals are indicated with arrows. d, The effect of different concentration of Zn on the rate of Nf1-23a GTP hydrolysis by KRas. Each assay was measured with 3 independent replications. The concentration of P_i in each assay was normalized against both the buffer + Zn and Nf1 + Zn control. Error bars represent the mean ± s.e.m. of n = 3 independent assays of the same sample. Significance calculated by one-way ANOVA followed by pairwise 2-sided t-tests, applying Bonferroni correction with α = 0.05. ***p < 0.00009; n.s. not significant. e, Nf1-23a GTP hydrolysis by KRas is only inhibited by Zn. Hydrolysis was measured in the presence of 50 μM metal using a GTPase assay kit (Abcam) to detect the amount of phosphate ions (P_i) in solution, with 3 independent replications. The concentration of P_i in each assay was normalized against both the buffer + metal and Nf1 + metal control. Error bars represent the mean ± s.e.m. of n = 3 independent assays of the same sample. Significance calculated by one-way ANOVA followed by pairwise 2-sided t-tests, applying Bonferroni correction with α = 0.05. **p = 0.0023; n.s. not significant.