Kaleidagraph

The dissociation constant (KD) of hsCRIP2 for Cu⁺ was determined through competition assays with the chromogenic ligand bathocuproine disulfonate BCS ([Cu⁺(BCS)₂]³⁻ β₂’ formation constant 10^20.8 M⁻², ε_{483 nm} 13000 M⁻¹ cm⁻¹) (95 (link)) as previously shown (96 (link)). Immediately before the assay, CRIP2 was fully reduced by incubation with 5 mM TCEP on ice for 3 h, and the reducing agent was removed using a 3 kDa Centricon (Millipore Sigma). Cu⁺ solutions were generated in situ from CuSO₄ in the presence of ascorbate. Briefly, 10 μM Cu⁺, 25 μM BCS in buffer 25 mM HEPES pH 8, 150 mM NaCl, 10 mM ascorbic acid were titrated with 1 - 30 μM purified CRIP2, incubated 5 min at room temperature, and the 300-800 nm absorption spectra were recorded. CRIP2-Cu⁺ ${\mathrm{K}}_{\mathrm{D}}$ was calculated by curve-fitting the experimental data to the equilibrium equations [1] and [2] (97 (link)), where M is the metal, L is the competing ligand and P are the Cu-sites in the protein. Reported errors are asymptotic standard errors provided by the fitting software (KaleidaGraph; Synergy).

Statistical analyses were done using Wilcoxon or two‐tailed Student's t‐tests. Significance probability values were P < 0.05. Statistical tests were performed in Excel (Microsoft) and KaleidaGraph (Synergy Software). Statistical analysis and mean ± standard deviation (s.d.) were performed on at least three independent biological replicates done in similar conditions. The measurement of cilia length by electron microscopy and data shown in Appendix Fig S6C and D were performed two times independently. The number of biological replicates and sampling sizes are indicated in figure legends and source data.