Fig. 3.

Stacking histidines of the zinc coordination center affect ECM association. a Diagram of the Shh structure (PDB: 3D1M) with relevant residues indicated. b Hh response in LightII cells grown on the conditioned and decellularized ECM of Hek293t cells transfected with the indicated Shh-C199* constructs. n ≥ 2, *p < 0.05, ****p < 0.0001. c The effect of mutations of the transition state-stabilizing residues H135 and H181 to alanine (A) or tyrosine (Y) on the zinc-dependent accumulation in the ECM was analyzed on a Western Blot of the extracted ECM from transfected Hek293t cells cultured in 0.18 mM calcium with or without 10 μM zinc. d zinc dose-response analysis of H135 and H181 mutations assessed by Western blot of the lysate and ECM of Hek293t cells transfected with the indicated mutants and cultured in 0.18 mM calcium and increasing concentrations of zinc (0.1, 1, 10 μM). Uncropped blots can be found in the supplement. e Quantification of band intensities shown in D as a fraction of ECM over lysate, normalized to the respective mock treatment. n = 2. Uncropped, full-length blots are presented in Supplementary Figure 5