(A) Cartoon representation of Siah1 surrounding the I151L mutation site (yellow sticks, blue label) that lies on a “helix-turn-helix” element that connects the dimer interface to the linker between the 2 Zn fingers (indicated by dashed black double-headed arrow). Ile151 is partly buried but also forms part of a prominent pocket (“D”; brown dashed circle). Loss-of-function mutation sites are colored red, and a red circle emphasizes how they cluster across the SBD dimer interface (part of second SBD is in gray). Three crystal forms are overlaid (see Methods and Results) to demonstrate conformational heterogeneity in the second Zn finger (ZnF-2). For clarity, ZnF-2 is represented by a single helix (each structure is labeled by its PDB code, see Methods), but the motion is simple rigid-body rotation about the axis labeled “R,” circled in orange. The RING domain is N-terminal to ZnF-2, and its structure has not been determined. (B) Western blot analysis of PIAS1 expression in 293T cells transfected with PIAS1-V5 alone or in combination with either Siah1-myc or Siah1-I251L-myc. A cycloheximide (CHX) chase was performed for the indicated times. Blots were probed with antibodies against V5 or myc. Tubulin served as a loading control. Dashed lines denote the different degradation rate of PIAS1 coexpressed with WT Siah1 versus PIAS1 coexpressed with Siah1-I251L. The graph shows quantification of PIAS1 degradation from 5 independent experiments. *P < 0.05, compared with Siah1 + PIAS1. (C) Immunohistochemical staining of PIAS1 in adrenal gland sections of 2 patients with primary aldosteronism harboring either I251L or R29I. The line represents the delimitation between the surrounding tissue (S) and the tumoral tissue (T). Data are shown as mean ± SEM. Statistical analysis was performed using t test. Scale bar: 50 μm