Structural modeling of the variants and expression studies in HEK293 cells. (A) Left panel: Mutation sites in Znf 1 of IKZF5. The structure of Znf 4 from CTCF (PDB entry 5kkq³³ ) is portrayed as a template to understand the effect of the mutations Y89C (site equivalent to Y358) and R96W (site equivalent to K365). To model the conformation of R96, K365 in the CTCF structure was replaced by an arginine in a similar conformation to that of R369 in PDB entry 5kl6.³⁴  WT side chains are shown in ball-and-stick representation, whereas the mutated side chains are shown as partially transparent cylinders. Right panel: The role of C140 in Znf 3. We used the coordinates for Znf 6 from PDB entry 5kkq without any changes as all the side-chains shown are conserved between the 2 sequences. (B) Western blot analysis of HEK293 transfected cells with myc-tagged IKZF5 WT and mutants after extraction of chromatin-bound proteins. HIST3H3 (histone H3) serves as loading control. HEK293 transfected with empty vector (Mock) express no IKZF5. The 5 causal mutants are highlighted in red. (C) Quantification of chromatin-bound IKZF5 after transfections and normalization against HIST3H3. The 5 causal mutants are highlighted in red. (D) Immunofluorescence (z-stack images by a structured illumination microscope) using an antibody against IKZF5 (green), phalloidin (actin; red), and 4′,6-diamidino-2-phenylindole (nucleus, blue) showing punctate, nuclear staining for WT IKZF5, whereas R96W and other mutants (supplemental Figure 4) remain largely outside the nucleus after transfecting HEK293 cells. Scale bar, 10 µm.