Extended Data Fig. 2: SPTBN1 variants alter interaction with critical cytoskeleton partners.
From: Pathogenic SPTBN1 variants cause an autosomal dominant neurodevelopmental syndrome
a, Immunofluorescence images, representative of three independent experiments, show HEK 293 T/17 cells transfected with mCherry-αIISp and with either WT or mutant GFP-βIISp plasmids. Cells were stained for actin (phalloidin) and DAPI. Scale bar, 10 μm. b, Immunofluorescence images of DIV8 mouse βIISp-WT (top) and βIISp-Het (bottom) cortical neurons transfected with indicated GFP-βIISp plasmids. Scale bar, 10 μm. GFP-positive aggregates are detected in neurons expressing these subsets of CH ___domain variants regardless of the level of endogenous βII-spectrin. Images are representative of n = 15 neurons per transfection derived from three independent experiments. c, Western blot from a binding assay to assess interaction between mCherry-αIISp and GFP-βII-spectrin proteins representative of n = 3 biological replicates from three independent experiments. Lysates from HEK 293 T/17 cells expressing mCherry-αII-spectrin were incubated with GFP-βII-spectrin proteins coupled to GFP beads. The presence of mCherry-αII-spectrin in eluates from GFP beads was evaluated by blotting with anti-GFP and anti-mCherry antibodies. d, Coomassie blue staining showing the presence of purified full-length βII-spectrin and F-actin in the supernatant (S) and pellet (P) fractions from an actin co-sedimentation assay. Blot is representative of three independent experiments each with n = 1 biological replicate. e, Co-IP assay in HEK 293 T/17 cells to assess interaction between 220-kDa ankyrin-B (AnkB)-2HA and GFP-βII-spectrin proteins. The presence of 220-kDa AnkB-3xHA and GFP-βII-spectrin proteins in initial lysates and eluates from beads coupled to rabbit IgG isotype control of a rabbit anti-GFP antibody was detected by blotting with anti-GFP and anti-HA antibodies. Blot is representative of four independent experiments, each with n = 1 biological replicate. Western blot images were cropped from Source Data Extended Data Fig. 2.
