Fig 1: Giant ANK2 is responsible for neuron-specific PPIs(A) Depiction of the ANK2 gene with ASD-associated mutations leading to splicing defects or early decay. Giant ANK2 includes exon 37, which is alternatively spliced to produce short ANK2. The epitope recognized by the ANK2 antibody used in this study is marked with an asterisk.(B) Volcano plot of an IP-MS experiment for ANK2 in iNs, showing ANK2 in red, significant interactors (log2 FC > 0 and FDR = 0.1) in green, and non-interactors in blue; known InWeb interactors identified as interactors or non-interactors are highlighted in yellow or white, respectively; interactors replicated by immunoblotting are highlighted in orange. Statistics were derived from two replicates using a two-tailed one-sample moderated t test.(C) Experimental workflow to identify giant ANK2-specific PPIs.(D) Top: CRISPR-Cas9 editing strategy to generate a cell line exclusively expressing the short ANK2 isoform, obtained by deletion of the giant ANK2 exon splicing acceptor. Bottom: validation of giant ANK2 KO cell line. The deletion was confirmed by PCR on genomic DNA using primers flanking the deletion site (left), and by immunoblotting (right) showing the absence of the giant ANK2 isoform in homozygous KO iNs (-/-) compared with WT (+/+) and heterozygous KO (-/-) cells. Giant and canonical ANK2 isoforms are indicated with an asterisk.(E) C3 and CACNA2D1 western blots on ANK2 IPs in WT and giant ANK2 KO iNs. FC was calculated as a ratio of the intensity of the bands detected in the IP versus IN lanes. L, ladder; IN, input; IP, immunoprecipitation. Molecular weights are in kilodaltons. Fifty micrograms of total lysate were loaded in the IN lanes, and 10% of the total immunoprecipitates were loaded in the IP lanes.