Fig 1: Fine mapping actinin interactions with the RNA-binding protein IGF2BP2(A) Representative immunoblot probed with antibodies to IGF2BP2, PCPB1, PCPB2, and SERBP1 in streptavidin affinity-purified iPSC-CM lysates. Double band represents two IGF2BP2 splice products.(B) Representative immunoblot probed with antibodies to IGF2BP2, PCPB1, PCPB2, and SERBP1 in anti-HA immunoprecipitated iPSC-CM lysates.(C) M2H (conducted in HEK293T cells) results demonstrating that IGF2BP2 interacts with full-length actinin, but not PCBP1, PCPB2, or SERBP1.(D) Representative confocal micrograph demonstrating partial colocalization of actinin (anti-actinin; red) with IGF2BP2 (anti-IGF2BP2; green) with DAPI DNA (blue) co-stain in iPSC-CMs (scale bars: main image, 10 µm; inset, 5 µm).(E) M2H study conducted to fine map the direct interaction between actinin and IGF2BP2. IGF2BP2 was divided into RRM (residues 1–160) and KH (residues 161–600) domains to fine map the interaction.(F) M2H results to map the direct interaction between the KH domain of IGF2BP2 and Actinin. Actinin was divided into AB (residues 1–275), rod (residues 276–750), and CaM domains (residues 751–895).(G) M2H results to map IGF2BP2’s interaction within actinin rod domain through deletions of each spectrin repeat (SR).(H) Ribbon structure PyMOL of actinin’s SR2 and SR3 domains (SR2 highlighted in red).(I) Visualization of electrostatic potential of SR2 and SR3 domains. Actinin residue E445 circled in diagram and shaded yellow.(J) M2H alanine scanning mutagenesis results within actinin SR2 domain with IGF2BP2 demonstrates partial disruption by E445A.(K) M2H results demonstrating preservation of wild-type (WT) actinin interaction with E445A actinin.All data are n = 3 and mean ± SEM; significance assessed by ANOVA using Holm-Sidak correction for multiple comparisons (C, E, F, G, J, and K) and defined by p > 0.05 (ns), p = 0.0001 (****). See also Figure S3.