Fig. 4

Sodium-calcium exchanger (NCX-1) is identified as a key downstream target for VILIP-1. a Schematic workflow illustrating immunoprecipitation-mass spectrometry (left panel) and proximity labeling experiments using VILIP-1-V5-miniTurbo under varying Ca²⁺ concentrations (right panel) to identify VILIP-1 interactors. b MaSigPro (R package) identified four dynamic VILIP-1-proximity labeling patterns in response to increased Ca²⁺ concentrations. Proteins in Cluster 1 were identified as VILIP-1’s candidate interactors based on their progressive enrichment in VILIP-1-V5-miniTurbo-mediated biotinylation profiles with increasing Ca²⁺ concentrations. c Venn diagram illustrating the overlap between interactors identified by co-immunoprecipitation and proximity labeling, confirming high-confidence candidates. d Western blot analysis of biotinylated proteins from VILIP-1-V5-miniTurbo proximity labeling, showing Ca²⁺-dependent enrichment of NCX-1. e Confocal microscopy images confirming the localization of biotinylated VILIP-1-interacting proteins (within a 10 nm radius), NCX-1, in neonatal rat cardiomyocytes (NRCMs) expressing VILIP-1-V5-miniTurbo. Cells were treated with 500 μM biotin for 13 min at 37 °C, and biotinylated proteins were visualized using Neutravidin–Alexa Fluor 488 staining. f Representative immunoblots showing co-immunoprecipitation of VILIP-1 and NCX-1 in rat atrial lysate, demonstrating their interaction. g Western blot analysis of in vitro GST pull-down experiments from VILIP-1-GST and HA-NCX-1, confirming the direct interaction between VILIP-1 and NCX-1. h Confocal microscopy images displaying colocalization of VILIP-1 and NCX-1 in rat atrial cardiomyocytes