Coimmunoprecipitation of t-SNARE heterodimers with the isolated C2 domains of otoferlin. (A) The assays were performed using 20 µM of each C2 domain using an anti-syntaxin monoclonal antibody. T denotes the total C2 domain loaded into the sample, and Ca and E denote the presence of 1 mM calcium or 0.1 mM EGTA, respectively. H-chain denotes the heavy chain of the anti-syntaxin antibody (HPC-1). (B) Quantitation of the coimmunoprecipitation data using densitometry. (C) Representative gel in which each isolated C2 domain was assayed for its ability to cofloat with vesicles in an Accudenz gradient in the presence of 0.1 mM EGTA or 1 mM free calcium. (D) Calcium-triggered C2 domain–induced liposome aggregation as measured by OD₄₀₀ is shown. Turbidity of samples containing liposomes was monitored in either 1 mM free calcium or 0.1 mM EGTA. Error bars indicate mean ± SD (n = 3).