Anti IgG F(c)

Otoferlin is phosphorylated by CaMKIIδ in vitro. (A) Otoferlin fragments C2ABC (aa 1616 in NP_001093865, 70 kDa) and C2DEF (aa 9081932, 118 kDa), were expressed in E. coli and subjected to an in vitro phosphorylation assay with CaMKIIδ and Ca2+/calmodulin. Reactions were stopped after 5 min of incubation and proteins were run on a Coomassie gel. Note the slight shift in mass of the fragments between experiment (lane 2) and control without kinase (lane 3). Coomassie stained bands corresponding to otoferlin C2DEF and C2ABC were cut off the gel and processed for mass spectrometric analysis of otoferlin phosphorylation (Supplementary Figure S2). (B) Three independent experiments as in (A) revealed 10 serine/threonines in otoferlin that were reproducibly phosphorylated by CaMKIIδ. The putative otoferlin domain topology (in mouse isoform 1; NP_001093865) predicts six C2 domains (C2A to C2F; purple), a coiled coiled domain (orange), a FerB domain (yellow), and a transmembrane domain (TM) (dark gray). Five of the phosphorylation sites are located in C2 domains. Figure provided by CiteAb. Source: Front Synaptic Neurosci, PMID: 29046633.