Figure 1.

Expression of native synaptotagmin-12 in various tissues and neuronal cultures. (A) Lysates of COS7 cells transfected with cDNAs encoding full-length synaptotagmins 1–13 (Syt 1–13) were analyzed with indicated polyclonal antibodies to various synaptotagmins and valosin-containing protein (VCP; used as a loading control). (B–F) Various tissues, cultured cell lines, and primary neuronal cultures were analyzed by immunoblotting with antibodies to synaptotagmin-12 (Syt 12), -1 (Syt 1), and VCP as a loading control. Equal amounts of total protein were loaded into each lane. (B) Synaptotagmin-12 is expressed in adult mouse brain and adrenal medulla. (C) Expression of synaptotagmin-12 could not be detected in cultured cell lines. (D) Analysis of indicated rat tissues isolated at different stages of development ranging from embryonic day 18 to postnatal day 40 (as shown on the right side of each image), shows that expression of synaptotagmin-12 is restricted to postnatal brain and displays progressive increase during development. (E and F) Relative expression levels of synaptotagmin-1 and -12 were determined by quantitative immunoblotting of total brain homogenates isolated from mouse at different stages of postnatal development and cortical neurons isolated from newborn mouse pups and cultured in vitro for 5–15 d. (E) Raw immunoblotting data. (F) Quantification of expression with I¹²⁵-labeled secondary antibodies. The relative expression levels of each protein were plotted as the percentage of expression in adult brains (n ≥ 4). Similar results were obtained with cultured hippocampal neurons (not depicted). Data are shown as the mean ± the SEM.