Fig. 1

SGIP1α is expressed in cultured primary neurons and binds to Syt1. a Domain structures of SGIP1 and SGIP1α. SGIP1α contains additional 28 amino acids in the MP domain and 20 amino acids between the PRD and μHD. b Whole cell lysates from HEK293T, HEK293T with FLAG-SGIP1 and FLAG-SGIP1α and cultured hippocampal neurons were resolved by SDS-PAGE and transferred to nitrocellulose and immunoblotted with SGIP1 antibody. The red dotted line indicates the size of FLAG-SGIP1. c, d Hippocampal neuron lysate was pull-downed with purified GST-SGIP1α (c) or purified GST- SGIP1α-μHD (d) immobilized on glutathione-sepharose. Bound proteins were eluted from the beads by boiling and resolved by SDS-PAGE and either stained with Coomassie blue or transferred to nitrocellulose. Replicate blots were probed with Syt1 antibody as indicated. The asterisk indicates the band of Syt1. e Purified GST-Syt1-C2AB fusion protein immobilized on beads were incubated with cell extracts derived from HEK293T cells transfected with HA-C1 or HA-SGIP1α. Bound proteins were detected by immunoblotting for HA. f GST-Syt1-C2AB fusion protein immobilized on beads were incubated with cell extracts derived from HEK293T cells transfected with HA-C1 or HA-SGIP1α-μHD or HA- SGIP1α ΔμHD. Bound proteins were detected by immunoblotting for antibody against HA. IB, Immunoblot; IP, Immunoprecipitation; TCL, Total cell lysates