Fig. 4
From: Synaptotagmins interact with APP and promote Aβ generation
APP interacts with Syt-1 and Syt-9 via its linker region between the E1 and KPI domain. a In vitro GST pull-down assay of Syt-1, −2 and −9. GST-tagged APP-ectodomain fragments were used to pull down V5-tagged Syt-1, −2 and −9 from stably expressing CHO cells. Western blot analysis with anti-V5 antibodies revealed specific interaction of Syt-1, −2 and −9 with both GST-APP-ectodomain and GST-E1 + KPI fragments. Analysis was based on 4 different sets of experiments. b In vitro GST pull-down assay showing interaction of Syt-1 and Syt-9 with the linker region of APP between the E1 and KPI domain. The GST-tagged APP-289 fragment (containing the linker region but not the KPI domain) pulled down both Syt-1 and Syt-9 while the GST-E1 domain did not. Analysis was based on three different sets of experiments. c Co-immunoprecipitation of the APP695 isoform with Syt-1 and Syt-9. CHO cells stably expressing APP695 were transfected with V5-tagged Syt-1 or Syt-9. Western blot analysis shows specific co-immunoprecipitation of APP695 with V5-tagged Syt-1 and Syt-9 (n = 3 for each condition). d In vitro GST pull-down assay showing that the Syt-1 N-terminal region interacts with APP. Purified GST-Syt1 N-terminal region pulled down full-length APP while no interaction was observed with control GST. Bottom panel shows separate colloidal blue stained gel of purified GST and GST-Syt1 N-terminal purified proteins used for the APP pull-down assay. Analysis was based on four different sets of experiments