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Fig. 6 | BMC Biology

Fig. 6

From: Identification of the novel activity-driven interaction between synaptotagmin 1 and presenilin 1 links calcium, synapse, and amyloid beta

Fig. 6

Mutations in Syt1 at Ca2+–binding aspartate residues disrupt Ca2+-dependent Syt1-PS1 interaction. a Western blot analysis of the Syt1 co-immunoprecipitated with PS1 from 1 % CHAPSO lysed Syt1 KD PC12 cells transiently transfected with Syt1.wt or Syt1.3D-A encoding vectors, following 15-minute 50 mM KCl (KCl+) or water control (KCl-) treatment. Inp.- input cell lysate shows a comparable level of Syt1 in cells transfected with wild type and 3D-A mutant Syt1. IgG (control) or PS1 CT antibody was used for pull-down. b PS70 CHO cells were transfected with Syt1.wt or Syt1.3D-A expression plasmids, treated for 15 minutes with 5 μM Ca2+ ionophore (A23187), and lysed in 1 % CHAPSO buffer. PS1-Syt1 complexes were immunoprecipitated using anti-PS1 NT, anti-PS1 CT or anti-PS1 loop antibodies. The immunoprecipitation efficiency was determined by re-probing the membrane with PS1 antibodies used for pull-down. c Quantitative analysis of the Syt1 co-immunoprecipitated with PS1 from PC12 (n = 3) and CHO (n = 6) in high calcium condition after KCl and calcium ionophore treatment, respectively. More Syt1.wt than Syt1.3D-A was co-immunoprecipitated with PS1 in the above conditions. All the data are presented as mean ± SEM. Statistical significance was determined using the unpaired student t-test, *** p < 0.001. Syt1 synaptotagmin 1, PS1 presenilin 1, wt wild type, CT C-terminus, NT N-terminus, CHO Chinese hamster ovary

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