Fig. 6.

SV2A-R231Q, W300A, and W666A restore expression of synaptotagmin 1. A, top: a representative Western blot analysis of conventional cultures of hippocampal neurons expressing the indicated SV2A-EGFP fusion construct or EGFP alone. Blots were probed for SV2A (left) and synaptotagmin 1 (right). Anti-actin labeling was used as loading control. Band net intensity was normalized to the net intensity of actin in the same lane. Bottom: average values normalized to WT in the same blot. The graphs represent data from four to seven independent experiments. Error bars represent SE. All three mutants restored synaptotagmin 1 expression levels to those seen in neurons expressing WT SV2A. B: comparison of synaptotagmin 1 levels at synapses expressing EGFP, SV2A, SV2A-R231Q, SV2AW300A, or SV2A-W666A. Synapses, detected with an anti-synaptophysin antibody, were labeled with a polyclonal antibody directed against synaptophysin 1. Normalized fluorescent labeling intensity was normalized to WT within each experiment. The table lists the normalized averages from three independent cultures. Data were assessed for significant differences by one-way ANOVA. N indicates the total number of images analyzed.