Figure 1.

Synaptic distribution of green fluorescent protein-ProSAP-interacting protein 1 (GFP-ProSAPiP1) in primary hippocampal neurons. (A) GFP-ProSAPiP1 is clearly visible at the expected molecular weight (~ 125 kDa) in infected primary hippocampal culture at DIV28, whereas no signal is observed in the uninfected control (Uninf.). Post-synaptic density (PSD) fraction from rat hippocampus was loaded as reference for endogenous ProSAPiP1, which is present in all lanes (~ 85 kDa). β3-Tubulin and β-Actin serve as loading control. (A,B) Protein levels of endogenous ProSAPiP1 were significantly increased after overexpression of GFP-ProSAPiP1 at DIV28. (B) Statistical analysis was performed using unpaired two-sided t-test. *p < 0.05; n = 3 lysates from three independent cultures for each condition. (C,D) Both ProSAPiP1 puncta intensity and ProSAPiP1 cluster size were significantly increased after overexpression of GFP-ProSAPiP1 in primary hippocampal culture at DIV28. Statistical analysis was performed using unpaired two-sided t-test. *p < 0.05; n = 10 neurons from two independent cultures. (E) Exemplary hippocampal neurons, infected with GFP-ProSAPiP1 (green) and immunostained for Bassoon (red) on DIV14 and DIV28 as indicated. Statistical analysis shows a significant increase of synaptic GFP-ProSAPiP1 signals from DIV14 to DIV28 (42.9% on DIV14; 70.3% on DIV28) and was performed using unpaired two-sided t-test. ***p < 0.001; n = 10 neurons from two independent cultures. (F) On DIV28, 62.1% of GFP-ProSAPiP1 signals co-localized with VGluT1 and 31.1% with VGAT, respectively. (G) Primary hippocampal cultures were infected with either FUGW empty vector (Vector) or GFP-ProSAPiP1 (both green) and stained for Bassoon (red) on DIV28 as indicated. No significant difference was observed. Scale bar: 10 μm. n = 15 neurons from three independent cultures.