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, Figure 1. Sec22b and Stx interact with the lipid transfer proteins E-Syt2 and E-Syt3

, Immunoblots of material recovered after GFP-Trap pull-down from HeLa cell lysates. Cells 737 were transfected for the co-expression of FLAG-Sec22b and Myc-E-Syt2 (A) or FLAG-Sec22b and 738

-. Myc, Cell lysates were 739 subjected to GFP-Trap pull-down. Total cells lysate (Input) and trapped material (Bound) were 740 processed for SDS-PAGE and Western blotting. Blots were probed with antibodies directed against 741 the tags (GFP, FLAG and Myc) as indicated. Both Myc-E-Syt2 and Myc-E-Syt3 were selectively 742 recruited by eGFP-Stx3. (B) GFP-Trap using Sec22b-pHL as a, the presence of either eGFP-Stx3, or eGFP (negative control)

-. Myc-e-syt2-or-myc, Matrix-bound material was processed as in A, B with the indicated 744 antibodies. (D) Stx1/3 immunoprecipitation from rat embryonic cortex. Naive rabbit IgGs were used 745 as negative control. Total cells lysate (Input) and immunoprecipitated material (IP) were processed 746 for SDS-PAGE and Western blotting. Blots were probed with antibodies directed against the 747 endogenous proteins, p.748

, Immunoblots of material recovered after GFP-Trap pull-down from NGF-treated PC12 cell lysates

, Cells were transfected for co-expression of Myc-E-Syt2 and the indicated GFP-tagged SNAREs and 750 processed as in A-C. Blots were revealed with antibodies against the indicated six target proteins

, Only Sec22b-pHL, but not its Longin-deleted version Sec22b?L-pHL or the other tested SNAREs, 752 could recruit Myc-E-Syt2. GFP-Trap pull-down of eGFP was used as control for non-specific binding

, Quantification of the ratio between Myc-E-Syt2 signal and the GFP signal given by the 754 immunoprecipitated GFP-tagged vSNAREs (left graph) and tSNAREs (right graph, p.755

, Quantification of the ratio between Myc-E-Syt2 signal and the GFP signal (left graph) and between 756

, endogenous SNAP25 signal and the GFP signal (right graph) given by the immunoprecipitated 757

, One-way ANOVA followed by Dunnett's multiple comparison post-758 test, Sec22b-pHL vs Sec22b?L-pHL

, Figure 2. E-Syt2 and Sec22b are abundantly in close proximity in neurites and growth 761 cones

, Duolink proximity ligation assay (PLA) for protein interactions in situ was performed in HeLa cells (A) 763

, Representative confocal images are shown for the indicated 764 antibody combinations using mouse anti-Sec22b, 3DIV hippocampal neurons (C)

, Lower 768 panel is a higher magnification of region outlined in A1. C1-3, PLA dots. C4-6, MAP2 769 immunofluorescence staining superimposed to fields shown in C1-3. Lower panels are a higher 770 magnification of regions outlined in C1 and C4. Scale bar, 10 µm. (B, D, E) Quantification of PLA is 771 expressed as dots per HeLa cell (B), and in 3DIV hippocampal neurons as dots per µm 2 of surface 772 area in neurites (D), growth cones (E) or in cell body (F), Negative controls consisted in using anti-Sec22b or anti-E-Syt2 antibody only

, It is higher in neurites and growth cones as compared to cell bodies in 775 neurons. One-way ANOVA followed by Dunn's multiple comparison post-test

, Analysis of E-Syt2 / Sec22b colocalization using super-resolution microscopy

, Representative confocal images of a 3DIV hippocampal neuron labeled for endogenous E-Syt2 780 (green) and Sec22b-pHL (red) and plasma membrane (gray)

, Germ Agglutinin (WGA) was used to label the plasma membrane (grey), p.782

, Super-resolution (3D STED) 3D reconstruction of the inset in (A) showing localization of endogenous 783

, Statistical association was assessed through spatial 784 distribution analysis using Ripley's function (Icy SODA Plugin). 3D STED reconstruction of Sec22b 785 (in green) and E-Syt2, E-Syt2 and Sec22b-pHL in the growth cone. (C)

, Topological scheme illustrating the measured distance (d) between associated Sec22b-E-Syt2

, d was estimated to an average of 67.12nm+/-1.22 in four different growth cones 789 for 4229 clusters and its median was: 33.6nm

, Duolink proximity ligation assay (PLA) for protein interactions in situ was performed in HeLa cells 794 either non transfected or overexpressing eGFP-E-Syt3 (A) and in HeLa cells expressing siRNAs 795 targeting E-Syt1, E-Syt2 and E-Syt3 simultaneously (E)