Fig. 3

Proximity ligation assay was used to assess interactions (represented by white dots) between IRIS-1 with VAMP2 (A with quantification in B) and IRIS-2 with VAMP2 (C with quantification in D) under high potassium stimulation. Proximity ligation assay was used to assess the SNARE complex formation under high potassium stimulation in SH-SY5Y cells treated with siRNA negative control and siRNA targeting CD59, IRIS-1, and 2. The following complexes were assessed: VAMP2 and SNAP-25 (E with quantification in F), VAMP2 and Syntaxin1 (G with quantification in H), Syntaxin 1 and SNAP-25 (I with quantification in J). n = 3, biological repeats in (A-J). (K) Subcellular fractionation of SH-SY5Y cells analyzed through western blot showing that IRIS-1 and 2 localizes within the same cellular compartments as VAMP2. The purity of fractions were assessed with specific antibodies form disulfide isomerase (PDI), specific for membranes, and β-tubulin, enriched in cytosolic fraction. n = 3. (L) Noradrenaline secretion from SH-SY5Y cells with or without CD59, IRIS-1, and 2 knockdown. Data are represented as a fold change of secreted noradrenaline in cells stimulated with 100 mM potassium (evoked noradrenaline release) compared to cells stimulated with 5 mM potassium (baseline noradrenaline secretion, represented as a dotted line). The amount of secreted noradrenaline in 5 mM and 100 mM potassium was normalized to total protein content, representing the number of cells. Statistics (in B, D, F, H, J, and L): two-tailed student T-test. The thickness of the Z-stack varies between 19.8 and 39.6 μm