Feline Lindhout

VAP–SCRN1 interaction regulates dynamic endoplasmic reticulum remodeling and presynaptic function 141 4 Supplementary Figure 1. VAPs modulate bouton maintenance and are associated with brain-enriched SCRN1 proteins A. Localization of exogenous HA-VAPA or HA-VAPB in neurons (DIV18) co-expressing GFP- Sec61β and immunostained for bassoon. Zooms represent (1) an axonal structure with bas- soon-positive presynaptic boutons (arrowheads), and (2) a dendritic structure. Scale bars: 10 μm (full size) and 5 μm (zoom). B. Quantifications of bouton density in hippocampal neurons (DIV18) co-expressing RFP and pSu- per empty vector or VAPA/B shRNAs. N = 2, n = 80 boutons. C. Quantifications of bouton size in hippocampal neurons (DIV18) co-expressing RFP and pSuper empty vector or VAPA/B shRNAs. N = 2, n = 380–400 boutons. D. Western blot of endogenous SCRN1 expression in indicated adult rat neuronal and non-neuronal tissues. Cereb., cerebellum. Hippoc., hippocampus. Spin., spinal. E. Scaled representation of SCRN1-associated proteins identified with pull-down assay followed by mass spectrometry analysis of purified BioGFP or BioGFP-SCRN1 from HEK293T cell lysates. Selected candidates all showed > 10 enrichment of PSM compared to control. F. Localization of exogenous GFP-SCRN1 in hippocampal neurons (DIV18) immunostained for vGlut. Zoom represents an axon structure with presynaptic sites (arrowheads). Scale bars: 10 μm (full size) and 5 μm (zoom). G. COS7 cells expressing BioGFP-SCRN1, BioGFP-SCRN2, or BioGFP-SCRN3 and immunostain- ed for SCRN1. Scale bar: 10 μm. H. Western blot of lysates from HEK293T cells expressing BioGFP-SCRN1, BioGFP-SCRN2, or BioGFP-SCRN2 and immunostained for indicated antibodies. Arrowheads represent (1) BioG- FP-SCRN1 expression, (2) endogenous SCRN1 expression, (3) full-length BioGFP-SCRN proteins, and (4) N-terminal cleaved Bio GFP-SCRN2 and Bio GFP-SCRN3. Actin was used as loading control. I. Cortical neurons (DIV4) co-expressing RFP with pSuper empty vector (control) or SCRN1 shRNA #1. Scale bar: 10 μm. J. Quantifications of fluorescence intensity of endogenous SCRN1 in cortical neurons (DIV4) co-ex- pressing RFP with pSuper empty vector (control) or SCRN1 shRNA #1, #2, or #3. N = 2, n = 13–14 cells. Data information: Data represent mean ± SEM; **P < 0.01; ***P < 0.001, by Mann–Whitney U-test. Source data are available online for this Figure.