Feline Lindhout

VAP–SCRN1 interaction regulates dynamic endoplasmic reticulum remodeling and presynaptic function 131 4 VAPA shRNA #2 (5′-GGAAACTGATGGAAGAGTG-3′; Teuling et al, 2007), and VAPB shRNA #1 (5′-GGTGATGGAAGAGTGC-3′; Teuling et al, 2007); and SCRN1 shRNA #1 (5′-GATCCTTCCAGGTCCATAT-3′), SCRN1 shRNA#2 (5′-GCACTTACATCTCAATTGA-3′), and SCRN1 shRNA #3 (5′-CAGGCTTGGTTTAGAACGA-3′). Antibodies The following primary antibodies were used for this study: rabbit anti-VAPA (homemade #1006-04; Teuling et al, 2007) and anti-VAPB (homemade #1006-00; Teuling et al, 2007); mouse anti-synaptotagmin (SySy, 105311, clone 604.2); rabbit anti-SCRN1 (SySy, 289003; used in Fig 1E), rabbit anti-SCRN1 (Abcam, ab105355; used in Fig S1D), and rabbit anti- SCRN1 (Sigma, HPA024517, RRID:AB_2184811; used for all other experiments; validation SCRN1 antibodies in Fig S1G–J); guinea pig anti-vGlut (Millipore, ab5095); rat anti-HA (Roche, 1867423; used for immunostainings); mouse anti-HA (BioLegend, mms-101p, clone 16B12; used for immunoblots); mouse anti-actin (Chemicon, MAB1501R, clone C4); rabbit anti-GFP (Abcam, ab290); mouse anti-Myc (Santa Cruz, SC40, clone 9E10); and mouse α-Tubulin (Sigma, T5168, clone B-5-1-2, RRID:AB_477579). The following secondary antibodies were used for this study: anti-rabbit Alexa 488 (Life Technologies, A11034), anti-rabbit Alexa 568 (Life Technologies, A11036), anti-rat Alexa 568 (Life Technologies, A11077), anti-guinea pig Alexa 568 (Life Technologies, A11075), anti-mouse Alexa 647 (Life Technologies, A21236), anti-mouse anti-HRP (Dako, P0260), anti-rabbit anti-HRP (Dako, P0399), anti-mouse IRDye 680LT (Li-Cor, 926-68020), and anti-rabbit IRDye 800CW (Li-Cor, 926-32211). Tissue extracts, cell extracts, and immunoblotting To generate tissue extracts for Western blot and mass spectrometry analysis, different brain regions (cerebellum, cortex, hippocampus, midbrain, brainstem, and spinal cord) or whole brains were dissected from adult female rats. Samples were homogenized in ice-cold homogenization buffer (150 mM NaCl, 50 mM Tris, 0.1% SDS, 0.2% NP-40, pH 7.8) supplemented with 1× complete protease inhibitor cocktail (Roche), sonicated, and centrifuged (15 min, 900 g, 4°C). Protein concentrations of supernatant were measured using a BCA protein assay (Pierce). Next, 20 μg protein per sample was resuspended in SDS sample buffer and boiled for 5 min at 95°C. To generate cell extracts for Western blot analysis, transfected HEK293T cells were washed and harvested in ice-cold PBS. Cells were centrifuged (5 min, 300 g, 4°C), and the pellet was resuspended in ice-cold lysis buffer (100 mM Tris, 150 mM NaCl, 1% Triton, pH 7.5) supplemented with 1× complete protease inhibitor cocktail (Roche). Cell lysates were centrifuged (5 min, 20,000 g, 4°C), and supernatant was resuspended in SDS sample buffer and boiled for 10 min at 100°C. Samples were resolved on SDS–page gels and transferred to nitrocellulose membranes (Bio-Rad) or polyvinylidene difluoride membranes (Millipore). Membranes were blocked for 30 min with PBS-T (PBS with 0.05% Tween) with 2% BSA. Next, membranes were sequentially incubated with primary and secondary antibodies diluted in PBS-T with 2%