Fig. 6: Inhibition of the function of Syt I by STAND-A36 expression in vitro.
From: An ultra-stable cytoplasmic antibody engineered for in vivo applications
a Binding of purified STAND-A36 to indicated GST-fused proteins, as determined by an enzyme-linked immunosorbent assay (n = 3 independent samples per group). Statistical analysis: two-tailed one-way ANOVA: absorbance, F (2, 6) = 3497, P < 0.00001; Tukey’s multiple comparison test: GST-C2A vs. GST-C2B: P < 0.00001, GST-C2A vs. GST: P < 0.00001, GST vs. GST-C2B: P = 0.98987). b Western blot analysis of total mouse brain homogenates using purified STAND-A36 as a primary antibody. c Intracellular interaction of STAND-A36, but not of STAND-M4, with Syt I expressed in 293 T cells co-transfected with Syt I-expressing vector and indicated vectors. Immunoprecipitates obtained using anti-HA antibody were analysed using western blotting with an anti-Flag antibody to detect scFv and anti-Syt I antibodies. d Decreased interaction of Syt I with the Syx1B-SNARE domain by the expression of STAND-A36. The cell lysate described in c was incubated with purified GST or GST-Syx1-SNARE, then subjected to immunoprecipitation with the anti-GST antibody. Immunoprecipitates were analysed using western blotting with an anti-Syt I antibody. e Quantification of the relative amount of Syt I protein co-immunoprecipitated with GST-Syx1-SNARE described in d (n = 8 independent experiments per group). Statistical analysis: Mann-Whitney U test, P = 0.0021645. f–h STAND-A36 expression in cultured dopamine neurons inhibited high potassium-induced dopamine release. f Fluorescent images of dopaminergic neurons (DIV 14) expressing STAND-A36 (upper panels) and STAND-M4 (lower panels) (Left: anti-HA antibody to detect STAND proteins; middle: anti-TH antibody to detect dopaminergic neurons, right: merged images of left and middle panels). Arrows show dopaminergic neurons, which did not express STAND proteins. Scale bar, 20 μm. g Transfection efficiency of STAND proteins in dopaminergic neurons. h Measurement for high potassium-induced dopamine release normalised with a low potassium-induced one in cultured dopamine neurons. Statistical analysis: two-tailed t-test, P = 0.0311. Error bars represent standard error of the mean. Source data are provided as a Source Data file.
