Figure 4
From: Ubiquitin-proteasome dependent degradation of GABAAα1 in autism spectrum disorder
SYVN1 is the E3 ligase associated with GABA A α1. (A) GABAAα1 co-precipitated with SYVN1. Lysates from primary cortical neurons (DIV 14) were subjected to immunoprecipitation (IP) using an anti-GABAAα1 antibody followed by western blotting (WB) with SYVN1 or GABAAα1 antibody. In reverse IP, neuronal lysates were immunoprecipiated with an anti-SYVN1 antibody followed by western blotting with GABAAα1 or SYVN1 antibody. Lysate represents 10% of the amount used in the IP. IgG, IgG control. Results are representative of three independent experiments. (B) Inhibition of proteasomal activity increased the expression of GABAAα1 in the ER. Primary cortical neurons (DIV 14) were treated with vehicle (control; DMSO) or MG132 (20 μM) for 9 h, and the co-localization of GABAAα1 and ER marker (PDI) was examined by immunofluorescence method. (C) SYVN1 knockdown increased GABAAα1 protein levels in cortical neurons. Primary cortical neurons (DIV 14) were transfected with control siRNA or SYVN1siRNA, and the protein levels of SYVN1 and GABAAα1 were examined at 48 h after transfection. The upper panel shows representative autoradiogram of SYVN1, GABAAα1, and GAPDH, and the lower panel represents fold change in normalized GABAAα1 protein levels. Results are mean ± SE for at least three independent neuronal preparations. (D) Reduced interaction between GABAAα1 with SYVN1 in ASD subjects. Lysates from middle frontal gyrus of ASD and control subjects were subjected to IP using an anti-GABAAα1 antibody followed by WB with SYVN1 or GABAAα1 antibody. Lysate represents 10% of the amount used in the IP. IgG, IgG control.