Preparation of Stable HEK293 Cell Lines expressing GABAARs
To test the target selectivity of α5-SOP002, a stable cell line of HEK293 cells expressing α5β2γ2 subunits of the GABAAR was developed using the previously established method based on antibiotic selection (Brown et al., 2016). HEK293 cells (2 × 106) were transfected using Lipofectamine LTX (catalog no. 15338-100, Invitrogen) with the α5 pcDNA3.1(+) construct, incorporating the G418 disulfate (Neomycin) resistance gene and β2 pcDNA3.1(+) construct, incorporating the Zeocin resistance gene. Cells were subsequently plated at the ratios of 1:3, 1:5, 1:7, 1:10, 1:15, and 1:20, and selected with G418 (Neomycin; catalog no. G5013, Sigma) and Zeocin (catalog no. R25001 Gibco) antibiotics (both at 800 μg/ml) until colonies were formed. After 7 days, ∼5–20 single colonies were selected and gradually scaled up. The clone expressing the highest level of GABAAR α5 and β2 subunits, as well as the previously established α2β2-HEK293 (Brown et al., 2016) stable cell line were further transfected with the γ2 pcDNA3.1(+) construct, incorporating the Hygromycin resistance gene, in order to produce triple cell lines. Expression of all three subunits was characterised by immunoblotting and immunocytochemistry. The α1β2γ2-HEK293 was characterised previously (Fuchs et al., 2013).