Figure 2
Detection of colocalized GABAB by a proximity ligation assay (PLA): Samples were incubated with primary antibodies and secondary probes, ligated and amplified. The red fluorescent PLA signal corresponds to spatial proximity, while BR2-mCit alone emits yellow fluorescence. Analysis was performed by a confocal laser scanning microscope. (A) Conceptualization of PLA using Merck Duolink PLA Kit. (B) Representative coexpression of BR1 and BR2 to detect heterodimeric assembly. Primary antibodies were previously validated (Fig. 1d,e). (C) Negative control: Separate CFPS of BR1 and BR2-mCit translocated into different microsomes. Primary antibodies were targeting BR2 and the fused His-tag of the nicotinic acetylcholine receptor subunit delta (nAChRd). (D) Negative control: Coexpression of nAchRd with BR2-mCit. Primary antibodies were targeting the nAchRd His-Tag and BR2-mCit. (E) Negative Control: Coexpression of glucagon-like peptide 1 receptor (GLP1R) with BR2-mCit. Primary antibodies were directed against the GLP1R His-Tag and BR2-mCit. (F) Negative control: CFPS approach without coding DNA-templates. Primary antibodies were previously validated (Fig. 1d,e).
