rabbit polyclonal gabaar α1 subunit antibody  (Alomone Labs)

Journal: Cell research

Article Title: Gephyrin-mediated formation of inhibitory postsynaptic density sheet via phase separation.

doi: 10.1038/s41422-020-00433-1

Figure Lengend Snippet: Fig. 1 Phase separation of the GPHN-E/GlyR-βLD complex. a Schematic diagrams showing the pentameric subunit assembly of GlyR or GABAAR. The gephyrin binding core sequences in the cytoplasmic TM3–4 loops of the receptors are highlighted by an orange line and the amino acid sequences are shown. The figure also shows the domain organization of gephyrin. The positions of mutations introduced into the recombinant receptor loops and gephyrin proteins, as well as three potential phosphorylation sites in the gephyrin C-domain are indicated. b Representative SDS-PAGE of sedimentation experiments (upper panel) and quantification of relative (line graph, left y-axis) and absolute (bar graph, right y-axis) amount of proteins recovered in the supernatant (S, gray columns) and pellet (P, blue columns) (lower panel) in the assay. Proteins were mixed at the indicated concentrations. Data from three different batches of experiments were presented as means ± SD. c DIC and fluorescence images showing that mixtures of Cy3-GPHN-E and 488-GlyR-βLD at the 1:1 molar ratio and indicated concentrations formed phase-separated droplets. The boxes show a 5× zoom-in analysis of a droplet at the 5 μM of the protein concentration. Scale bars, 10 μm. d Time-lapse imaging showing the fusion of two small droplets into a larger one. The concentration of the protein mixture was 20 μM. Scale bars, 5 μm. e Time-lapse imaging showing that membrane-tethered 488-GlyR-βLD gradually formed clusters on lipid bilayers upon the addition of 100 nM Cy3-GPHN-E. Scale bars, 2 μm. f Fluorescence images showing that the clustering patterns of membrane-tethered 488- GlyR-βLD on lipid bilayers depended on the concentration of Cy3-GPHN-E. Cy3-GPHN-E was added at the indicated concentrations. Scale bars, 2 μm. g Top panel: representative fluorescence images showing that co-expression of mCherry-GPHN-E with GFP-GlyR-βLD in HeLa cells led to the formation of many puncta with the two proteins colocalized together in each punctum. These puncta are not enriched with membranes (Fig. S4a). The dashed box is magnified and shown at right. Scale bars, 20 μm. Scale bars for the zoomed-in images are 2 μm. Bottom panel: fluorescence intensity line-scanning plots showing that both proteins were concentrated and colocalized together in the two bright puncta shown in the zoomed-in images.

Article Snippet: The genes encoding GABAAR α1-3 subunits (from Addgene plasmid # 49168, # 49169, and custom-synthesized based on the sequence of NM_000808, respectively) were each subcloned into pET.32M3C with an N-terminal His6-tag and a dimeric (or trimeric) GCN4 coiled-coil domain69 (referred to as His6-GlyR-βLD or His6-GABAAR-αLD).

Techniques: Binding Assay, Recombinant, Phospho-proteomics, SDS Page, Sedimentation, Protein Concentration, Imaging, Concentration Assay, Membrane, Fluorescence, Expressing

Journal: Cell research

Article Title: Gephyrin-mediated formation of inhibitory postsynaptic density sheet via phase separation.

doi: 10.1038/s41422-020-00433-1

Figure Lengend Snippet: Fig. 7 A schematic model showing GABAAR/GlyR binding-induced iPSD formation by gephyrin. a Clustering of GABAAR/GlyR on the synaptic plasma membrane and formation of the gephyrin sheet beneath the synaptic membrane via receptor binding-induced phase separation. Note that the width of the gephyrin E-domain dimer is ~11 nm, and the distance between the two GABAAR receptors in iPSD is also ~11 nm.28 b A top-down view from the extracellular side of the synaptic membranes showing the phase separation-mediated GABAAR/ GlyR clustering and organization by the gephyrin condensates. c A schematic diagram showing progressive steps of zoomed-in views of an inhibitory synapse to show the formation of the iPSD sheet via phase separation.

Article Snippet: The genes encoding GABAAR α1-3 subunits (from Addgene plasmid # 49168, # 49169, and custom-synthesized based on the sequence of NM_000808, respectively) were each subcloned into pET.32M3C with an N-terminal His6-tag and a dimeric (or trimeric) GCN4 coiled-coil domain69 (referred to as His6-GlyR-βLD or His6-GABAAR-αLD).

Techniques: Binding Assay, Clinical Proteomics, Membrane