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. 2016 Mar 24;7:11080. doi: 10.1038/ncomms11080

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(a,b) BRAG1's C terminus interacts with PSD-95 in vitro. (a) Protein overlay assay demonstrating the interaction between WT BRAG1 and recombinant PSD-95. Top: HEK293 cell lysates expressing FLAG-WT BRAG1, FLAG-BRAG1 C-terminal deletion mutant (FLAG-BRAG1ΔCt) or no plasmid (untransfected) were run on the gel, and the membrane was overlaid with recombinant GST-PSD-95, and stained with antiserum UCT80 against PSD-95. Middle and bottom: the blot was also probed with anti-FLAG to detect total BRAG1 in the lysates and with anti-tubulin as a loading control. (b) Left: GST-PSD-95 and GST were separated by SDS–polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane, which was overlaid with a HEK293 cell lysate overexpressing FLAG-WT BRAG1 and stained with antiserum C3 against BRAG1. BRAG1 binds to GST-PSD-95, but not GST (indicated by the *). Right: protein overlay assay with FLAG-BRAG1ΔCt. GST-PSD-95 and GST were run on the gel, and the membrane was overlaid with a HEK293 cell lysate overexpressing FLAG-BRAG1 ΔCt and stained with anti-FLAG to detect BRAG1. WT BRAG1 but not BRAG1ΔCt binds to GST-PSD-95. Molecular weight markers in kDa are shown on the left. (c–e) Insets: sample traces of AMPAR- and NMDAR-mediated synaptic responses recorded at −60 (left) and +40 mV (right), respectively. Scale bars, 20 pA, 20 ms. Data represent averaged evoked EPSCs recorded for AMPA (left graphs), NMDA (centre graphs) and AMPA/NMDA ratios (right graphs) simultaneously from pairs of untransfected (control) CA1 neurons and neurons transfected with BRAG1-ΔCt (c; AMPA: n=18, P=0.24; NMDA: n=18, P=0.24; AMPA/NMDA: n=15, P=0.12) or BRAG1 (d,e). In d and e, shortly after BRAG1 transfection, TAT-BRAG1-Ct peptide (d; AMPA: n=10, P=0.67; NMDA: n=7, P=0.25; AMPA/NMDA: n=7, P=0.97) or TAT-Ct control peptide (e; AMPA: n=7, P=0.031; NMDA: n=8, P=0.42, AMPA/NMDA: n=7, P=0.047) was added to the culture media. * Indicates significance (P≤0.05).