glun1  (OriGene)

Journal: bioRxiv

Article Title: A GluN2B disease-associated variant promotes degradation of NMDA receptors via autophagy

doi: 10.1101/2025.01.12.632651

Figure Lengend Snippet: (A) Structure of the rat tetrameric GluN1_GluN2B NMDA receptor in complex with glutamate, with the GluN1 subunit in gray, and the GluN2B subunit in blue (PDB:9ARI) . The amino-terminal domain (ATD), ligand-binding domain (LBD), and transmembrane domain (TMD) are shown in ribbon format with the R519 residue selected for study in yellow spheres. (B) Residues involved in the glutamate binding site of the WT GluN2B subunit are represented a sticks, with the glutamate ligand in pink (upper). Mutagenesis of the R519 residue to a glutamine eliminates the electrostatic interaction with glutamate (lower). (C) Effects of R519Q on total GluN2B protein expression levels 48 hrs post transient transfection of HEK293T with GluN1 and GluN2B constructs at a 1:1 ratio to express WT or GluN2B_R519Q variant NMDARs (n=7). β-actin serves as the soluble total protein loading control. ( D) Surface biotinylation assay to monitor the influence of the R519Q DAV on the surface expression of NMDARs 48 hrs post transient transfection. Na + /K + ATPase served as a membrane protein loading control (n=8). (E) HEK293T cells stably expressing WT or R519Q GluN2B NMDARs were subjected to cyclohexmide (100 µg/mL) for the indicated times to determine the stability and rate of degradation (n=5). (F) Immunofluorescence images of GluN2B (green) and calnexin (red) an ER marker to assess ER accumulation (scale bar = 20 μm). Pearson’s coefficients are reported (n>30) and statistical significance was determined using a Mann-Whitney test. All data normalized to the appropriate loading control and data are presented as mean ± SEM. Statistical significance was determined using an unpaired two-tailed Student’s t-test between two groups or an analysis of variance (ANOVA) followed by a post-hoc Dunnett’s test for comparison in multiple groups. Significance level defined as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: The pcDNA3.1-GRIN1 (OHu22255D, NM_007327, human) and the pcDNA3.1-GRIN2B (OHu26128D, NM_000834, human) and pcDNA3.1-CCPG1 (OHu07897C, NM_004748.5) were obtained from GenScript.

Techniques: Ligand Binding Assay, Residue, Binding Assay, Mutagenesis, Expressing, Transfection, Construct, Variant Assay, Control, Surface Biotinylation Assay, Membrane, Stable Transfection, Immunofluorescence, Marker, MANN-WHITNEY, Two Tailed Test, Comparison

Journal: bioRxiv

Article Title: A GluN2B disease-associated variant promotes degradation of NMDA receptors via autophagy

doi: 10.1101/2025.01.12.632651

Figure Lengend Snippet: (A) Validation of HEK293T ATG7 KO line. Immunoblot was used to quantify levels of ATG7 in HEK293T and ATG7 KO lines. p62 was used to assess impaired autophagic flux (n=3). (B) Effects of R519Q on total GluN2B protein expression levels 48 hrs post transient transfection with GluN1 and GluN2B constructs at a 1:1 ratio in ATG7 KO HEK293T to express WT or GluN2B_R519Q variant NMDARs (n=6). β-actin served as the soluble total protein loading control. (C) Surface biotinylation assay to monitor the influence of the R519Q DAV on the surface expression of NMDARs 48 hrs post transient transfection (n=6). Na + /K + ATPase served as a membrane protein loading control. (D) Inhibition of the proteasome with MG132 (10 µM) and the lysosome with Baf-A1 (1 µM) for 6 hrs on the effect of the GluN2B subunit in ATG7 KO HEK293T cells stably expressing recombinant WT or R519Q NMDARs (n=3). β-actin served as the soluble total protein loading control. (E) Ratio of the normalized accumulation of R519Q/WT GluN2B upon treatment with Baf-A1 and MG132. (F) Immunofluorescence images of GluN2B (green) and calnexin an ER marker (red) to assess ER accumulation. Mander’s coefficients are reported, where M1= ratio of GluN2B/Calnexin and M2= ratio of Calnexin/GluN2B (scale bar: 20 μm, n>=30) and statistical significance was determined using a Mann-Whitney test. All data normalized to the appropriate loading control and data are presented as mean ± SEM. Statistical significance was determined using an unpaired two-tailed Student’s t-test between two groups or an analysis of variance (ANOVA) followed by a post-hoc Tukey test for comparison in multiple groups. Significance level defined as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: The pcDNA3.1-GRIN1 (OHu22255D, NM_007327, human) and the pcDNA3.1-GRIN2B (OHu26128D, NM_000834, human) and pcDNA3.1-CCPG1 (OHu07897C, NM_004748.5) were obtained from GenScript.

Techniques: Western Blot, Expressing, Transfection, Construct, Variant Assay, Control, Surface Biotinylation Assay, Membrane, Inhibition, Stable Transfection, Recombinant, Immunofluorescence, Marker, MANN-WHITNEY, Two Tailed Test, Comparison

Journal: bioRxiv

Article Title: A GluN2B disease-associated variant promotes degradation of NMDA receptors via autophagy

doi: 10.1101/2025.01.12.632651

Figure Lengend Snippet: ( A ) Canonical LC3b interacting motif consensus sequence pointing to the LIR motif identified in the GluN2B subunit and subsequent alanine substitution. Aromatic residue shown in red, hydrophobic residue in blue. (B) Sequence alignment of Homo sapiens GluN2A and GluN2B CTD amino acid sequence in the region of interest. The LIR motif is in red in the GluN2B subunit. The serine residue that is phosphorylated by CAMKII is shown in green for each subunit. (C) Amino acid sequence alignment of mammalian species demonstrating species conservation of the GluN2B CTD region of interest surrounding the LIR motif. (D) Effects of LIR motif disruption on WT, WT_F1307A and R519Q, R519Q_F1307A total GluN2B protein expression levels 48 hrs post transient transfection of HEK293T cells transfected with a 1:1 ratio of GluN1 and GluN2B constructs to express WT or GluN2B_R519Q variant NMDARs (n=4). β-actin served as the soluble total protein loading control. (E) Surface biotinylation assay to monitor the influence of the LIR domain disruption on the WT and R519Q DAV surface expression of NMDARs 48 hrs post transient transfection (n=3). Na + /K + ATPase served as a membrane protein loading control. (F) Effects of LIR motif disruption on total expression of GluN2B subunits expressed in ATG7 KO cells. 48 hrs post transient transfection of GluN1 and GluN2B constructs with a 1:1 ratio of GluN1 and GluN2B constructs to express WT, WT_F1307A, R519Q, R519Q_F1307A variant NMDARs (n=4). β-actin served as the soluble total protein loading control. (G) Surface biotinylation assay to monitor the influence of the LIR motif disruption on WT and the R519Q variant GluN2B subunits on the surface expression of NMDARs in ATG7 KO cells 48 hrs post transient transfection (n=4). Na + /K + ATPase served as a membrane protein loading control. All data normalized to the appropriate loading control and data are presented as mean ± SEM. Statistical significance was determined using an unpaired two-tailed Student’s t-test between two groups or an analysis of variance (ANOVA) followed by a post-hoc Tukey test for comparison in multiple groups. Significance level defined as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: The pcDNA3.1-GRIN1 (OHu22255D, NM_007327, human) and the pcDNA3.1-GRIN2B (OHu26128D, NM_000834, human) and pcDNA3.1-CCPG1 (OHu07897C, NM_004748.5) were obtained from GenScript.

Techniques: Sequencing, Residue, Disruption, Expressing, Transfection, Construct, Variant Assay, Control, Surface Biotinylation Assay, Membrane, Two Tailed Test, Comparison