Journal: Cell reports

Article Title: Regulation of NMDA receptor trafficking and gating by activity-dependent CaMKIIα phosphorylation of the GluN2A subunit

doi: 10.1016/j.celrep.2021.109338

Figure Lengend Snippet: (A) ITC experiments comparing the binding of GluN2A C-terminal peptides, either WT (blue), or the S1459A phospho-deficient (orange), S1459E phospho-mimetic (green), or S1462E PDZ binding-deficient (purple, negative control) mutant, to the purified SNX27 PDZ domain protein. The thermodynamic parameters of binding are given in . (B) GST pull-down experiments from HEK293T cells co-transfected with plasmids encoding myc-SNX27 and GST alone or GST-GluN2A C-tails including WT, S1459A (SA), S1459E (SE), and the V1464E PDZbm mutant (PDZd). Total cell lysates (input) and bound proteins (pull-down) were resolved by SDS-PAGE and analyzed by western blotting with specific antibodies against myc, VPS35, VPS26, and GST. (C–E) Quantification of the levels of myc-SNX27 (C), VPS35 (D), and VPS26 (E) binding to GST-GluN2A C-tails. Data represent mean ± SEM of band intensities normalized to WT values (n = 3–4; from three independent experiments). *p < 0.05, ***p < 0.001, ****p < 0.0001 using one-way ANOVA with a Tukey’s multiple comparison test. (F) GST pull-down experiments revealed that the H112A mutation in myc-SNX27, which disrupts binding to PDZbm, failed to bind both WT and the S1459E phospho-mimetic GST-GluN2A C-tails in HEK293T cells. (G) HEK293T cells were co-transfected with plasmids encoding myc-SNX27, GST-GluN2A C-tails (WT or S1459A), together with pEGFP or pEGFP-tCaMKIIa (constitutively active truncated CaMKIIα). Cells were lysed and pulled down with GSH-Sepharose. Bound proteins and total lysates were analyzed by immunoblotting with specific antibodies against myc, GST, GluN2A pS1459, and GFP. (H) Quantification of the level of myc-SNX27 binding to GST-GluN2AC-tails. Data represent mean ± SEM of band intensities normalized to WT values(n = 3; from three independent experiments). *p < 0.05 using two-way ANOVA with a Sidak’s multiple comparison test.

Article Snippet: Briefly, HEK293 cells were transfected with cDNAs encoding human GluN1 and SEP-tagged rat GluN2A subunits, plus empty pEGFP and mouse neuroligin-1B (Addgene #15261, in the pCAGGS expression vector) in a ratio of 1:1:1:0.5:1, using a calcium-phosphate co-precipitation protocol.

Techniques: Binding Assay, Negative Control, Mutagenesis, Purification, Transfection, SDS Page, Western Blot, Comparison

Journal: Cell reports

Article Title: Regulation of NMDA receptor trafficking and gating by activity-dependent CaMKIIα phosphorylation of the GluN2A subunit

doi: 10.1016/j.celrep.2021.109338

Figure Lengend Snippet: KEY RESOURCES TABLES

Article Snippet: Briefly, HEK293 cells were transfected with cDNAs encoding human GluN1 and SEP-tagged rat GluN2A subunits, plus empty pEGFP and mouse neuroligin-1B (Addgene #15261, in the pCAGGS expression vector) in a ratio of 1:1:1:0.5:1, using a calcium-phosphate co-precipitation protocol.

Techniques: Western Blot, Recombinant, Protease Inhibitor, In Situ, Suspension, Plasmid Preparation, Software