Journal: The Journal of Neuroscience

Article Title: GluN2B-Containing NMDA Receptors Regulate AMPA Receptor Traffic through Anchoring of the Synaptic Proteasome

doi: 10.1523/jneurosci.3567-14.2015

Figure Lengend Snippet: Figure 3. GluN1 and GluN2A total and surface levels are decreased in GluN2B / cortical neurons. Membrane extracts from cortical cultures from GluN2B /, heterozygous GluN2B /, and GluN2B / mice at 7 DIV (A) or 15 DIV (B) were used to evaluatetotalGluN1andGluN2AproteinlevelsbyWesternblot.C,D,TotalGluN1andGluN2Aproteinlevelswerenormalizedwith transferrinreceptor(TransfR)levelsandpresentedaspercentageoftheproteinlevelsdetectedinwild-typeneurons7DIV.Dataare presentedasmean SEMfromthreetofourindependentexperiments.*p0.05,**p0.01,***p0.0001,one-wayANOVA followed by Bonferroni’s multiple-comparisons test. Relative mRNA levels for GluN1 (E) and GluN2A (F) in 15 DIV mouse cortical culturesweredeterminedbyquantitativereal-timePCR.Dataarepresentedasmean SEMfromfiveindependentexperiments forGluN1mRNAlevelsandfourindependentexperimentsforGluN2AmRNAlevels.p0.05,pairedttest.G,Totalextractsandcell surface biotinylated-purified proteins isolated from high-density cortical culture 15 DIV from GluN2B / and / mice were assayed by Western blot using an antibody against GluN1. H, Transferrin receptor protein levels were used as a loading control. GluN1 levels are normalized to GluN1 levels in wild-type neurons. Data are presented as mean SEM from three independent experiments. **p 0.01, paired t test.

Article Snippet: Primary: anti-GluN1 (1:500 WB, #MAB363), anti-GluN2A (1:1000, #AB1555P), anti-GluA1 (1:1000 WB or 1:100 ICC, #AB1504), anti-GluA2 (1:1000 WB or 1:500 ICC, #MAB397), anti-phospho-GluA1 Ser845 (1:1000, #AB5849), anti-phospho-GluA2 Ser880 (1:1000, #07- 294), phospho-stargazin (Ser239/240) (1:500, #ab3713), phosphoCaMKII / subunit (Thr286/287) (1:1000, #06 – 881) were from Millipore; anti-GluN1 (1:1500 ICC, #MAb 54.1) was from Invitrogen; N-terminal of GluA1 (1:50) was a kind gift from Dr. Andrew Irving; anti-PSD-95 (D27E11) (1:2000 WB or 1:100 ICC, #3450) and antiphospho-protein kinase C (PKC) substrates (1:1000, #2261S) were from Cell Signaling Technology; anti-SynGAP (1:1000, #PA1-046) and antiPSD-95 (1:500, #MA 1.046 C7E3-1B8) were from Affinity Bioreagents; anti-Synaptophysin (1:10000, #101 011) was from Synaptic Systems; anti-CaMKII (K-19) (1:500, #sc-100366) was from Tebu-Bio, Santa Cruz Biotechnology); CaMKII (6G9) (1:2000, #C265) was from SigmaAldrich; anti-phospho-GluA1 Ser 831 (1:5000, #2041) was from Tocris Bioscience; anti-phospho-GluA1 T840 was a kind gift from R. Huganir (JH2811); anti-Transferrin receptor antibody (1:1000, #13-6800) was from Invitrogen; anti-vesicular glutamate transporter 1 (VGLUT1; 1:5000, #AB5905) was from Millipore; anti-MAP2 (1:5000, #ab5392) was from Abcam; and anti- -actin (1:5000, #1378996) was from Roche Molecular Biochemicals.

Techniques: Membrane, Purification, Isolation, Western Blot, Control