Journal: The Journal of Neuroscience
Article Title: GluN2B-Containing NMDA Receptors Regulate AMPA Receptor Traffic through Anchoring of the Synaptic Proteasome
Figure Lengend Snippet: Proteomic analysis of the PSD fraction of GluN2B −/− cortical neurons. A , PSDs were successfully purified from high-density rat cortical cultures (14–15 DIV). Western blot for markers of the presynaptic (Synaptophysin) and postsynaptic fractions (PSD-95, SynGAP, NMDAR receptor subunits GluN1 and GluN2A, and AMPAR subunit GluA1) validated PSD isolation of PSDs. Transferrin receptor (Transf R) and β-actin were used as controls. Hom, Homogenate; S1, non-nuclear fraction; P2 crude synaptosomes. Equal amounts (13 μg) of each fraction were applied. B , PSD-localized proteins (PSD-95, GluN1, SynGAP) are increased relative to the homogenate in the PSD fraction, whereas presynaptic synaptophysin (Synapto.) or the trans membrane receptor of transferrin are decreased compared with the P2 fraction, consistent with their absence or low expression, respectively, at the PSD. Actin levels did not change in any of the fractions analyzed. Data are presented as mean ± SEM from two independent experiments. C , Setup of the 8-plex iTRAQ experiments. Isolated PSDs from GluN2B +/+ , GluN2B +/− , and GluN2B −/− cortical neurons, 10 μg each, were digested with trypsin and samples (three wild-types, two heterozygous, and three knock-outs) were tagged with a set of 8-plex iTRAQ reagents. Peptides were pooled together and fractionated by 2D liquid chromatography and subject to tandem mass spectrometric analysis. After using peptides for protein identification, the individual contribution of each sample to each peptide can be measured by the intensity of the reporter ion peaks and used for relative quantification. Example spectra correspond to one of the GluN1 peptides identified in the PSD preparations. D , Chymotrypsin-like proteasome activity was measure in protein extracts from GluN2B +/+ and GluN2B −/− cortical neurons. Proteasome activity is significantly decreased ( p
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), phospho-CaMKII α/β 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 anti-phospho-protein kinase C (PKC) substrates (1:1000, #2261S) were from Cell Signaling Technology; anti-SynGAP (1:1000, #PA1-046) and anti-PSD-95 (1:500, #MA 1.046 C7E3-1B8) were from Affinity Bioreagents; anti-Synaptophysin (1:10000, #101 011) was from Synaptic Systems; anti-CaMKII β (K-19) (1:500, #sc-100366) was from Tebu-Bio, Santa Cruz Biotechnology); CaMKII α (6G9) (1:2000, #C265) was from Sigma-Aldrich; 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-β-actin (1:5000, #1378996) was from Roche Molecular Biochemicals.
Techniques: Purification, Western Blot, Isolation, Expressing, Two-dimensional Liquid Chromatography, Activity Assay