psd 93 mouse n18 30 igg1  (NeuroMab)

Journal: eNeuro

Article Title: Myosin Va Brain-Specific Mutation Alters Mouse Behavior and Disrupts Hippocampal Synapses

doi: 10.1523/ENEURO.0284-20.2020

Figure Lengend Snippet: Synaptic transport of proteins to PSD is altered in Flr mice. WT FLR and Flr hippocampal synaptosome extracts were used for quantitative Western blot analyses of synaptic proteins. A1 , A2 , Levels of full-length MyoVa and TLS/FUS were reduced in Flr extracts. B1 , B2 , Expression levels of major MAGUKs (PSD-95, PSD-93, SAP102) and ( C1 , C2 ) SAPAP1, SHANK1, SHANK3, Homer1b/c, and IP3R were significantly reduced in Flr. D1 , D2 , mGluRs, NMDAR, AMPAR subunit levels, and FMRP were not changed in Flr when compared with WT FLR . Homogenate (Homo) and synaptosome (Syn) was prepared from whole hippocampus. Protein levels in Flr were quantified and plotted as % of WT FLR ; n = 3 samples per group/each sample pool three to five animals. Unpaired t test was used for statistical analysis. Error bars represent ±SEM; * p ≤ 0.05, ** p ≤ 0.001.

Article Snippet: Primary antibodies used were: rabbit anti-MyoVa (Sigma, LE18), mouse anti-TLS (Santa Cruz, sc-47 711), rabbit anti-mGluR1 (Millipore, 07-617), rabbit anti-mGluR5 (Neuromics, RA16100), mouse anti-fragile X mental retardation protein (FMRP; Millipore, 1C3), mouse anti-PSD-95 (NeuroMab, K28/43), mouse anti-PSD-93 (NeuroMab, N18/30), mouse anti-SAP102 (NeuroMab, N19/2), mouse anti-SAPAP1 (NeuroMab, N238/29), mouse anti-Shank1 (NeuroMab, N22/21), mouse anti-Shank3 (NeuroMab, N367/62), mouse anti-GluN1 (NeuroMab, N308-48), rabbit anti-GluN2A (Millipore, A12W), mouse anti-GluN2B (NeuroMab N59/20 and N59/36), mouse anti-GluA2 (NeuroMab, L21/32), mouse anti-homer 1 (NeuroMab, L113/130), mouse anti-IP3R (NeuroMab, N18/30), rabbit anti-tubulin β III (Abcam, ab6046), and mouse anti-actin (Sigma, AC-74).

Techniques: Western Blot, Expressing

Journal: Scientific Reports

Article Title: PSD-95 deficiency disrupts PFC-associated function and behavior during neurodevelopment

doi: 10.1038/s41598-019-45971-w

Figure Lengend Snippet: PSD-95 deficiency alters specific AMPAR and NMDAR-subunit protein expression levels in the mPFC at postnatal day 35, but not postnatal day 21. ( A ) Left panel shows representative blots for AMPAR subunits (GluA1 & GluA2) and NMDAR subunits (GluN1, GluN2A, GluN2B, and GluN3A) in control (Con) versus PSD-95 knockout (KO) mice at P21. All proteins were normalized to actin. Right panel displays summary graphs of protein expression levels in arbitrary units (a.u.) of GluA1 (p = 0.28, Con, n = 7, KO, n = 8), GluA2 (p = 0.39, n = 6), GluN1 (p = 0.23, Con, n = 7, KO, n = 10), GluN2A (p = 0.85, Con, n = 8, KO, n = 10), GluN2B (p = 0.51, Con, n = 7, KO, n = 10), GluN3A (p = 0.15, Con, n = 8, KO, n = 9) in Con vs. KO mice. ( B ) Left panel shows representative blots for AMPAR subunits, NMDAR subunits and actin at P35 in con vs. KO mice. Right panel displays summary graphs of protein expression levels in a.u. in GluA1 (p = 0.04, n = 7), GluA2 (p = 0.51, Con, n = 8, KO, n = 5) GluN1 (p = 0.002, Con, n = 10, KO, n = 8); GluN2A (p = 0.18, Con, n = 9, KO, n = 8) GluN2B (p = 0.02, n = 7), GluN3A (p = 0.08, Con, n = 9, KO, n = 8) in Con vs. KO mice. PSD-95 deficiency causes a significant increase in SAP-102 that leads to an increase in interaction of GluN2B in the mPFC. ( C ) Upper panel shows representative western blots of SAP-102 and PSD-93 in control versus PSD-95 knockout mice. Both proteins were normalized to actin. Lower panel displays summary graphs of protein expression levels of SAP-102 and PSD-93 in Con vs. KO mice (SAP-102, p = 9 × 10 −7 , n = 7; PSD-93, p = 3 × 10 −6 , Con, n = 7, KO, n = 6). ( D ) Upper panel shows blots of SAP-102 and GluN2B co-immunoprecipitation in Con vs. KO mice. GluA1 was used as a negative control. Lower panel displays a summary graph of protein levels of GluN2B and SAP-102 interaction (GluN2B/SAP-102, p = 0.03, n = 5). All full length/uncropped western blots are presented in Supplementary Fig. . *p < 0.05, **p < 0.01, ****p < 0.0001, n.s., not significant.

Article Snippet: Membranes were blocked with 5% non-fat dry milk in TBST (0.05% Tween-20 in 1X Tris-buffered saline) for 1 hr and incubated in the following dilutions of primary antibodies for 1 hr: mouse monoclonal anti-GluN1 (1:2000, ThermoFisher Scientific Cat# 32-0500, RRID: AB_2533060), rabbit monoclonal anti-GluN2A (1:1000, Millipore Cat# 04-901, RRID: AB_1163481), mouse monoclonal anti-GluN2B (1:1000, Millipore Cat# 05-920, RRID: AB_417391), rabbit polyclonal anti-GluN3A (1:1000, Millipore Cat# 07-356, RRID:AB_2112620), mouse monoclonal anti-GluA1 N-terminus (1:1000, Millipore Cat# MAB2263, RRID: AB_11212678), mouse monoclonal anti-GluA2 (1:1000, Millipore Cat# MABN71, RRID: AB_10806492), rabbit polyclonal anti-PSD-95 (1:5000, Millipore Cat# AB9708, RRID:AB_2092543), rabbit polyclonal anti-SAP-102 (1:1000, ThermoFisher Scientific Cat# PA5-29116, RRID:AB_2546592), and mouse monoclonal anti-PSD-93 (1:1000, Millipore Cat# MABN497).

Techniques: Expressing, Knock-Out, Western Blot, Immunoprecipitation, Negative Control