MA1-046

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of PSD95 was performed by loading 25 µg of mouse brain (lane 1) and rat brain (lane 2) cell lysates onto an SDS polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked at 4ºC overnight. The membrane was probed with a PSD95 monoclonal antibody (Product # MA1-046) at a dilution of 1:200 overnight at 4°C, washed in TBST, and probed with an HRP-conjugated secondary antibody for 1 hr at room temperature in the dark. Chemiluminescent detection was performed using Pierce ECL Plus Western Blotting Substrate (Product # 32132). Results show a band at ~95 kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockout of DLG4 was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, AssayID CRISPR53838_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of DLG4 was performed by loading 50 µg of U-87MG wild type (Lane 1), U-87MG CAS9 (Lane 2) and U-87MG DLG4 KO (Lane 3) whole cell extracts. The samples were electrophoresed using Novex® NuPAGE® 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with Anti-PSD-95 Monoclonal Antibody (7E3-1B8)(Product # MA1-046) using 1:500 dilution and Goat anti-Mouse IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4,000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using SuperSignal™ West Dura Extended Duration Substrate (Product # 34076). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to DLG4. An uncharacterized band was observed at ~50 kDa in HeLa wild type sample.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-PSD-95 Monoclonal Antibody (7E3-1B8) (Product # MA1-046) and a 80 kDa band corresponding to PSD-95 was observed across Mouse Brain and Rat Brain. Tissue extracts (30 µg lysate) of Mouse Skeletal Muscle (Lane 1), Mouse Liver (Lane 2), Mouse Brain (Lane 3) and Rat Brain (Lane 4) were electrophoresed using NuPAGE™ 10% Bis-Tris Protein Gel (Product # NP0302BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23002) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:2000 dilution) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence staining of PSD-95 in rat hippocampal cells with MA1-046 yields a staining pattern coincident with NMDA receptor.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Rat hippocampal neurons (21 days in vitro) were stained using a monoclonal PSD-95 antibody (red, Product # MA1-046) and a monoclonal MAP2 antibody (green, MA5-12826). DAPI staining was shown in blue.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of PSD95 using Anti-PSD95 Monoclonal Antibody (7E3-1B8) (Product # MA1-046) shows staining in C6 Cells. PSD95 staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing PSD95 (Product # MA1-046) at a dilution of 1:20 over night at 4°C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35503, Goat Anti-Mouse). Images were taken at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of PSD95 using Anti-PSD95 Monoclonal Antibody (7E3-1B8) (Product # MA1-046) shows staining in Hela Cells. PSD95 staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing PSD95 (Product # MA1-046) at a dilution of 1:20 over night at 4°C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35503, Goat Anti-Mouse). Images were taken at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence staining of PSD-95 in rat hippocampal cells with MA1-046 yields a staining pattern coincident with NMDA receptor.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow cytometry analysis of PSD95 in Neuro-2a cells compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10^6 cells/mL, fixed with 2% paraformaldehyde and washed with PBS. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with a PSD95 monoclonal antibody (Product # MA1-046) at a dilution of 2 µg/test for 60 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody and re-suspended in PBS for FACS analysis.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow cytometry analysis of PSD95 in SH-SY5Y cells compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10^6 cells/mL, fixed with 2% paraformaldehyde and washed with PBS. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with a PSD95 monoclonal antibody (Product # MA1-046) at a dilution of 2 µg/test for 60 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody and re-suspended in PBS for FACS analysis.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow cytometry analysis of PSD95 in U87-MG cells compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10^6 cells/mL, fixed with 2% paraformaldehyde and washed with PBS. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with a PSD95 monoclonal antibody (Product # MA1-046) at a dilution of 2 µg/test for 60 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody and re-suspended in PBS for FACS analysis.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1 PSD-95 is in a complex with ephrin-B3 at synapses a) ST imulated E mission D epletion (STED) imaging of PSD-95 and ephrin-B3 at synapses. DIV21 tdTomato transfected rat cortical neurons were labeled with the indicated antibodies. PSD-95 and ephrin-B3 were imaged at super-resolution using STED (~80 nm). VGlut1 and tdTomato were imaged at conventional resolution (~250 nm) using confocal. Images are high contrast examples of PSD-95, ephrin-B3 and vGlut1 immunostaining. Arrows indicate PSD-95, ephrin-B3 and vGlut1 co-clusters in dendritic spines. Scale bar: 2 um. b) PSD-95, ephrin-B3 and vGlut1 are found in most (>=80%) spines (248 spines on 15 neurons, two separate experiments). c) PSD-95-ephrin-B3 co-clusters are found predominantly in vGlut + spines (162 PSD-95-ephrin-B3 containing spines out of 202 vGlut1 + spines). Single PSD-95 (14 out of 202 vGlut + spines) or ephrin-B3 (20 out of 202 vGlut + spines) clusters are rarely found at synapses. d-f) Co-immunoprecipitation (co-IP) of ephrin-B3 and PSD-95 from P21 wild type and Efnb3 -/- mouse whole brain lysates. The efficacy of ephrin-B3 IP (e) and input (f) are shown for each condition. (g-i) PSD-95-ephrin-B3 co-IP from synaptosomes of wild type and Efnb3 -/- animals (g). Control immunoblots indicating the amount of ephrin-B3 IP (h) and input (i) . (j) PSD-95 selectively co-IPs with ephrin-B3 in synaptosomes isolated from P21 wild type brains despite the efficient IP (k) and presence (l) of each of the three ephrin-B f

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 Ephrin-B3 regulates subcellular PSD-95 localization (a) Western blots of non-synaptic (S1), crude synaptosomal (P2) and pure synaptosomal (Syn.) fractions from cortices of P10 and P21 wild type and Efnb3 -/- mice probed with the indicated antibodies. Less synaptic enrichment of PSD-95 is observed in synaptosomes from Efnb3 -/- mice. (b) Model and predicted outcomes for the organotypic slice culture experiment. (c-d) Representative two-photon images of cortical pyramidal neurons from wild type and Efnb3 -/- organotypic slices transfected with PSD-95-GFP and the indicated ephrin-B3 rescue constructs. Right panels in c and d are the high contrast representations of tdTomato and PSD-95-GFP. Filled arrowheads indicate PSD-95-GFP localized to a spine; open arrowheads show PSD-95-GFP localized in the dendritic shaft. PSD-95-GFP is diffusely localized in dendritic shafts of Efnb3 -/- neurons (c) and ephrin-B3 shRNA-transfected wild type neurons (d) . Scale bar in c, d : 5 um. (e) The ratio of average GFP pixel intensities in dendritic shafts to the GFP intensities per pixel in puncta was used to measure the fraction of diffuse PSD-95-GFP in c-d (ANOVA, F (4, 93) = 7.155, p-values: wild type// Efnb3 -/- : 0.0105, wild type// Efnb3 -/- + S332D: 0.0233, Efnb3 -/- // Efnb3 -/- + wt-eB3: 0.0285, Efnb3 -/- // Efnb3 -/- + S332A: 0.0015, Efnb3 -/- + wt-eB3// Efnb3 -/- + S332D: 0.0487, Efnb3 -/- + S332A// Efnb3 -/- + S332D: 0.0027; wild type (n = 22), Efnb3 -/- (n = 31), Efnb3 -/- +

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 Ephrin-B3 links MAPK signaling to PSD-95-ephrin-B3 interaction and localization ( a) Sequence alignment of intracellular domains (ICD) of ephrin-B (eB1-3) family. ERK binding D-Domain in the juxtamembrane region of ephrin-B3 (yellow box) and a putative MAPK phosphorylation motif (green box). Red boxes indicate the positions of point mutations in the D-domain (L293A) and in the putative MAPK phosphorylation motif (S332A/S332D). ( b) Immunoprecipitation, using antibodies to Flag, of HEK 293T lysates transfected with PSD-95-GFP and indicated flag-tagged ephrin-B3 (flag-eB3) constructs. (c) Quantification of co-IP experiments shown in b (ANOVA, F (3, 23) = 9.842. WT-S332D: p = 0.0475, WT-L293A: p = 0.0034, S332A-S332D: p = 0.0087, S332A-L293A: p = 0.0006; n = 6 blots from separate transfections). (d) In vitro generated flag-tagged wild type and mutant ephrin-B3 ICDs used to pull down PSD-95-GST. Direct interaction between the two proteins is disrupted by S332D ephrin-B3 ICD. (e) Quantification of pull-down experiments from d (ANOVA, F (2, 9) = 2.823, Fisher's LSD post-hoc, p = 0.0176 (WT-S332D), p = 0.0257 (S332A-S332D); n = 4 independent in vitro binding experiments). (f) Representative high contrast STED images of DIV21 neurons transfected with tdTomato, ephrin-B3 (eB3) shRNA and indicated flag-eB3 rescue constructs. Arrowheads indicate co-localized, flag-ephrin-B3/PSD-95/vGlut1 clusters. Scale bars : , 1 um. (g) Quantification of spine localization of flag-ephrin

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 ERK phosphorylation of S332 regulates subcellular localization of ephrin-B3 (a) Schematic representation of the experiments. (b) Representative immunoblots of lysates from DIV14 rat cortical neurons treated with TTX (1uM, 4 hr.) and U0126 (10uM, 4 hr.) to block activity and ERK activation, respectively. Depolarization with KCl (55mM, 1 hr.) was used to elevate ERK activity. ( c ) Ratio of pS332 to ephrin-B3 signals was used to quantify the effect of neuronal activity on S332 phosphorylation (ANOVA, F (3, 12) = 19.27, Tukey's post-hoc, TTX-KCl: p = 0.0012, TTX+U0126-KCl: p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 11. Altered composition of the PSD in mice with heterozygous deletion of synGAP . Ratios of amounts of the indicated proteins to PSD-95 in each lane were measured as described in 'Materials and methods' and are reported as mean +- S.E. For all blots except those for neuroligin-1, PSD-95 was detected with a secondary Ab labeled with AlexaFluor680 and the binding protein was detected with secondary Ab labeled with IRDye 800. On the neuroligin-1 blot, both PSD-95 and neuroligin-1 were detected with AlexaFluor680; the two bands were well-separated in each lane. Representative sets of visualized bands for WT and HET from the same blot are shown below the graphs. Individual points represent the ratio of the indicated protein to PSD-95 in a single lane (n refers to these technical replicates). The WT and HET PSD preparations were each made from six animals; thus, each of the two preparations represents six biological replicates. ( A ) SynGAP to PSD-95 ratio. Data were collected for 22 lanes from two blots containing 5 ug total PSD fraction per lane. One blot contained six lanes WT and six lanes HET samples, the other contained five lanes of each. The mean ratio of synGAP to PSD-95 was 0.234 +- 0.012 for WT (n = 11) and 0.179 +- 0.005 (n = 11) for HET (-24%). Means were compared by unpaired, one-tailed t-test with Welch correction, p = 0.0007. Effect size, d = 1.75. ( B ) TARP g -2,3,4,8 to PSD-95 ratio. Data were collected for 24 lanes from two blots containing 10 u