- 711020 - Invitrogen Antibodies SLC1A2 antibody

Submitted references Semaphorin 4D is upregulated in neurons of diseased brains and triggers astrocyte reactivity.

Evans EE, Mishra V, Mallow C, Gersz EM, Balch L, Howell A, Reilly C, Smith ES, Fisher TL, Zauderer M
Journal of neuroinflammation 2022 Aug 6;19(1):200

ALS-associated genes in SCA2 mouse spinal cord transcriptomes.

Scoles DR, Dansithong W, Pflieger LT, Paul S, Gandelman M, Figueroa KP, Rigo F, Bennett CF, Pulst SM
Human molecular genetics 2020 Jun 27;29(10):1658-1672

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of PC-12 (Lane 1), U-87 MG (Lane 2), SH-SY5Y (Lane 3), Neuro-2a (Lane 4), K-562 (Lane 5), HEL (Lane 6). The blots were probed with Anti-EAAT2 Recombinant Rabbit Polyclonal Antibody (Product # 711020, 1-2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A 62 kDa band corresponding to EAAT2 was observed across cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12% Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-GLT-1 Polyclonal Antibody, (Product # 711020) and band at ~55 kDa corresponding to GLT-1 was observed in Mouse and Rat Brain and Cerebellum but not in Kidney and Lung which are reported to be negative. Tissue extracts (30 µg lysate) of Mouse Brain (Lane 1), Mouse Cerebellum (Lane 2), Mouse Kidney (Lane 3), Mouse Lung (Lane 4), Rat Brain (Lane 5), Rat Cerebellum (Lane 6), Rat Kidney (Lane 7) and Rat Lung (Lane 8) were electrophoresed using Novex® NuPAGE® 4-12% % Bis-Tris gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (2µg/ml) and detected by chemiluminescence with Goat anti- Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 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)
Main image
Experimental details: Immunofluorescence was performed on fixed and permeabilized U-87 MG cells for detection of endogenous EAAT2 using Anti-EAAT2 Recombinant Rabbit Polyclonal Antibody (Product # 711020, 2 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Panel a) shows representative cells that were stained for detection and localization of EAAT2 protein (green), Panel b) is stained for nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). Panel c) represents cytoskeletal F-actin staining using Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d) is a composite image of Panels a, b and c clearly demonstrating membrane localization of EAAT2. Panel e) represents control cells with no primary antibody to assess background.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 5 SEMA4D triggers receptor-mediated astrocyte reactivity, including changes in astrocyte morphology, expression of key transporters for glutamate recycling and energy metabolism and impairs astrocyte function of glucose uptake. a Primary human astrocyte cultures were stained for PLXN receptors (blue), compared to isotype control antibodies (red). Antibody blocking effect was determined by incubation with rSEMA4D or control protein, in presence/absence of anti-SEMA4D antibody/VX15 (human IgG4) or isotype-matched control antibodies for 48 h. Cultures were stained for b EAAT-2, and c GLUT-1 and MCT-4 transporters. d In a separate experiment, blocking of receptors was assessed using anti-PLXNB1 (mouse IgG1) and/or anti-PLXNB2 (mouse IgG2a) or isotype-matched control antibodies and the same conditions as above. Quantification is shown as; mean + SEM of replicates for each treatment. e Glucose uptake was measured in human astrocyte cultures treated as above. rSEMA4D was added at time 0 and antibodies were added at t = 0 (solid lines and circles) for inhibition or t = 24 h (dotted lines and triangles) to evaluate reversal of activity. Quantification for each condition is shown as average + SEM from 3 wells/condition/timepoint. f Morphologic changes showing length and number of primary processes. For b to f , multivariate regression analysis was performed to determine the effect treatment conditions and dependent variables with treatment type. The significance levels are reporte

711020

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