- MA5-32618 - Invitrogen Antibodies TNFRSF1B antibody

Sadri M, Hirosawa N, Le J, Romero H, Martellucci S, Kwon HJ, Pizzo D, Ohtori S, Gonias SL, Campana WM
Glia 2022 Feb;70(2):256-272

Pawar AS, Eirin A, Tang H, Zhu XY, Lerman A, Lerman LO
Cytokine 2020 Mar 30;130:155080

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of TNFR2 in different lysates using a Monoclonal antibody (Product #MA5-32618) at a dilution of 1:500. Positive control: Line 1: MCF-7, Line 2: Jurkat.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of TNFR2 in different lysates using a Monoclonal antibody (Product #MA5-32618) at a dilution of 1:500. Positive control: Line 1: MCF-7, Line 2: Jurkat.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of Tumor necrosis factor receptor superfamily member 1B was achieved by transfecting U-937 with Tumor necrosis factor receptor superfamily member 1B specific siRNAs (Silencer® select Product # S14268, S14269). Western blot analysis (Fig. a) was performed using Membrane enriched extracts from the Tumor necrosis factor receptor superfamily member 1B knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with TNFR2 Recombinant Rabbit Monoclonal Antibody (JM113-01) (Product # MA5-32618, 1:1000 ) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to Tumor necrosis factor receptor superfamily member 1B.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-TNFR2 Recombinant Rabbit Monoclonal Antibody (JM113-01) (Product # MA5-32618) and a 60 kDa band corresponding to Tumor necrosis factor receptor superfamily member 1B was observed. Membrane enriched extracts (30 µg lysate) of THP-1 (Lane 1), SW480 (Lane 2), COLO 205 (Lane 3) and NK-92 (Lane 4) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein 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 (1:1000) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000) 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: Immunocytochemical analysis of TNFR2 in MCF-7 cells using a TNFR2 Monoclonal antibody (Product # MA5-32618) as seen in red. The nuclear counter stain is DAPI (blue). Cells were fixed in paraformaldehyde, permeabilised with 0.25% Triton X100/PBS.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunocytochemical analysis of TNFR2 in Hela cells using a TNFR2 Monoclonal antibody (Product # MA5-32618) as seen in red. The nuclear counter stain is DAPI (blue). Cells were fixed in paraformaldehyde, permeabilised with 0.25% Triton X100/PBS.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunocytochemical analysis of TNFR2 in SW480 cells using a TNFR2 Monoclonal antibody (Product # MA5-32618) as seen in red. The nuclear counter stain is DAPI (blue). Cells were fixed in paraformaldehyde, permeabilised with 0.25% Triton X100/PBS.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of Tumor necrosis factor receptor superfamily member 1B was performed using 70% confluent log phase THP-1 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with TNFR2 Recombinant Rabbit Monoclonal Antibody (JM113-01) (Product # MA5-32618) at 1:100 in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790), (1:2000), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing Plasma Membrane localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow Cytometric analysis of TNFR2 in HL-60 cells using a TNFR2 Monoclonal Antibody (Product # MA5-32618) at a dilution of 1:50, as seen in red compared with an unlabelled control (cells without incubation with primary antibody; black). Alexa Fluor 488-conjugated goat anti rabbit IgG was used as the secondary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: 1 FIGURE TNFR1 is selectively sequestered into SC EVs. (a) SC EVs isolated by UC from conditioned media (CM) of primary cultured SCs were analyzed by NTA and had an average particle size of 153 +- 2 nm; n = 3 independent experiments. (b) Transmission electron microscopy (TEM) using negative staining of EVs. Note large crescent shaped particles and smaller particles. Scale bar, 200 nm. (c) Immunoblot analysis of whole SC lysates (SC-L) and SC-derived EVs to detect the exosome biomarkers, Flotillin-1, TSG101, CD9, ALIX, and CD81. GM130 is a Golgi biomarker not found in exosomes. (d) Immunoblot analysis to detect the SC biomarkers, p75 NTR and myelin protein zero, P0, and non-myelinating marker, GFAP. Images represent n = 3-5 independent experiments. (e) TNFalpha receptors, TNFR1 (55-kDa) and TNFR2 (65-kDa), and P0 in extracts of SCs cultured in complete medium (SC-L1) or in DMEM with 10% FBS depleted of EVs (SC-L2), in SC-EVs, and in bone marrow derived macrophages (BMDMs) (2 mug/lane) were determined by immunoblot analysis. (f) Immunoblot of TNFR1 levels in EVs derived from SCs treated with and without TNFalpha. TSG101 (44-kDa) shows load control in cells and presence in EVs. (g) Quantification of TNFR1 levels in SC EV immunoblots (2 mug). Data are expressed as mean +- SEM ; ( n = 4-5/group)

MA5-32618