- 43-7700 - Invitrogen Antibodies CYLD antibody

Submitted references Necroptosis mediates TNF-induced toxicity of hippocampal neurons.

Liu S, Wang X, Li Y, Xu L, Yu X, Ge L, Li J, Zhu Y, He S
BioMed research international 2014;2014:290182

Direct activation of RIP3/MLKL-dependent necrosis by herpes simplex virus 1 (HSV-1) protein ICP6 triggers host antiviral defense.

Wang X, Li Y, Liu S, Yu X, Li L, Shi C, He W, Li J, Xu L, Hu Z, Yu L, Yang Z, Chen Q, Ge L, Zhang Z, Zhou B, Jiang X, Chen S, He S
Proceedings of the National Academy of Sciences of the United States of America 2014 Oct 28;111(43):15438-43

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Jurkat cell lysate separated by SDS-PAGE and probed with CYLD Monoclonal Antibody

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of CYLD was achieved by transfecting U-87 MG with CYLD specific siRNAs (Silencer® select Product # S590). Western blot analysis (Fig. a) was performed using Whole cell extracts from the CYLD knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with CYLD Monoclonal Antibody (733) (Product # 43-7700, 1:1000 dilution) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution). 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 CYLD.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-CYLD Monoclonal Antibody (733)(Product # 43-7700) and a 107kDa band corresponding to CYLD was observed across cell lines tested. Whole cell extracts (30 µg lysate) of U-87 MG (Lane 1), HEK-293 (Lane 2) and Hep G2 (Lane 3) were electrophoresed using NuPAGE™ 10% Bis-Tris Protein Gel (Product # NP0301BOX). 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 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)
Main image
Experimental details: Jurkat cell lysate separated by SDS-PAGE and probed with CYLD Monoclonal Antibody

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of CYLD was done on 70% confluent log phase SHSY5Y cells. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton™ X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with CYLD Mouse Monoclonal Antibody (Product # 43-7700) at 1 µg/mL in 1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d is a merged image showing cytoplasmic localization. Panel e is a no primary antibody control. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of CYLD showing staining in the cytoplasm of paraffin-embedded mouse brain tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10 mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a CYLD Mouse Monoclonal antibody (Product # 43-7700) diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

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

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 4 RIP1 and its deubiquitinase CYLD are required for TNF- alpha -induced necrosis of HT-22 cells. (a) HT-22 cells were transfected with the negative control or RIP1 siRNAs. After 60 h, cells were treated with control or TNF- alpha /z-VAD for another 20 h and then cell viability was determined by measuring ATP levels. Data were represented as mean +- standard deviation of duplicates. * P < 0.01, ** P < 0.001 versus NC-T + Z. (b) The knockdown efficiency of RIP1 RNAi. Cell lysates were collected 60 h after transfection and subjected to western blot analysis of RIP1 and beta -actin levels. (c) HT-22 cells were transfected with the negative control or CYLD siRNAs. Forty-eight hours after transfection, cells were treated with control or TNF- alpha /z-VAD for another 20 h and then cell viability was determined by measuring ATP levels. Data were represented as mean +- standard deviation of duplicates. * P < 0.01, ** P < 0.001 versus NC-T + Z. (d) The knockdown efficiency of CYLD RNAi. Cell lysates were collected 60 h after transfection and subjected to western blot analysis of CYLD and beta -actin levels. All experiments were repeated three times with similar results.

43-7700

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