- PA5-78704 - Invitrogen Antibodies ABHD5 antibody

Submitted references Cancer-derived exosomal TRIM59 regulates macrophage NLRP3 inflammasome activation to promote lung cancer progression.

Liang M, Chen X, Wang L, Qin L, Wang H, Sun Z, Zhao W, Geng B
Journal of experimental & clinical cancer research : CR 2020 Aug 31;39(1):176

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of ABHD5 in rat kidney extract (lane 1) and A431 whole cell lysate (lane 2). Sample was incubated with ABHD5 polyclonal antibody (Product # PA5-78704) at a dilution of 0.5 µg/mL. Signal development was performed using a chemiluminescence (ECL) kit.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-ABHD5 Rabbit Polyclonal Antibody (Product # PA5-78704) and a 40kDa band corresponding to ABHD5 was observed across cell lines and tissues tested except in Mouse Skeletal Muscle which is reported negative for expression. Whole cell extracts (30 µg lysate) of 3T3-L1 (Lane 1), 3T3-L1 differentiated to adipocytes (Lane 2), Mouse Adipose (Lane 3), Rat Adipose (Lane 4), Mouse Brown Fat (Lane 5) and Mouse Skeletal Muscle (Lane 5) 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 (0.5ug/ml dilution) and detected by chemiluminescence Goat Anti-Rabbit IgG Secondary Antibody, HRP conjugate (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: Immunocytochemistry analysis of Abhd5 using anti-Abhd5 antibody (Product # PA5-78704) . Abhd5 was detected in a section of A431 cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent for 15 mins. The cells were blocked with 10% goat serum and then incubated with 2μg/mL rabbit anti-Abhd5 antibody (Product # PA5-78704) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Rabbit IgG was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of ABHD5 was performed using 70% confluent log phase 3T3-L1 cells differentiated to adipocytes. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with ABHD5 Polyclonal Antibody (Product # PA5-78704) at 1:100 dilution in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) 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 Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing increased ABHD5 expression along with localization to surface of fat globules as well as cytoplasm. Panel e shows untreated cells with lower expression of ABHD5. Panel f 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: Fig. 4 ABHD5 deficiency promotes NLRP3 inflammasome activation in macrophages. a . Western blot analysis of ABHD5 expression in macrophages transfected with scrambled control shRNA or ABHD5 shRNA for 36 h. Immunoblot of the IL-1beta, the pro-caspase-1 and cleaved caspase-1 in the supernatants (SNs) or cell lysates of ABHD5-silenced THP-1 macrophages, primed with LPS, and then stimulated with Nigericin (Nig. ), ATP or Alum. beta-actin served as a loading control. Quantification of Western blotting were performed with the Image J software. Numbers below each blot indicate relative band intensity normalized to beta-actin. b . ELISA of IL-1beta in supernatants from THP-1 macrophages silenced of ABHD5, primed with LPS for 8 h, and followed by stimulation with ATP, Nig., Alum, poly(dA:dT) or flagellin for 30 min. c . ELISA of IL-1beta in supernatants from BMDMs silenced of ABHD5, primed with LPS for 8 h, and followed by stimulation with ATP, Nig., Alum, poly(dA:dT) or flagellin for 30 min. d . RT-PCR analysis of IL-1beta mRNA expression in macrophages transfected with shRNA as indicated and stimulated as indicated. e. ELISA of IL-1beta in supernatants from THP-1 cells infected with lentiviral vectors expressing ABHD5 or GFP control, primed with LPS for various times, and followed by stimulation with ATP for 30 min. f . ELISA of IL-1beta in supernatants from THP-1 cells infected with lentiviral vectors expressing TRIM59 or GFP control, primed with LPS, and followed by stimulation wi

PA5-78704

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