- PA5-95826 - Invitrogen Antibodies PYCARD antibody

Sun X, Duan J, Gong C, Feng Y, Hu J, Gu R, Xu B
Journal of the American Heart Association 2022 Jul 5;11(13):e025266

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of extracts of various cell lines, using PYCARD Polyclonal antibody (Product # PA5-95826) at 1:1000 dilution. Secondary antibody: HRP Goat Anti-Rabbit IgG (H+L) at 1:10000 dilution. Lysates/proteins: 25ug per lane. Blocking buffer: 3% nonfat dry milk in TBST. Exposure time: 30s.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western Blot analysis of PYCARD in extracts of various cell lines using PYCARD Polyclonal Antibody (Product # PA5-95826) at a dilution of 1:1000. A HRP Goat Anti-Rabbit IgG (H+L) secondary antibody was used at a dilution of 1:10,000. Lysates/proteins: 25 µg per lane. Blocking buffer: 3% nonfat dry milk in TBST.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockout of PYCARD was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR1008587_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of PYCARD was performed by loading 20 µg of THP-1 wild type (Lane-1), THP-1 Cas9 (Lane-2) and THP-1 PYCARD KO (Lane-3) whole cell extracts. The samples were electrophoresed using NuPAGE™ Novex™ 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 PYCARD Polyclonal Antibody (Product # PA5-95826, 1:2000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:10,000 dilution) using the iBright™ FL1500 (Product # A44115). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to PYCARD. Uncharacterized band was observed in ~100 kDa in all samples.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-PYCARD Polyclonal Antibody (Product # PA5-95826) and a 21kDa band corresponding to Apoptosis-associated speck-like protein containing a CARD was observed across the cell lines tested and tissues tested. Whole cell extracts (30 µg lysate) of MCF7 (Lane 1), THP-1 (Lane 2), HL-60 (Lane 3), K-562 (Lane 4), HEK-293 (Lane 5), HeLa (Lane 6), Jurkat (Lane 7), Mouse Spleen (Lane 8), Rat Spleen (Lane 9), Mouse Liver (Lane 10), Rat Liver (Lane 11) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). 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 (1:100) 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)
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Experimental details: Immunocytochemistry-Immunofluorescence analysis of PYCARD was performed in U-2 OS cells using PYCARD Polyclonal Antibody (Product # PA5-95826).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunocytochemistry-Immunofluorescence analysis of PYCARD was performed in U-2 OS cells using PYCARD Polyclonal Antibody (Product # PA5-95826).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry analysis of PYCARD in paraffin-embedded human placenta using PYCARD Polyclonal Antibody (Product # PA5-95826) at a dilution of 1:200.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunoprecipitation analysis of PYCARD was performed in 200 µg extracts of U937 cells using PYCARD Polyclonal Antibody (Product # PA5-95826). Western blot was performed from the immunoprecipitate using PYCARD Polyclonal Antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 NLRP3 (NOD-like receptor protein 3) inflammasome pathway was inhibited by colchicine treatment. A and B , Representative immunoblots and the corresponding analysis of NLRP3 inflammasome pathway in cardiac tissues at the end point of experiment. GAPDH shows as loading control. C , Representative immunofluorescence images of cardiac tissues staining with ASC (green), cTNT (cardiac troponin T) (red), and DAPI (blue). Frequency of ASC speck is shown on the right (n=6-8 each group). Five images per mouse are evaluated. Scale bar=50 mum. Data are shown as mean+-SEM. All data statistical significance are determined by 1-way ANOVA (Tukey post-test). cTNT indicates cardiac troponin T; DCM, dilated cardiomyopathy; IL-1beta, interleukin-1beta; and NLRP3, NOD-like receptor protein 3. * P

PA5-95826