Rabbit anti asc

Manufactured by Abcam

Rabbit anti-ASC is a primary antibody that recognizes the ASC (Apoptosis-associated speck-like protein containing a CARD) protein. ASC is an adaptor protein involved in the activation of the NLRP3 inflammasome. This antibody can be used for the detection of ASC in various applications such as Western blotting, immunocytochemistry, and immunohistochemistry.

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4 protocols using rabbit anti asc

Western Blotting Analysis of Neuroinflammatory Markers

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As previously described by us,
⁴⁰ (link) proteins were extracted from the MCA‐supplied brain regions and loaded onto SDS‐polyacrylamide gel for electrophoresis. Gel transfer to a PVDF membrane was performed under 200 V for 1 h. Membranes were blocked with 5% skimmed milk. Membranes were incubated with primary antibodies (1:1000, rabbit anti‐NLRP3, Merck; 1:500, rabbit anti‐ASC, Abcam; 1:1000, rabbit anti‐IL‐1β, Abcam; 1:1000, rabbit anti‐IL‐18, Proteintech; 1:1000, rabbit anti‐caspase‐1, Abcam; 1:500, rabbit anti‐GSDMD, Affinity; 1:2000, rabbit anti‐G3BP1, Proteintech; 1:500, rabbit anti‐TIA1, Proteintech; 1:1000, rabbit anti‐DDX3X, Proteintech; 1:1000, rabbit anti‐IgG antibody, Cell Signaling Technology; 1:10,000, rabbit anti‐SYN, Abcam; 1:1000, rabbit anti‐PSD‐95, Cell Signaling Technology; 1:1000, rabbit anti‐BDNF, Abcam; 1:500, rabbit anti‐Ang‐1, Proteintech; 1:500, rabbit anti‐Ang‐2, Proteintech; 1:500, rabbit anti‐VEGF, Abcam; 1:1000, mouse β‐actin, Santa Cruz) for 24 h at 4°C. Next, membranes were incubated with a secondary antibody (1:4000, goat anti‐rabbit IgG, goat anti‐mouse IgG, Cell Signaling Technology) for 2 h at room temperature. Western blot images for each of the antibodies were analyzed using an image analysis program (ImageJ 1.42, National Institutes of Health) to quantify protein expression in terms of relative image density.

Wang Q., Kohls W., Wills M., Li F., Pang Q., Geng X, & Ding Y. (2023). A novel stroke rehabilitation strategy and underlying stress granule regulations through inhibition of NLRP3 inflammasome activation. CNS Neuroscience & Therapeutics, 30(1), e14405.

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Protein Expression Analysis in Lung

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The protein expression of total STAT6, NLRP3, ASC, total p38 MAPK, p-p38 MAPK (Thr180/Tyr182), p62 and LC3 in the lung tissues and cells were analyzed by Western blot analysis. Primary antibodies included rabbit anti-total STAT6 (Bioss antibodies, Boston, MA), rabbit anti-ASC and rabbit anti-NLRP3 (Abcam, Cambridge, MA), rabbit anti-p-p38 MAPK, rabbit anti-p62 and rabbit anti-LC3 (Cell signaling technology, Danvers, MA). The anti-mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or β-Tubulin antibodies were used as loading internal controls. Protein expression was quantitatively analyzed by ImageJ software and data was presented as ratio of densitometric density of target protein to internal loading controls.

Hu L., Shao C., Pan L, & Jiang Z. (2022). Lack of STAT6 enhances murine acute lung injury through NLRP3/p38 MAPK signaling pathway in macrophages. BMC Immunology, 23, 25.

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Inflammasome Activation in Lung Fibroblasts

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The following antibodies were used in explant cultures and were obtained from R&D Systems: anti-IL1β, anti-TNFα, goat IgG, and rat IgG. Gel-purified Escherichia coli LPS (O55:B5) was purchased from Sigma-Aldrich. The Caspase-1 Inhibitor I (Ac-Tyr-Val-Ala-Asp-CHO; YVAD) was purchased from Calbiochem. The following antibodies were used for immunoblotting and immunofluorescence: rat anti-CD68 (Acris), rabbit anti-caspase 1 p10 (Santa Cruz Biotechnology), rabbit anti-NALP1/NLRP1 (Santa Cruz Biotechnology), rabbit anti-ASC (Abcam), and rabbit anti-Cryopyrin (NLRP3, Santa Cruz Biotechnology). ProLong Gold with DAPI mounting media and Alexa-conjugated secondary antibodies (AF488, AF555) were obtained from Invitrogen. The nuclear stain DRAQ5 was purchased from Thermo Scientific. Fetal lung mesenchymal cells were treated with recombinant IL1β (rIL1β, R&D Systems), recombinant TNFα (rTNFα, R&D Systems), recombinant IL18 (rIL18, MBL International Corporation), and Pam₃Cys-Ser-(Lys)₄ (Millipore). Luciferase activity was measured with a luciferase assay system from Promega. Cells were cultured in media from Corning’s Cellgro and 10% FBS (Thermo Scientific). DNase I from bovine pancreas type IV (DNase) and collagenase from clostridium histolyticum type XI were purchased from Sigma-Aldrich.

Stouch A.N., McCoy A.M., Greer R.M., Lakhdari O., Yull F.E., Blackwell T.S., Hoffman H.M, & Prince L.S. (2016). Interleukin-1β and Inflammasome Activity Link Inflammation to Abnormal Fetal Airway Development. Journal of immunology (Baltimore, Md. : 1950), 196(8), 3411-3420.

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Molecular Mechanisms of Hippocampal Inflammation

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The hippocampus was weighed and homogenized in RIPA buffer containing protease inhibitors and phosphatase inhibitors. After the lysates were centrifuged at 12,000 g for 10 min at 4°C, supernatants were collected and protein concentrations were analyzed using bicinchoninic acid assay. Next, loading buffering 5X was added, and proteins were boiled for 5 min in a metal bath. Protein samples were run on SDS-PAGE, transferred onto polyvinylidene difluoride (PVDF) membranes, and then blocked with 3% BSA. The membranes were incubated with the following primary antibodies over night at 4°C: rabbit anti-SIRT3 (1:1000, CST), rabbit anti-p-NF-κB p65 (1:800, CST), rabbit anti-NF-κB p65 (1:1000, CST), rabbit anti-NLRP3 (1:800, Abcam), rabbit anti-ASC (1:1000, Abcam), rabbit anti-cleaved-Capspase-1 (1:1000, Abcam), rabbit anti-cleaved-IL-1β (1:1000, Abcam), rabbit anti-cleaved-IL-18 (1:1000, Abcam), rabbit anti-GSDMD-N (1:800, Abcam), rabbit anti-GSDMD (1:1000, Abcam), rabbit anti-PSD-95 (1:1000, CST), rabbit anti-Synapsin-1 (1:1000, CST), rabbit anti-GAPDH (1:1000, Abcam). HRP-conjugated Affinipure Goat Anti-Rabbit IgG (1:5000, proteintech) were incubated for 90 min at room temperature. The membranes were digitally scanned by an imaging system (Bio-Rad, USA) and then quantified using ImageJ software. The relative expression levels of all proteins were normalized to GAPDH. n=6 per group.

Xia B., Tong Y., Xia C., Chen C, & Shan X. (2020). α-Cyperone Confers Antidepressant-Like Effects in Mice via Neuroplasticity Enhancement by SIRT3/ROS Mediated NLRP3 Inflammasome Deactivation. Frontiers in Pharmacology, 11, 577062.

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