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Sc 271054

Manufactured by Santa Cruz Biotechnology
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Sc-271054 is a laboratory product offered by Santa Cruz Biotechnology. It is a research-use-only item intended for scientific applications. The core function of this product is to serve as a tool for researchers in their investigations. No further details about the intended use or specifications of Sc-271054 can be provided in an unbiased and factual manner.

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Lab products found in correlation

Ab9722, by Abcam (4 mentions) Sc 56036, by Santa Cruz Biotechnology (2 mentions) Thp ascgfp, by InvivoGen (2 mentions) Ag 20b 0014 c100, by Adipogen (2 mentions) Nbp2 12446, by Novus Biologicals (1 mentions) Ab23875, by Abcam (1 mentions) Ab109272, by Abcam (1 mentions) Ab80039, by Proteintech (1 mentions) Ecl plus chemiluminescence reagent kit, by Cytiva (1 mentions) Pvdf membrane, by Bio-Rad (1 mentions) Anti myc agarose affinity gel, by Merck Group (1 mentions) Anti hmgb1, by Abcam (1 mentions) Anti caspase 1, by Santa Cruz Biotechnology (1 mentions) Anti α tubulin, by Merck Group (1 mentions) Anti flag, by Merck Group (1 mentions) Lsm 800, by Zeiss (1 mentions) Ab1872, by Abcam (1 mentions) Ab210491, by Abcam (1 mentions) Ab6671, by Abcam (1 mentions) Ab208113, by Abcam (1 mentions)

10 protocols using sc 271054

1

Quantifying NLRP3 Inflammasome Activation in CAFs

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Production of NLRP3, ASC, Casp-1,IL1β, Phospho-NF-κB p65, NF-κB p65 in CAFs was determined by Western blotting using anti-NLRP3 antibody (NBP2-12446, Novus, 1:500), anti-ASC antibody (sc-271054, Santa Cruz Biotechnology, 1:200), anti- Casp-1 antibody (sc-56036, Santa Cruz Biotechnology, 1:200) and anti-IL1β antibody (ab9722, Abcam, 1:10000), anti- Phospho-NF-κB p65 antibody (3033s, Cell and Signaling, 1:1000), and anti-NF-κB p65 antibody (8242s, Cell and Signaling, 1:1000). Anti-GAPDH (AC-15) (Sigma-Aldrich, St Louis, MO, USA) was used as the loading control. Sample lysates and Western blots were collected as previously described.16 (link) The results normalized to GAPDH were quantified by using Image J. (NIH, Bethesda, MD, USA).
Nie S., Wang X, & Wang H. (2019). NLRP3 Inflammasome Mediated Interleukin-1β Production in Cancer-Associated Fibroblast Contributes to ALA-PDT for Cutaneous Squamous Cell Carcinoma. Cancer Management and Research, 11, 10257-10267.
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2

Dissecting the Inflammasome Pathway

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siRNAs targeting RSK1, RSK2, RSK3, PKN1, PKN2, MEFV and scrambled siRNA were purchased from Invitrogen. The three siRNAs for RSK knockdown were 5’-CACUGAUUCUGAAGGCGAA-3’ (s12273); 5’-GAGAUUUGUUUACACGCUU-3’ (s12279), 5’-GGUUUUCCCUAAGAAGAUG-3’ (1054). The siRNA for MEFV was 5’-CUUUCUGAAACAAACUGAAF-3’ (s502557). The two siRNAs for PKN were 5’-GGACAGUAAGACCAAGAUU-3’ (s11113) and 5’- GACGAGAAGAUGUUAGUAA-3’ (S11116). For siRNA gene knockdown experiments, 50–250 pmol siRNA were transfected into THP1-ASC-GFP cells (thp-ascgfp, Invivogen) by electroporation and plated in a chamber slide (1 X 106 cells per well) or 6 well plate (2 X 106 cells per well). The plated cells were treated with 500 nm PMA for 3h and medium replaced with fresh RPMI medium containing 10% FBS. After 72 h, the transfected cells were infected with Y. pestis (MOI 30). For quantification of ASC specks, ten random fields were selected and cells with an ASC speck were counted from GFP positive cells. For the ASC oligomerization assay, cells were lysed and then insoluble pellets were chemically crosslinked with 2.5 mM disuccinimidyl suberate (DSS) (21658,Thermo Scientific) for 30 min at RT and eluted by SDS sample loading buffer and analyzed by immunoblot with anti-ASC (sc-271054, Santa Cruz Biotechnology) antibody.
Park Y.H., Remmers E.F., Lee W., Ombrello A.K., Chung L.K., Shilei Z., Stone D.L., Ivanov M.I., Loeven N.A., Barron K.S., Hoffmann P., Nehrebecky M., Akkaya-Ulum Y.Z., Sag E., Balci-Peynircioglu B., Aksentijevich I., Gül A., Rotimi C.N., Chen H., Bliska J.B., Ozen S., Kastner D.L., Shriner D, & Chae J.J. (2020). Ancient familial Mediterranean fever mutations in human pyrin and resistance to Yersinia pestis. Nature immunology, 21(8), 857-867.
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3

Western Blot Analysis of Inflammatory Markers

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Western blotting was conducted as previously described [31 (link)]. Proteins from brain samples and cultured BV2 cells were lysed using RIPA lysis buffer. Equal amounts of protein (40 μg/10 μL) were loaded onto sodium dodecyl sulfate–polyacrylamide gels. The proteins were electrophoresed until sufficiently separated and then transferred to PVDF membranes. The membranes were incubated overnight at 4 °C with primary antibodies against rabbit anti-STING (1:1000, Proteintech, Cat.No.19851-1-AP), rabbit anti-TBK1 (1:1000, Proteintech, Cat.No. 28397-1-AP), rabbit anti-TBK1 (phospho S172) (1:2000, Abcam, ab-109272), rabbit anti-AMPK (phospho T183 and T172) (1:1000, Abcam, ab-23875), rabbit anti-AMPK (1:1000, Abcam, ab-80039), rabbit anti- iNOS (1:500, Proteintech, Cat.No. 18985-1-AP), rabbit anti-IL-1β (1:1000, Abcam, ab-9722), rabbit anti-NLRP3(1:1000, Abcam, ab210491), mouse anti-ASC (1:2000, Santa Cruz, sc-271054), goat anti-caspase-1 (1:2000, Santa Cruz, sc-22165), and mouse anti-β-actin (1:5000, Proteintech, Cat.No. 60008-1-Ig). The membranes were processed with horseradish-peroxidase-conjugated secondary antibodies at room temperature for 1 h. Bands were visualized using the ECL Plus chemiluminescence reagent kit (Amersham Bioscience, Arlington Heights, IL). The band densities were quantified with the Image J software (NIH).
Peng Y., Zhuang J., Ying G., Zeng H., Zhou H., Cao Y., Chen H., Xu C., Fu X., Xu H., Li J., Cao S., Chen J., Gu C., Yan F, & Chen G. (2020). Stimulator of IFN genes mediates neuroinflammatory injury by suppressing AMPK signal in experimental subarachnoid hemorrhage. Journal of Neuroinflammation, 17, 165.
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4

Immunoblotting Analysis of NLRP3 Inflammasome

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Western blot analysis was performed. In brief, proteins from the H9c2 cells were extracted using sucrose buffer (20 mM HEPES, 1 mM EDTA, 255 mM sucrose, cocktail o protease inhibitors (Roche), pH 7.4). After boiling for 5 min at 95 °C in a 2× loading buffer, 30μg of total protein was separated by a 10% or 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins of these samples were then electrophoretically transferred at 100 V for 1 h onto a PVDF membrane (Bio-Rad, USA). The membrane was blocked with 5% nonfat milk in Tris-buffered Saline-Tween 20. After washing, the membrane was probed with 1:1000 dilution of primary mouse or rabbit antibodies against Nlrp3 (cst15101s, Cell Signaling Technology), caspase-1 (sc-56036, Santa Cruz Biotechnology), ASC (sc-271054, Santa Cruz Biotechnology), or GAPDH (60004-1-Ig, Proteintech Group) overnight at 4 °C followed by incubation with second-antibody (Abcam 6721, goat-rabbit; Abcam 6789, goat-mouse;). The immuno-reactive bands were detected by a Syngene instrument (Syngene, USA). Densitometry analysis of the images was performed using the GeneTools from Syngene Software.
Zhang Y.Z., Zhang Y.L., Huang Q., Huang C., Jiang Z.L., Cai F, & Shen J.F. (2019). AdipoRon Alleviates Free Fatty Acid-Induced Myocardial Cell Injury Via Suppressing Nlrp3 Inflammasome Activation. Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy, 12, 2165-2179.
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5

Immunoblotting Analysis of Protein Complexes

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Cell lysates were prepared in lysis buffer (50 mM Tris-HCl (pH 7.5), 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 10 mM sodium fluoride, 10 mg/ml aprotinin, 10 mg/ml leupeptin, 150 mM NaCl, and 10 mg/ml pepstatin A) containing 1% NP-40. Soluble proteins were subjected to immunoprecipitation with anti-Flag or Anti-Myc Agarose Affinity Gel (Sigma-Aldrich) antibodies. An aliquot of the total lysates (5%, v/v) was included as control. Immunoblotting analysis was performed with anti-Myc, anti-HA, anti-Flag, anti-α-Tubulin (Sigma-Aldrich), anti-HMGB1 (ab92310, Abcam, Cambridge, UK), anti-caspase-1 (sc-514, Santa Cruz Biotechnology), anti-ASC (sc-271054, Santa Cruz), and anti-NLRP3 (AG-20B-0014-C100, Adipogen, San Diego, CA, USA) antibodies, respectively. The antigen-antibody complexes were visualized by chemiluminescence (5200, Tanon, Inc., Shanghai, China). The Band Analysis tools of Gel Image System software version 4.2 (Tanon) were used to select and determine the background-subtracted density of the bands in all the gels and blots. Level of protein was first normalized to respective tubulin control. The control (wild-type) condition was normalized to 1 and all other experimental conditions were compared with this.
Wei C., Wang Y., Du Z., Guan K., Cao Y., Yang H., Zhou P., Wu F., Chen J., Wang P., Zheng Z., Zhang P., Zhang Y., Ma S., Yang R., Zhong H, & He X. (2016). The Yersinia Type III secretion effector YopM Is an E3 ubiquitin ligase that induced necrotic cell death by targeting NLRP3. Cell Death & Disease, 7(12), e2519-.
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6

Immunofluorescence Assay for Caspase-1, ASC, and Nrf2

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After incubation in hypoxia condition for 6 h, cells were fixed with 4% buffered paraformaldehyde for 10 min, permeabilized with 0.5% TritonX-100 for 20 min at RT, rinsed with PBS three times, and blocked with goat serum for 30 min. Cell slides were then incubated with primary antibody against caspase-1, ASC (sc-271054; Santa Cruz) and Nrf2 at 4°C overnight. Consequently, the slides were washed with PBS and incubated with AlexaFluor 555 (4413S; CST) and 488-labeled (4408S; CST)secondary antibodies for 1 h at RT in the dark. All samples were then incubated with DAPI for 5 min and blocked with an antifluorescence quencher. Images were captured using a laser scanning confocal microscope (LSM800;ZEISS).
Pang Y., Zhang P.C., Lu R.R., Li H.L., Li J.C., Fu H.X., Cao Y.W., Fang G.X., Liu B.H., Wu J.B., Zhou J.Y, & Zhou Y. (2020). Andrade-Oliveira Salvianolic Acid B Modulates Caspase-1–Mediated Pyroptosis in Renal Ischemia-Reperfusion Injury via Nrf2 Pathway. Frontiers in Pharmacology, 11, 541426.
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7

Inflammatory Signaling Pathway Analysis

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Protein expression was analyzed by western blotting as previously described (Chai et al. 2014 (link); Yuan et al. 2015 (link)). For western blot analysis, primary antibodies against NFκB (rabbit polyclonal antibody, ab16502), TNFα (rabbit polyclonal antibody, ab6671), NLRP3 (rabbit polyclonal antibody, ab210491), IL-1β (rabbit polyclonal antibody, ab9722), IL-6 (rabbit polyclonal antibody, ab208113) and caspase1 (rabbit polyclonal antibody, ab1872) were purchased from Abcam (UK). TLR4 (mouse monoclonal antibody, sc-293072) and ASC (mouse monoclonal antibody, sc-271054) were purchased from Santa Cruz (USA). IL-18 (rabbit polyclonal antibody, TA324190) was purchased from ORIGENE (USA). The goat anti-mouse IgG-HRP (ZB2305) and goat anti-rabbit (ZB2301) IgG-HRP secondary antibodies were purchased from ZSGB-Bio, Beijing, China. The targeted proteins were visualized with the Super Signal West Femto Chemiluminescent Substrate (Thermo Scientific Pierce) and the intensity of the visualized bands were analyzed using Quantity One software (Bio-Rad). β-actin (mouse monoclonal antibody, TA-09, Zhongshan Jinqiao Biotech company, Beijing, China) was used as an internal control. Data are expressed as a ratio to β-actin.
Gong Q., He L., Wang M., Zuo S., Gao H., Feng Y., Du L., Luo Y, & Li J. (2019). Comparison of the TLR4/NFκB and NLRP3 signalling pathways in major organs of the mouse after intravenous injection of lipopolysaccharide. Pharmaceutical Biology, 57(1), 555-563.
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8

Western Blot Analysis of NLRP3 Inflammasome Proteins

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The protein expressions of NACHT, LRR, and PYD Domains-Containing Protein 3 (NLRP3), apoptosis-associated Speck-like protein containing a CARD (ASC), pro-IL-1β and mature IL-1β, and β-actin in the hippocampal homogenates were evaluated using a western blotting method. The protein concentration of the homogenates was equalized, and the samples were separated by 10% polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes. To minimize the non-specific binding, the membrane was blocked in 5% bovine serum albumin for 1 h. The membranes were incubated with primary antibodies, such as NLRP3 (1:500, ab214185, Abcam), ASC (1:500, sc-271054, Santa Cruz), pro-IL-1β and mature IL-1β (1:1,000, ab9722, Abcam), or β-actin (1:2500, PA1-183, Thermo-Fisher Scientific), overnight at 4°C. After being washed, the membranes were incubated with an HRP-conjugated anti-rabbit or anti-mouse antibody (GeneTex, Inc., Irvine, CA) for 1 h. The western blotting results were visualized with an enhanced chemiluminescence (ECL) advanced kit. The intensity was analyzed with ImageJ version 1.46 (NIH, Bethesda, MD, USA).
Lee J.S., Kim W.Y., Jeon Y.J., Lee S.B., Lee D.S, & Son C.G. (2019). Antidepressant-Like Activity of Myelophil via Attenuation of Microglial-Mediated Neuroinflammation in Mice Undergoing Unpredictable Chronic Mild Stress. Frontiers in Pharmacology, 10, 683.
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9

Dissecting the Inflammasome Pathway

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siRNAs targeting RSK1, RSK2, RSK3, PKN1, PKN2, MEFV and scrambled siRNA were purchased from Invitrogen. The three siRNAs for RSK knockdown were 5’-CACUGAUUCUGAAGGCGAA-3’ (s12273); 5’-GAGAUUUGUUUACACGCUU-3’ (s12279), 5’-GGUUUUCCCUAAGAAGAUG-3’ (1054). The siRNA for MEFV was 5’-CUUUCUGAAACAAACUGAAF-3’ (s502557). The two siRNAs for PKN were 5’-GGACAGUAAGACCAAGAUU-3’ (s11113) and 5’- GACGAGAAGAUGUUAGUAA-3’ (S11116). For siRNA gene knockdown experiments, 50–250 pmol siRNA were transfected into THP1-ASC-GFP cells (thp-ascgfp, Invivogen) by electroporation and plated in a chamber slide (1 X 106 cells per well) or 6 well plate (2 X 106 cells per well). The plated cells were treated with 500 nm PMA for 3h and medium replaced with fresh RPMI medium containing 10% FBS. After 72 h, the transfected cells were infected with Y. pestis (MOI 30). For quantification of ASC specks, ten random fields were selected and cells with an ASC speck were counted from GFP positive cells. For the ASC oligomerization assay, cells were lysed and then insoluble pellets were chemically crosslinked with 2.5 mM disuccinimidyl suberate (DSS) (21658,Thermo Scientific) for 30 min at RT and eluted by SDS sample loading buffer and analyzed by immunoblot with anti-ASC (sc-271054, Santa Cruz Biotechnology) antibody.
Park Y.H., Remmers E.F., Lee W., Ombrello A.K., Chung L.K., Shilei Z., Stone D.L., Ivanov M.I., Loeven N.A., Barron K.S., Hoffmann P., Nehrebecky M., Akkaya-Ulum Y.Z., Sag E., Balci-Peynircioglu B., Aksentijevich I., Gül A., Rotimi C.N., Chen H., Bliska J.B., Ozen S., Kastner D.L., Shriner D, & Chae J.J. (2020). Ancient familial Mediterranean fever mutations in human pyrin and resistance to Yersinia pestis. Nature immunology, 21(8), 857-867.
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10

Immunoprecipitation of Kir6.1, NLRP3, and ASC

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The total cell lysates were prepared from WT and CKO astrocytes and then incubated with anti-Kir6.1 (ab241996, Abcam, 1:500 dilution), anti-NLRP3 (AG-20B-0014-C100, Adipogen, 1:800 dilution) or anti-ASC (sc-271054, Santa Cruz Biotechnology, 1:1000 dilution) antibodies at 4 °C overnight, followed by an incubation with 20 μl protein A/G plus agarose (sc-2003, Santa Cruz Biotechnology, USA) for 4 h at 4 °C. After washing the beads, the bound proteins were eluted and analyzed by immunoblotting.
Li F., Jiang S.Y., Tian T., Li W.J., Xue Y., Du R.H., Hu G, & Lu M. (2022). Kir6.1/K-ATP channel in astrocytes is an essential negative modulator of astrocytic pyroptosis in mouse model of depression. Theranostics, 12(15), 6611-6625.
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