RIPA (GBCBIO, G3424) was used to lyse cells and extract total protein to prepare protein samples. The protein lysates were separated by conventional electrophoresis, transferred onto the membrane, and washed with PBS, followed by blocking with 1% BSA for 2 h. Then, primary antibodies Box (ab32503, Abcam, Cambridge, UK), Bcl-2 (ab32124, Abcam, Cambridge, UK), Caspase-1 (ab32503, Abcam, Cambridge, UK), Cleaved Caspase-3 (ab32503, Abcam, Cambridge, UK), Caspase-8 (ab207802, Abcam, Cambridge, UK), Caspase-9 (ab2302, Abcam, Cambridge, UK), Caspase-11 (ab25901, Abcam, Cambridge, UK), Cyclin D1 (ab202068, Abcam, Cambridge, UK), ERK 1/2 (ab180673, Abcam, Cambridge, UK), p-ERK 1/2 (ab16663, Abcam, Cambridge, UK), WNT (ab17942, Abcam, Cambridge, UK), β-catenin (ab223500, Abcam, Cambridge, UK), GSDMD (ab15251, Abcam, Cambridge, UK) and β-actin (ab32572, Abcam, Cambridge, UK) (abcam, ab32503, ab32124, ab207802, ab2302, ab25901, ab202068, ab180673, ab16663, ab17942, ab223500, ab15251, ab32572, ab219800, ab8227) each at 1:1000 dilution were incubated with the membrane overnight at 4°C. The next day, HRP (ab32572, Abcam, Cambridge, UK) and labeled IgG secondary antibody (ab150077, Abcam, Cambridge, UK) each at 1:5,000 were further incubated with the membrane at room temperature for 1 h. ECL Luminescence Kit (Thermo, 32209) was used to develop the signal in a dark room.
Ab180673
Manufactured by Abcam
Sourced in United Kingdom
Ab180673 is an anti-Galectin-3 antibody. Galectin-3 is a protein involved in various cellular processes. This antibody can be used to detect and study Galectin-3 expression in samples.
Automatically generated - may contain errors
Lab products found in correlation
Ab209845, by Abcam (6 mentions)
Ab138483, by Abcam (4 mentions)
Ab207802, by Abcam (3 mentions)
Ab22684, by Abcam (2 mentions)
Ab214185, by Abcam (2 mentions)
Ab219800, by Abcam (2 mentions)
Ab32503, by Abcam (2 mentions)
Ag 20b 0014, by Adipogen (2 mentions)
Epr16891, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Ecl kit, by Beyotime (1 mentions)
Ab207323, by Abcam (1 mentions)
Nbp1 45433, by Novus Biologicals (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Ab1872, by Abcam (1 mentions)
Bca protein quantification kit, by Thermo Fisher Scientific (1 mentions)
Af4012, by Affinity Biosciences (1 mentions)
Gel doc 2000, by Bio-Rad (1 mentions)
Ab9722, by Abcam (1 mentions)
12 protocols using ab180673
1
Western Blot Analysis of Apoptosis Markers
2
Western Blot Analysis of Caspase Activation
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The treated cells were lysed in RIPA buffer containing 1 mM DTT, 11 μg/ml DNase I, and protease inhibitor cocktail (Roche) and incubated on ice for 30 min. Sixty micrograms of protein sample were separated by electrophoresis on a denatured 10% SDS-PAGE gel and blotted onto a PVDF membrane (Millipore). The primary antibodies (Anti-Caspase-1, 1:1,000, ab138483, Abcam; Anti-cleavage Caspase-1, 1:1,000, ab207802, Abcam; Anti-Caspase-11, 1:1,000, ab22684, Abcam Anti-cleavage Caspase-11, 1:1,000, ab180673, Abcam) were incubated overnight at 4°C. Rabbit monoclonal to GAPDH (EPR16891, Abcam) was used as a loading control. Afterward, the membranes were rinsed and subsequently incubated with appropriate secondary antibodies. An ECL kit (Beyotime Biotechnology, Shanghai, China) was used to detect the bands of Western blots.
3
Western Blot Analysis of Inflammasome Proteins
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Brain or spleen tissue were washed with PBS and lysed in 1× RIPA buffer with protease and phosphatase inhibitors. Protein concentrations were determined by the Bradford assay, and equal amounts of protein were subjected to SDS/PAGE. Then, the samples were transferred onto nitrocellulose membranes. The blots were incubated overnight at 4 °C with the following antibodies: GSDMD (1:1000, ab209845, Abcam, Cambridge, England), Caspase-1 (1:1000, NBP1-45433, NOVUS, Colorado, USA), Caspase-11 (1:1000, ab180673, Abcam, Cambridge, England), IL-1β (1:1000, #31202, CST, Southcarolina, USA), IL-18 (1:1000, ab207323, Abcam, Cambridge, England) and α-tubulin (1:1000, #9099, CST, Southcarolina, USA). The membranes were incubated with appropriate secondary antibodies for 2 h at room temperature. The blots were visualized with SuperSignal West Femto substrate (Pierce) on a ChemiDoc Imaging System (Bio-Rad) and analyzed by ImageJ.
4
Protein Quantification and Western Blotting
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Protein concentration was determined with a bicinchoninic acid (BCA) protein quantification kit (ThermoFisher Scientific, Rockford, IL, USA) following to the manufacturer’s instructions. After preparation of the gels, the sample was slowly added to the lane. After electrophoresis, the proteins were transferred and blocked. After blocking, the polyvinylidene difluoride (PVDF) membranes were incubated overnight with the primary antibodies anti-caspase-11 (ab180673; Abcam, Cambridge, UK), anti-GSDMD (AF4012; Affinity, Jiangsu, China), anti-caspase-1 (ab1872; Abcam), anti-NLRP3 (ab214185; Abcam), or anti-IL-1β (ab9722; Abcam). Subsequently, the membranes were incubated with the secondary antibody. Then, the membranes were exposed with a gel imager (Gel Doc2000; Bio-Rad, Hercules, CA, USA).
5
Caspase-11 Activation in B. thailandensis Infection
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TC-1 cells were seeded into 10 cm tissue culture dishes and infected when confluent with B. thailandensis or the bsaZ mutant (MOI 500) in a final volume 5 ml in the presence or absence of 100 ng/ml IFNγ. Three hours later, cell monolayers were extensively washed with D-PBS to remove extracellular bacteria and incubated for another 4 hours in medium containing gentamicin and kanamycin (200μg/mL). Biotin-VAD-FMK (15 μM, Santa Cruz) was added to the culture medium one hour before lysing cells in RIPA buffer. Active caspase-11 was pulled down by incubation overnight at 4 °C with streptavidin agarose (Sigma Aldrich) and analyzed by Western Blot with rabbit anti-caspase-11 antibody (Abcam, ab180673)
6
Western Blotting for Protein Analysis
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Western blotting (WB) was executed as previously reported to measure the protein levels in the tissues and cells [30 (link)]. Primary antibodies against Bax, Bcl-2, cleaved caspase-3, caspase-3, Nrf2, HO1, NQO1, NLRP3, caspase-1, caspase-11, GSDMD, G-CSF, RANTES, MCP1, and β-actin were used in the present study (Abcam; Cat #: ab32503, ab182858, ab214430, ab184787, ab92946, ab13243, ab34173, ab214185, ab138483, ab180673, ab219800, ab181053, ab189841, ab242013, and ab81283) at dilutions of 1 : 1000.
7
Investigating Pyroptosis Regulatory Pathways
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J774A.1 cells, a mouse monocyte/macrophage cell line, were propagated in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS) at 37°C with 5% CO2.
T. pyogenes (strain 0912) was cultured in Martin broth medium with 10% fetal bovine serum under aerobic conditions.
Primary antibodies included rabbit monoclonal anti-GSDMD (ab209845, Abcam), rat monoclonal anti-caspase-1(BL-645102, Biolegend), rabbit monoclonal anti-caspase-11 (ab180673, Abcam), mouse monoclonal anti-NLRP3(AG-20B-0014, Adipogen), and mouse anti-GAPDH antibody (GTX627408, GeneTex). Secondary antibodies included horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (ZB-2305, ZSGB-BIO), HRP-conjugated goat anti-rabbit IgG (ZB-2301, ZSGB-BIO) and HRP-conjugated goat anti-rat IgG secondary antibody (ZB-2307, ZSGB-BIO).
Chemicals included VX-765 (Capase-1 inhibitor) (S2228, Selleck), MCC950 (NLRP3 inhibitor) (S8930, Selleck), Necrostatin-1 (Nec-1) (receptor-interacting serine/threonine protein kinase 1 [RIP1] inhibitor) (A4213, APExBIO), Necrosulfonamide (NSA) (mixed lineage kinase domain-like protein [MLKL] and GSDMD inhibitor) (B7731, APExBIO), and Wedelolactone (Wed) (caspase-11 and NLRP3 inhibitor) (HY-N0551, MedChemExpress).
T. pyogenes (strain 0912) was cultured in Martin broth medium with 10% fetal bovine serum under aerobic conditions.
Primary antibodies included rabbit monoclonal anti-GSDMD (ab209845, Abcam), rat monoclonal anti-caspase-1(BL-645102, Biolegend), rabbit monoclonal anti-caspase-11 (ab180673, Abcam), mouse monoclonal anti-NLRP3(AG-20B-0014, Adipogen), and mouse anti-GAPDH antibody (GTX627408, GeneTex). Secondary antibodies included horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (ZB-2305, ZSGB-BIO), HRP-conjugated goat anti-rabbit IgG (ZB-2301, ZSGB-BIO) and HRP-conjugated goat anti-rat IgG secondary antibody (ZB-2307, ZSGB-BIO).
Chemicals included VX-765 (Capase-1 inhibitor) (S2228, Selleck), MCC950 (NLRP3 inhibitor) (S8930, Selleck), Necrostatin-1 (Nec-1) (receptor-interacting serine/threonine protein kinase 1 [RIP1] inhibitor) (A4213, APExBIO), Necrosulfonamide (NSA) (mixed lineage kinase domain-like protein [MLKL] and GSDMD inhibitor) (B7731, APExBIO), and Wedelolactone (Wed) (caspase-11 and NLRP3 inhibitor) (HY-N0551, MedChemExpress).
8
Western Blot Antibody Validation Protocol
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Western blot was performed using standard protocol as previously described [28] . The following primary antibodies were used: anti-GSDMD (ab209845, abcam), anti-IL-18 (ab243091, abcam) (1:2000), anti-IL-1β (ab254360, abcam) (1:2000), anti-caspase-1 (ab180673, abcam) (1:1000), anti-caspase-11(ab138483, abcam) (1:1000), anti-Notch 4 (A-12) (sc-393893, Santa Cruz) (1:1000), anti-DTX4 (25222-1-AP, Proteintech, 1:100), and anti-β-actin (ab8226, abcam) (1:10000) antibodies. β-actin was used as an internal control.
9
Analyzing Pyroptosis Signaling Pathways
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Primary antibodies against caspase-1 (ab179515), caspase-11 (ab180673), GSDMD (ab209845), GSDME (ab215191) and Biotin Goat polyclonal to Chlamydia trachomatis (ab20387) were obtained from Abcam (Cambridge, UK), Antibody for Chlamydia HSP60 (sc57840) was bought from Santa Cruz Biotechnology (California, USA), Antibodies for caspase-8 (4790), caspase-8 p18 (8592), β-Tubulin (2128) and GAPDH (5174) were obtained from Cell Signaling Technology (Danvers, MA, USA), while secondary antibodies (SA00001-15, SA00001-1) used for immunoblotting and Fluorescein (FITC)-conjugated Affinipure Rabbit Anti-Goat IgG(H+L) (SA00003-4) used for immunofluorescence staining were bought from Proteintech (Chicago, USA). IL-1β (CSB-E08054m) and IL-18 (CSB-E04609m) ELISA kits were obtained from CUSABIO (Wuhan, China). Caspase-1 inhibitor Ac-YVAD-cmk (SML0429) was purchased from Sigma (California, USA), Necrosulfonamide (AG-CR1-3705-M005) was bought from Adipogen (Epalinges, Switzerland). Caspase-11 inhibitor Wedelolactone (T3384) was obtained from Target Mol (Boston, MA). All inhibitors were dissolved in dimethyl sulfoxide (DMSO).
10
Western Blot Analysis of Apoptosis-Related Proteins
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Cells or heart tissues were lysed in ice-cold RIPA lysis buffer (Solarbio, Beijing, China) containing protease inhibitors (Roche) for 30 min. Proteins were separated by 12% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were probed with primary antibodies toward caspase-1 (Abcam, Cambridge, UK, ab138483), cleaved caspase-1 (Abcam, ab207802), caspase-11 (Abcam,ab22684), cleaved caspase-11 (Abcam, ab180673), USP46 (Abcam, ab88795), Tubulin (Cell Signaling Technology, Danvers, MA, USA, D65A4), GAPDH (Abcam, ab8245) and β-actin (Cell Signaling Technology, 8H10D10), respectively. After washing with PBS-Tween 20, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies, and protein bands were visualized using Pierce® ECL Western blotting substrate (Pierce, Rockford, IL, USA). Densitometric analysis was performed using Image J.
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