Total proteins were extracted with RIPA lysis containing protease inhibitors from mouse lung tissues and Raw264.7 cells. And, protein samples were separated through 10–13% SDS-PAGE, transferred to PVDF membranes. The membranes were incubated with primary antibodies: total-STAT1 (1:1000, PTM-5754, PTM Bio, Hangzhou, China), Phospho-STAT1 (1:1000, 340797, ZenBio, Chengdu, China), total-STAT6 (1:1000, 380957, ZenBio), Phospho-STAT6 (1:500, sc-136019, Santa Cruz), iNOS (1:1000, ab178945, Abcam), Arg1 (1:1000, #93668S, Cell signaling technology), Caspase1 (1:500, sc-392736, Santa Cruz), MT1/2 (1:500, sc-398788, Santa Cruz), β-Tublin (1:5000, M20005, Abmart, Shanghai, China) and GAPDH (1:5000, ab181602, Abcam) overnight at 4℃. After incubated with Goat Anti-Rabbit IgG second antibody (1:5000, M21003, Abmart), the membranes were visualized by Odyssey infrared imaging system (Tanon, Shanghai, China).
Sc 392736
Manufactured by Santa Cruz Biotechnology
Sourced in China, United States
Sc-392736 is a lab equipment product offered by Santa Cruz Biotechnology. It serves as a core function for laboratory applications, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Ab178945, by Abcam (2 mentions)
Sc 398788, by Santa Cruz Biotechnology (2 mentions)
Sc 47778, by Santa Cruz Biotechnology (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Sc 514414, by Santa Cruz Biotechnology (2 mentions)
Ab181602, by Abcam (1 mentions)
Phospho stat6, by Santa Cruz Biotechnology (1 mentions)
M20005, by Abmart (1 mentions)
M21003, by Abmart (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Complete phostop, by Roche (1 mentions)
Ab16046, by Abcam (1 mentions)
Goat anti rabbit or anti mouse igg fluorescent secondary antibody, by Thermo Fisher Scientific (1 mentions)
Bca protein assay kit, by Solarbio (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
β actin, by Transgene (1 mentions)
Ab209845, by Abcam (1 mentions)
Ripa buffer, by Solarbio (1 mentions)
13 protocols using sc 392736
1
Protein Expression Analysis in Mouse Lung
2
Macrophage Priming and PXR Agonist-Induced Activation
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Prior to performing inflammation activation experiments, isolated macrophages were pulsed with ultra-pure LPS (100 ng/ml for 30 minutes; Invivogen/Cedarlane). PMA-differentiated or LPS-pulsed mouse peritoneal macrophages were treated with their respective PXR agonists. Following the designated treatment period, culture supernatants were collected, cells were washed with ice-cold phosphate-buffered saline, and cell lysates were isolated following incubating the cells with lysis buffer (150 mM NaCl, 20 mM Tris, pH 7.5, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, and protease inhibitor cocktail; phosphatase inhibitor cocktail, Complete Minitab; Complete PhoStop, Roche/Sigma-Aldrich Canada). Total protein was quantified using the Precision Red Advanced Protein Assay (Cytoskeleton/Cedarlane, Burlington, Ontario, Canada), and sample protein concentration was equalized. Culture supernatant and cell lysate samples were resolved, transferred to polyvinylidene difluoride membranes (0.2-μm pores; Bio-Rad Laboratories, Mississauga, Ontario, Canada), and blotted with the following antibodies: anti–caspase-1 (sc-622, sc-56036, and sc392736; Santa Cruz Biotechnology, Dallas, TX). Densitometry was performed using ImageJ, and cleaved caspase-1 was expressed as a percentage of pro–caspase-1.
3
Immunofluorescence Analysis of FOXP2 and Caspase-1 in CRC Cells
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CRC cells were incubated in chamber slides for 24 h, fixed in 4% paraformaldehyde for 20 min, and permeabilized with 0.1% Triton X-100 for 1 h. Next, the cells were incubated with FOXP2 (ab16046, 1:100 Abcam) and caspase-1 (sc-392736, 1:100, Santa Cruz) primary antibodies overnight at 4°C. Finally, the cells were incubated with goat anti-rabbit or anti-mouse IgG fluorescent secondary antibody (Thermo Fisher Scientific), and the nuclei were stained with DAPI. The intensity of immunofluorescence was examined by confocal fluorescence microscopy at wavelengths of 488 and 594 nm.
4
Western Blot Protein Analysis Protocol
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All proteins were extracted by RIPA buffer (Solarbio Life Science, Beijing, China) mixed with protease inhibitors on ice plates. Then, protein concentration was qualified by a BCA protein assay kit purchased from Solarbio Life Science. Next, equal amounts of protein were loaded onto 10% SDS-PAGE gels and transferred to nitrocellulose membranes (Millipore). The membranes were blocked with 5% BSA (Sigma-Aldrich) and incubated with primary antibodies at 4°C overnight. On the second day, the membranes were washed with 1x TBS solution and incubated with the secondary antibody conjugated with HRP at normal temperature for 1 hour. Finally, the results were determined using the ChemiDocTM Imaging System (Bio-Rad, Hercules, CA, USA). The following primary antibodies were used: FOXP2 (20529-1-AP, 1:1000, ProteinTech), PCNA (ab29, 1:1000, Abcam), cyclin D1 (ab40754, 1:5000, Abcam), caspase-1 (sc-392736, 1:1000, Santa Cruz), caspase-3 (sc-7272, 1:1000, Santa Cruz), caspase-9 (ab32539, 1:1000, Abcam), GSDMD (ab209845, 1:1000, Abcam), GAPDH (1:1000, TransGen Biotech, Beijing), Flag-tag (D6W5B, CST), HA-tag (C29F4, CST), and β-actin (1:1000, TransGen Biotech, Beijing). The secondary protein was purchased from TransGen Biotech (Beijing).
5
Western Blot Analysis of Inflammatory Proteins
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The tissues were homogenized in cold T-Per lysis buffer (Pierce Biotechnology, Inc. USA). Approximately 30μg/lane of protein samples were separated by 10–15% SDS-PAGE gel and subsequently transferred to nitrocellulose membranes. After blockage in 5% skim milk powder in 0.1% Tris-buffered saline/Tween 20 (TBST) for 2 h and incubation with antibodies against NLPR3 (D4D8T, Cell Signaling Technology), caspase-1 (sc-392736, Santa Cruz Biotechnology), GSDMD (YT7991, Immunoway Biotechnology), PSMA7 (15219-1-AP, Proteintech Group) overnight at 4°C at a dilution of 1:500 or 1:1000. Then the membranes were incubated with a secondary horseradish peroxidase-conjugated antibody for 1 h at room temperature. Immunoreactive proteins were visualized using an enhanced chemiluminescence western blotting detection system (Santa Cruz Biotechnology). The chemiluminescence signal from the membranes was quantified by the GeneGenome HR scanner using GeneTools software (SynGene). To control sampling errors, the ratio of band intensity to β-actin was obtained to quantify the relative protein expression level.
6
Caspase-1 Expression in Renal Tissue
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The kidney was placed on a clean workbench, and the fibrous capsule was stripped using an aseptic technique. Next, 1 cm3 of renal tubular epithelial tissue was removed from the outer skin tissue, washed with normal saline, and fixed with 10% formalin. Sections 4 μm thick were incubated overnight with caspase-1 mouse monoclonal antibody (1:50, sc-392736, Santa Cruz Biotechnology) at 4 °C, and then conjugated with secondary goat anti-mouse IgG (Alexa Fluor® 488) (1:200, ab150113, Abcam, Cambridge, United Kingdom) in the dark for 1 h. After washing in PBS, the slides were incubated with 4’,6-diamidino-2-phenylindole (DAPI) (D9542, Sigma, United States) for 3 min, followed by glycerine loading. The fluorescence was evaluated under a fluorescence microscope (Olympus Life Sciences, Tokyo, Japan).
7
Protein Expression Analysis in Lung Tissues
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Total proteins were extracted with RIPA lysis containing protease inhibitors from murine lung tissues, Raw264.7 cells, and BMDMs. And protein samples were separated through 10–13% SDS-PAGE and transferred to PVDF membranes. The membranes were incubated with primary antibodies: iNOS (1 : 2000, ab178945, Abcam), Arg1 (1 : 1000, #93668S, Cell Signaling Technology), NLRP3 (1 : 1000, #13158S, Cell Signaling Technology), Caspase1 (1 : 200, sc-392736, Santa Cruz), MT-1/2 (1 : 200, sc-398788, Santa Cruz), GSDMD (1 : 1000, TA4012, Abmart, Shanghai, China), and β-actin (1 : 200, sc-47778, Santa Cruz) overnight at 4°C. Subsequently, the membranes were incubated with HRP-conjugated anti-rabbit or anti-mouse secondary antibodies (1 : 2000, Cell Signaling Technology) for 1 h at room temperature. Then, the blots were visualized by Odyssey infrared imaging system (Tanon, Shanghai, China).
8
Immunofluorescence Detection of Apoptosis Proteins
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The cells were fixed in 95% absolute ethanol for 15 min, incubated with 5% BSA to block non-specific staining, and then incubated with specific primary antibody against apoptosis-associated speck-like protein (ASC) (sc-514414, 1: 100, Santa Cruz Biotechnology) and caspase-1 (sc-392736, 1: 100, Santa Cruz Biotechnology) at 4 °C overnight in darkness. Next, the cells were incubated with fluorescent secondary antibody against IgG H & L (Life Technologies, Carlsbad, CA) at 37 °C for 2 h, followed by incubation with DAPI at room temperature for 15 min. Finally, the cells were observed under a confocal scanning microscope (LSM 700; Carl Zeiss MicroImaging, Inc., Thornwood, NY).
9
Western Blot Analysis of Inflammatory Markers
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Cells and tissue samples were lysed for protein extraction using RIPA assay. The concentration of each protein sample was determined by a BCA (bicinchoninic acid) kit. Equal amounts (50 mg) of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (Bio-Rad, Hercules, CA, United States). Membranes were blocked using 5% non-fat dry milk with TBST buffer for 1 h, then incubated overnight at 4° C with GPR43 (1:1000, ab131003, Abcam), MFN2 (1:500, ab205236, Abcam), PPARγ (1:1000, ab178860, Abcam), NOX-1 (1:1000, ab121009, Abcam), EBP50 (1:1000, ab3452, Abcam), p47phox (1:1000, ab166930, Abcam), NLRP3 (1:1000, 15101, Cell Signaling Technology, Danvers, MA, US), Caspase-1 (1:1000, sc-392736, Santa Cruz Biotechnology), IL-1β (1:1000, 12242, Cell Signaling Technology, Danvers, MA, US), and β-Actin (1:5000, sc-47778, Santa Cruz Biotechnology). After washing, membranes were probed further with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (1:5000, Santa Cruz Biotechnology). After washing with TBST for 15 min, immunoreactive bands were exposed by enhanced chemiluminescence method (Thermo Fisher Scientific, Waltham, MA, USA).
10
Apoptosis Assay by Flow Cytometry
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After the third passage cells (1.2 × 106 cells) underwent different treatments, the cells were incubated in 5 μL of FITC anticaspase-1 (SC-392736, Santa Cruz Biotechnology, USA) in a dark room at 37°C for 1 h, washed with 2 mL of apoptosis buffer, and centrifuged. Then, 5 μL of PI was added to the tube, followed by another incubation for 15 min. Consequently, the cells were resuspended in 500 μL of apoptosis buffer and analyzed by flow cytometry (FACSCanto II, BD, USA).
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