The cells were lysed in buffer containing 10 mM Tris-buffer (pH 7.6), 1% Triton X-100, 1% phosphatase inhibitor cocktail and 1 mM PMSF. The lysates were boiled in sodium dodecyl sulfate (SDS) sample buffer and were subjected to SDS–PAGE. Cytoplasmic extracts and nuclear extracts were obtained using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Sigma, 78833). Rabbit monoclonal antibodies against GAPDH, NLRP3, and ASC were purchased from Santa Cruz Biotechnology (CA, USA) and were diluted 1:1000. The anti-ATG5 antibody, anti-LC-3 antibody, anti-Caspase-1 antibody, anti-PCNA, and anti-p65 antibody were purchased from Cell Signaling Technology (Beverly, MA, USA) and were diluted 1:1000. The anti-TRIF antibody was purchased from Abcam and was diluted 1:1000. Horseradish peroxidase- conjugated goat anti-rabbit immunoglobulin G (Cell Signaling Technology, MA, USA) was used as the secondary antibody. Immunoreactive bands were identified using the ECL Western Blotting Detection System (Millipore Corporation, Billerica, MA, USA).
Horseradish peroxidase conjugated goat anti rabbit immunoglobulin g
Manufactured by Cell Signaling Technology
Sourced in United States
Horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G is a secondary antibody used in various immunoassay techniques. It is produced by conjugating horseradish peroxidase, an enzyme, to goat-derived antibodies that specifically recognize and bind to rabbit immunoglobulin G.
Automatically generated - may contain errors
Lab products found in correlation
Ecl kit, by Beyotime (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Caspase 3, by Cell Signaling Technology (2 mentions)
Anti lc3 antibody, by Cell Signaling Technology (1 mentions)
Anti p65 antibody, by Cell Signaling Technology (1 mentions)
Ecl western blotting detection system, by Merck Group (1 mentions)
Ne per nuclear and cytoplasmic extraction reagents, by Merck Group (1 mentions)
Nlrp3, by Santa Cruz Biotechnology (1 mentions)
Anti atg5 antibody, by Cell Signaling Technology (1 mentions)
Anti caspase 1 antibody, by Cell Signaling Technology (1 mentions)
Anti trif antibody, by Abcam (1 mentions)
Anti pcna, by Cell Signaling Technology (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Las 4000 luminescent image analyzer, by Fujifilm (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Ripa buffer, by Solarbio (1 mentions)
Anti gapdh, by Abcam (1 mentions)
Super ecl plus kit, by Keygen Biotech (1 mentions)
Phenylmethylsulfonyl fluoride, by Solarbio (1 mentions)
8 protocols using horseradish peroxidase conjugated goat anti rabbit immunoglobulin g
1
Western Blot Analysis of Inflammasome Proteins
2
Western Blotting Analysis of Intestinal Proteins
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Western blotting analysis was conducted as described by Xu et al. [29 (link)], with some modifications. Briefly, intestinal tissues were homogenized, and protein concentrations
were determined with the BCA Protein Assay Kit (Beyotime, Jiangsu, China). Then, protein from each sample (20 µg) was separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The
membranes were blocked with 5% skimmed milk in blocking buffer containing 0.1% TBST for 1.5 h at room temperature, and then incubated with primary antibodies against p62 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mTOR, and
β-actin (Cell Signaling Technology, Danvers, MA, USA) for 12 h at 4°C. After three consecutive washes with TBST, the membranes were incubated with the secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit
immunoglobulin G; Cell Signaling Technology). Antibody-bound protein bands were detected using enhanced chemiluminescence reagents (ECL-Kit; Beyotime) followed by autoradiography. The blots were scanned using a LAS-4000
Luminescent Image Analyzer (Fuji Film, Tokyo, Japan), and the antigen-antibody complexes were quantified with Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA).
were determined with the BCA Protein Assay Kit (Beyotime, Jiangsu, China). Then, protein from each sample (20 µg) was separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The
membranes were blocked with 5% skimmed milk in blocking buffer containing 0.1% TBST for 1.5 h at room temperature, and then incubated with primary antibodies against p62 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mTOR, and
β-actin (Cell Signaling Technology, Danvers, MA, USA) for 12 h at 4°C. After three consecutive washes with TBST, the membranes were incubated with the secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit
immunoglobulin G; Cell Signaling Technology). Antibody-bound protein bands were detected using enhanced chemiluminescence reagents (ECL-Kit; Beyotime) followed by autoradiography. The blots were scanned using a LAS-4000
Luminescent Image Analyzer (Fuji Film, Tokyo, Japan), and the antigen-antibody complexes were quantified with Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA).
3
TRPC6 Protein Expression Analysis in PC12 Cells
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PC12 cells were collected and treated with RIPA buffer (Beijing Solarbio Science & Technology Co., Ltd.) containing 1% (v/v) phenylmethylsulfonyl fluoride (Beijing Solarbio Science & Technology Co., Ltd.), 0.3% (v/v) protease inhibitor (Sigma-Aldrich; Merck KGaA) and 0.1% (v/v) phosphorylated proteinase inhibitor (Sigma-Aldrich; Merck KGaA). Western blots were performed as previously described (22 (link)). Membranes were incubated with primary antibodies against TRPC6 (cat. no. ab62461; 1:1,000; Abcam) and anti-GAPDH (cat. no. 2118; 1:5,000; Cell Signaling Technology, Inc.) overnight at 4˚C. Membranes were subsequently incubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (cat. no. ZB-2301; 1:5,000; Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.) for 2 h at room temperature, followed by three washes with TBS-Tween-20. Enhanced chemiluminescence (ECL; EMD Millipore) was used to determine protein concentrations according to the manufacturer's protocol. Protein signal was detected using a Super ECL Plus kit (Nanjing KeyGen Biotech Co., Ltd.). Relative protein expression was normalized to GAPDH. All experiments were repeated three times. ImageJ v1.43b software (National Institutes of Health) was used for densitometric analysis.
4
Protein Expression Analysis in Intestinal Samples
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Antibodies against related proteins [TLR4 (# 14358 S), MyD88 (# 4283 S), IκBα (# 4812 S), NF-κB p65 (# 8242 S), caspase-3 (# 14220 S), PCNA (# 13110 S), claudin (# 13255 S), and occludin (# 13255 S)] were purchased from Cell Signaling Technology (Danvers, MA, USA). The protein content of the samples was measured using a BCA Protein Assay Kit (Beyotime, Jiangsu, China). For Western blotting analysis, 50 μg of protein from each jejunum sample was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After SDS-PAGE, the separated proteins were transferred onto polyvinylidene difluoride membranes. The membranes were blocked with blocking buffer (5% nonfat dry milk) for 12 h at 4 °C and probed with appropriate primary and secondary antibodies (horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G, Cell Signaling Technology; 1:10,000 dilution in 1% milk). Signals were detected using enhanced chemiluminescence (ECL-Kit, Beyotime), followed by autoradiography. Photographs of the membranes were taken using the Luminescent Image Analyzer LAS-4000 system (Fujifilm Co.) and quantified with ImageJ 1.42q software (NIH, Bethesda, MD, USA).
5
PD-1 Inhibitor Protocol for T Cell Activation
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DMEM, RPMI-1640 medium, Leibovitz 15 medium, FBS, GlutaMAX™ and 2-mercaptoethanol were purchased from Gibco; Thermo Fisher Scientific, Inc. Penicillin-streptomycin was purchased from Sigma-Aldrich (Merck KGaA). FuGENE® HD Transfection Reagent was purchased from Promega Corporation and TRIzol® reagent was purchased from Invitrogen (Thermo Fisher Scientific, Inc). Anti-CD3 and anti-CD28 were purchased from BD Biosciences. Allophycocyanin-conjugated anti-human-CD274/PD-L1 antibody were purchased from eBioscience (Thermo Fisher Scientific, Inc.). Rabbit anti-Erk1/2, rabbit anti-phospho-Erk1/2, rabbit anti-AKT, rabbit anti-GAPDH and horseradish peroxidase-conjugated Goat anti-rabbit immunoglobulin G were purchased from Cell Signaling Technology, Inc. Human IFN-γ ELISA kits and human IL-2 ELISA kits were purchased from Cisbio (PerkinElmer, Inc.). A0-L (a PD-1 inhibitor; patent no. WO 2015/034820 A1; molecular weight, 475.58) was synthesized by Dr Wei Lv of East China Normal University. The company name and catalog number for ELISA kits and all antibodies are listed in Table SI .
6
Western Blot Analysis of Apoptosis and Inflammation Markers
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For cleaved caspase-3 and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor (IκB) expression analysis [11 (link), 18 (link), 21 (link)], supernatants of homogenized liver tissues (100 μg total protein) were separated by electrophoresis in a 10–12% polyacrylamide gel and transferred onto nitrocellulose membrane (Mini Trans-Blot Cell; Bio-Rad Laboratories, Hercules, CA, USA). The membranes were blocked with 5% albumin (BioShop Canada Inc., Burlington, ON, USA) in Tris-buffered solution containing 0.1% Tween-20 (TBST) for 1.5 h at room temperature and then incubated overnight at 4 °C with primary antibody (cleaved caspase 3, 1:500 dilution, Cell Signaling Technology Inc., Danvers, MA, USA; IκB, 1:5000 dilution, Abcam, Cambridge, UK) in TBST buffer, which was followed by the addition of a horseradish peroxidase-conjugated goat antirabbit immunoglobulin G (Cell Signaling Technology Inc.). The protein expression was visualized using Pierce enhanced chemiluminescence Western blotting reagent (Thermo Fisher Scientific, Rockford, IL, USA), followed by exposure to radiographic film. The densitometry of bands was quantified.
7
Western Blot Analysis of Apoptosis Markers
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Cells were collected and lysed with cold RIPA lysis buffer. Subsequently, the extracted proteins (50 µg) were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA). The membrane was then probed with primary antibodies against caveolin-1 (Cat No. #3267), AKT (Cat No. #4685), phosphorylated (p)-AKT (Cat No. #4060), Bad (Cat No. #9292), p-Bad (Cat No. #9291), Bcl-xl (Cat No. #2764), Bcl-2 (Cat No. #4223), cytochrome c (Cat No. #4280), caspase-9 (Cat No. #9502), caspase-3 (Cat No. #9662), and GAPDH (Cat No. #5174) (Cell Signaling Technology) at 4°C overnight. Subsequently, the membrane was washed and probed with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (Cell Signaling Technology). Protein bands were detected using an enhanced chemiluminescent substrate (Thermo Fisher Scientific, Inc.).
8
Immunoblotting for Stem Cell Markers
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Sample collection and immunoblotting were performed as previously described [21] . The primary antibodies used were rabbit polyclonal anti-Nestin antibody (Abcam, Cambridge, MA, USA), anti-α-SMA (Abcam, Cambridge, MA, USA), anti-Col I (ABclonal, Wuhan, China), anti-ST-2 (ABclonal, Wuhan, China), IL-33 (ABclonol, Wuhan, China) and mouse monoclonal anti-β-actin (Thermo Scienti c, Waltham, USA). The secondary antibodies used in this study were horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (Cell Signaling Technology, Danvers, MA, USA) antibody and goat anti-mouse immunoglobulin G (Cell Signaling Technology, Danvers, MA, USA).
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