The A. sobria–infected PMφs pellets (MOI=1, 10, 100) were lysed in RIPA buffer containing 1 mM phenylmethylsulfonylfluoride (PMSF, Solarbio, Beijing). The lysed PMφ pellets and A. sobria–infected PMφ supernatants were concentrated using methanol and chloroform as previously described (Wang et al., 2018a (link)) and quantified using BCA method with BCA Protein Assay Kit (Thermo Scientific, USA). Equal amounts of protein extraction (30 μg) were separated by SDS-PAGE electrophoresis and transferred onto PVDF membrane (Millipore, USA). The protein-contained membranes were blocked in 5% non-fat milk; incubated at 4°C overnight with primary antibodies, including IL-1β (1:2,000, R&D, USA), caspase-1 (p20) (1:1,000, Adipogen, Switzerland), NLRP3 (1:1,000, Adipogen, Switzerland), ASC (1:500, Wanleibio, Shenyang), and β-actin (1:5000, Proteintech, Wuhan); incubated at room temperature for 1 h with horseradish peroxidase-conjugated secondary detection antibodies, including rabbit anti-goat IgG, goat anti-mouse IgG (H+L), and goat anti-rabbit IgG (1:2,000, Proteintech, Wuhan); and visualized with Immobilon Western Chemiluminescent HRP substrate (Millipore, USA) on a ChemiScope Western Blot Imaging System (Clinx, Shanghai).
Goat anti rabbit igg
Manufactured by Proteintech
Sourced in United States, China
Goat anti-rabbit IgG is an antibody that binds to and recognizes rabbit immunoglobulin G (IgG) molecules. It is commonly used in various immunological techniques, such as Western blotting, ELISA, and immunohistochemistry, to detect and quantify the presence of rabbit IgG in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti mouse igg, by Proteintech (37 mentions)
Pvdf membrane, by Merck Group (11 mentions)
β actin, by Proteintech (7 mentions)
Anti β actin, by Proteintech (7 mentions)
Affinipure goat anti mouse igg, by Proteintech (6 mentions)
Sa00001 2, by Proteintech (6 mentions)
Bca protein assay kit, by Beyotime (6 mentions)
Ripa lysis buffer, by Beyotime (6 mentions)
Anti gapdh, by Proteintech (5 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Gapdh, by Proteintech (5 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (5 mentions)
β actin, by Cell Signaling Technology (4 mentions)
Sa00001 1, by Proteintech (4 mentions)
Bcl 2, by Proteintech (4 mentions)
Anti β actin antibody, by Proteintech (4 mentions)
Vimentin, by Abcam (4 mentions)
E cadherin, by Abcam (4 mentions)
N cadherin, by Abcam (4 mentions)
124 protocols using goat anti rabbit igg
1
Western Blot Analysis of Aeromonas sobria-Infected Macrophages
2
Proteasome and Apoptosis Pathway Analysis
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The proteins were extracted from lung tissue or cells and lysed by using RIPA buffer (high; cat. no. R0010; Beijing Solarbio Science & Technology Co., Ltd.) on ice for >30 min. Protein levels were quantified using BCA reagent. Samples were loaded 10 µl per lane and electrophoresed by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were blocked in 5% non-fat milk for 2 h at room temperature. The membranes were incubated with primary antibodies at 4˚C overnight. The primary antibodies used were: 20S proteasome β1 (1:500; cat. no. sc-374405; Santa Cruz Biotechnology, Inc.), iNOS (1:1,000; cat. no. AF0199; Affinity Biosciences), cleaved caspase 3 (1:1,000; cat. no. 9661; Cell Signaling Technology, Inc.), p-JNK (1:1,000; cat. no. ET1609-42; HUABIO, Inc.), total JNK Antibody (1:500; cat. no. ET1601-28; HUABIO, Inc.) and GAPDH (1:5,000; cat. no. 60004-1-lg; ProteinTech Group, Inc.). The secondary antibodies used were horseradish peroxide-conjugated goat anti-mouse IgG (1:5,000; cat. no. SA00001-1; ProteinTech Group, Inc.) and goat anti-rabbit IgG (1:5,000; cat. no. SA00001-2; ProteinTech Group, Inc.). The bands were visualized using the Chemiluminescent Substrate kit (cat. no. PE0010; Beijing Solarbio Science & Technology Co., Ltd.) combined with a Bio-Rad exposure system (Bio-Rad Laboratories, Inc.) and analyzed using ImageJ 1.53e.
3
Protein Extraction and Western Blot Analysis
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Proteins were extracted from the jejunum and ileum samples and IPEC-J2 cells, as previously described (Zhang et al., 2015 (link); Yang et al., 2017 (link)). The primary antibodies were rabbit anti-phospho-Ser473 (p)-Akt (1:1000, ab138726), rabbit anti-phospho-Tyr1068 (p)-EGFR (1:250, ab32430; Epitomics, Burlingame, CA), rabbit anti-LC3A/B (1:1000, Cell Signaling Technology, Danvers, MA), rabbit anti-total-EGFR (1:500, 18986-1-AP), rabbit anti-total-Akt (1:1000, 10176-2-AP), and mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:500, 60004-1-Ig, Proteintech Group, Chicago, IL). Horseradish peroxidase-conjugated AffiniPure goat anti-mouse IgG (1:5000, SA00001-1; Proteintech Group) or goat anti-rabbit IgG (1:5000, SA00001-2; Proteintech Group) were used as secondary antibodies.
4
Western Blot Analysis of Cell Proteins
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Protein samples were prepared in RIPA lysis buffer (Thermo Scientific, Rockford, IL, USA). Aliquots of 30 µg protein were fractionated by SDS-PAGE and transferred to PVDF membranes (Merck Millipore, Schwalbach, Germany). The membranes were blocked in skim milk, rinsed in PBS and incubated with primary antibodies overnight [E2F5 1:1000 (Abcam, Cambridge, UK, ab59769), PUMA 1:1000 (Cell Signaling Technology, Danvers, MA, USA, 98672), P21 1:1000 (Cell Signaling Technology, Danvers, MA, USA, 2947), GAPDH 1:1000 (Cell Signaling Technology, Danvers, MA, USA, 5174), and β-Actin 1:1000 (Cell Signaling Technology, Danvers, MA, USA, 3700)]. The secondary antibodies, including goat anti-rabbit IgG (1:2000, Proteintech, USA, srbAF488-1) and goat anti-mouse IgG (1:2000, Proteintech, USA, SA00001-1), were then incubated with the membranes. The relative densities of the bands were quantified using densitometry (Quantity One software, BioRad).
5
Hepatitis B Virus Protein Expression Analysis
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Cells were lysed 72 h after transfection. Cell and tissue samples were lysed in RIRA (Sigma-Aldrich) buffer with PMSF (Sigma-Aldrich). Lysates were centrifuged for 10 min at 12000 × g, and protein in the supernatant was quantified using the BCA method (Pierce). SDS-PAGE and western blot analysis were performed according to standard procedures. Mouse anti-HBs, anti-HBx, anti-HBp, and anti-HBc antibodies were kindly provided by Dr. Quan Yuan from Xiamen University. Goat anti-MAN1A1 (Santa Cruz; 1:500 dilution), rabbit anti-MAN1A2 (Proteintech; 1:200 dilution), rabbit anti-MAN1B1 (GeneTex; 1:500 dilution), mouse anti-MAN1C1 (Abcam; 1:500 dilution) antibodies were used as the primary antibodies. Mouse anti-β-actin (Proteintech; 1:1000 dilution) was used as a reference for protein quantification. Goat anti-mouse IgG (Proteintech; 1:10000 dilution), goat anti-rabbit IgG (Proteintech; 1:10000 dilution), and donkey anti-goat IgG (Santa Cruz; 1:10000 dilution) were used as the secondary antibodies followed by enhanced chemiluminescence (ECL; ThermoFisher Scientific) detection.
6
Immunohistochemical Analysis of Glioma
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Glioma specimens of different grades and normal brain tissue were fixed with formalin, embedded in paraffin, and then made into 4-µm sections. Sections were dewaxed and rehydrated pretreated for antigen retrieval in citrate buffer and quenched for endogenous peroxidase with 3% hydrogen peroxide (H2O2). Nonspecific antigenic sites were blocked with 10% normal goat serum, and sections were incubated overnight withMLLT11 I antibody (1:100, Abcam, ab109016) and CD163 I antibody (1:500, CST, #93498) at 4°C. These were then incubated with secondary antibody (goat anti-rabbit IgG, 1:5,000, Proteintech) and stained with diaminobenzidine tetrahydrochloride (DAB) and hematoxylin. IHC images were acquired, and the score ofMLLT11 and CD163 protein expression was calculated by image processing software ImageJ.
7
Western Blot Analysis of Yap and p-Yap
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Brain tissue and N2a cells were lysed in a radioimmunoprecipitation assay buffer with protease inhibitors on ice to extract total proteins. Electrophoresis was conducted to separate proteins on a sodium dodecyl sulfate-polyacrylamide gel. Then, proteins were transferred to polyvinylidene difluoride membranes and blocked for 2 hours in 5% non-fat milk. The membranes were incubated with primary antibodies at 4°C overnight, including rabbit anti-Yap (Cat# 14074T; 1:1000; Cell Signaling Technology, Beverly, MA, USA), rabbit anti-phospho-(p-)YAP (Cat# 13619S; 1:1000; Cell Signaling Technology), and mouse anti-GAPDH (Cat# sc-365062; 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA). After incubation for 2 hours with horseradish peroxidase-conjugated secondary antibodies (goat anti-mouse IgG, Cat# SA00001-1 or goat anti-rabbit IgG, Cat# SA00001-2; 1:10,000; Proteintech, Chicago, IL, USA), immunoreactive bands were visualized by enhanced chemiluminescence (Cat# WBKLS0500; Millipore, Billerica, MA, USA). GAPDH was used as an endogenous control. The integrated density values were calculated using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
8
Renal Protein Expression Analysis
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Renal protein was extracted from the medulla and cortex of rat kidneys. The protein concentration was measured by the Bradford method, and the supernatant was denatured at 95 °C for 5 min in Laemmli sample buffer. Samples were subjected to SDS-PAGE gels. After electrophoresis, proteins were electro-transferred to a polyvinylidene difluoride membrane (Merck), which was incubated in the blocking buffer (5% non-fat milk, 20 mM Tris-HCl, 150mMNaCl, PH = 8.0, 0.01%Tween 20) for 1 h at room temperature and was followed by incubation with anti-fibronectin (1:1000, ab23750, Abcam) or anti-Collagen-I (1:1000, sc-293,182, Santa Cruz) or anti-CTGF (1:1000, sc-373,936, Santa Cruz) anti-nNOS (1:1000, 4236 s, CST) or anti-HIF-1α (1:1000, ab2185, Abcam) or anti-VEGF (1:1000,A0280, Abclonal) overnight at 4 °C. Binding of the primary antibody was detected by an enhanced chemiluminescence method (BeyoECL Star, P0018A, Byotime) using horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit IgG,1:1000, Proteintech). The quantification of protein expression was performed using Quantity One Analyzer (Bio-Rad).
9
Protein Extraction and Western Blotting Protocol
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A protein extraction kit (Beyotime, Jiangsu, PR China) was used to extract proteins from hepatic cells and liver tissue. The bicinchoninic acid method (Beyotime) was used to estimate the total protein concentration. Western blotting was then carried out according to a previously published method [17 (link)]. In this study, the antibodies used were: anti-PINK1 (catalog no. 23274-1-AP, 1:1000, Proteintech, Rosemont, IL, USA), anti-Parkin (catalog no. 14060-1-AP, 1:1000, Proteintech), anti-protein kinase B beta (AKT2) (catalog no. 17609-1-AP, 1:1000, Proteintech), anti-phospho-AKT2 (pAKT2) (Ser473) (catalog no. 28731-1-AP, 1:1000, Proteintech), anti-insulin receptor (INSR) (catalog no. 20433-1-AP, 1:1000, Proteintech), anti-glucose transporter type 2 (GLUT2) (catalog no. 20436-1-AP, 1:1000, Proteintech), anti-insulin receptor substrate 2 (IRS2) (catalog no. 20702-1-1AP, 1:1000, Proteintech), and anti-β-actin (catalog no. 66009-1-Ig, 1:1000, Proteintech). The secondary antibodies comprised goat anti-rabbit IgG (catalog no. SA00001-2, 1:1000, Proteintech) or goat anti-rat IgG (catalog no. GB23302, 1:1000, Servicebio, Wuhan, PR China) conjugated to horseradish peroxidase (HRP). Immunoreactive bands were captured and analyzed using Image J software (NIH, Bethesda, MD, USA).
10
Western Blot Analysis of Apoptosis Markers
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Western blot analysis was performed as previous described [29 (link)]. Briefly, total protein was extracted from the cells using RIPA lysis buffer (Cwbio, Beijing, China), which was added with Protease Inhibitor Cocktail (1%, v/v) (Cwbio, Beijing, China). Then proteins were separated by 8% and 12% SDS-PAGE gels and electrotransfer onto Immobilon PVDF membranes (Merck KGaA, Darmstadt, Germany). After blocking the membranes with fat free milk, the membranes were incubated overnight at 4 °C with primary antibodies (Table 2 ). After incubation with secondary antibodies (goat anti-rabbit IgG, Proteintech, China), membranes were then imaged by an enhanced chemiluminescent detection kit (Cwbio, Beijing, China).
Antibodies used for Western blotting
| Name | Description | Manufacturer |
|---|---|---|
| Anti-β-actin | Rabbit monoclonal, 43 kDa | Proteintech (20,536–1-AP) |
| Anti-Caspase-3 | Rabbit monoclonal, 34 kDa | CST (#9662S) |
| Anti-Cleaved caspase-3 | Rabbit monoclonal, 16 kDa | CST (#9664S) |
| Anti-CHD8 | Rabbit monoclonal, 290 kDa | CST (#77,694) |
| Anti-p21 | Rabbit monoclonal, 21 kDa | Abcam (109,199) |
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