LIΔilo:hly was inoculated into BHI broth medium and cultured to the logarithmic phase of growth. The culture supernatant and bacterial precipitate were respectively collected after centrifugation. The total protein from the culture supernatant was extracted by TCA-acetone precipitation, and the total protein from the bacterial precipitate was extracted by ultrasonic breaking and TCA-acetone precipitation. The protein samples were subjected to SDS-PAGE electrophoresis and then transferred onto a PVDF membrane. The membrane was incubated with rabbit anti-listeriolysin O antibody (1:1000) (Abcam, Cambridge, United Kingdom) as the primary antibody, and horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (H + L) (1:1,000) (Beyotime Biotechnology Co., Ltd., Shanghai, China) as the secondary antibody. The HRP chemiluminescence substrate was applied for color development and the results were analysis after incubation.
Horseradish peroxidase hrp labeled goat anti rabbit igg h l
Manufactured by Beyotime
Sourced in United Kingdom, United States, China
Horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (H + L) is a secondary antibody conjugate. It is used to detect and quantify rabbit immunoglobulin G (IgG) in various immunoassay techniques.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Primary antibody dilution buffer, by Beyotime (1 mentions)
Anti mfn2, by Abcam (1 mentions)
Anti lamin b1, by Abcam (1 mentions)
Anti p65, by Cell Signaling Technology (1 mentions)
Anti phospho p65, by Cell Signaling Technology (1 mentions)
Anti nur77, by Abcam (1 mentions)
Celastrol, by MedChemExpress (1 mentions)
Polyvinylidene fluoride (pvdf), by Beyotime (1 mentions)
Anti phospho erk1 2, by Cell Signaling Technology (1 mentions)
Anti cox 2, by Abcam (1 mentions)
Anti erk1 2, by Cell Signaling Technology (1 mentions)
Anti p38, by Cell Signaling Technology (1 mentions)
Anti phospho jnk, by Cell Signaling Technology (1 mentions)
Anti jnk, by Cell Signaling Technology (1 mentions)
Anti phospho p38, by Cell Signaling Technology (1 mentions)
Anti inos, by Abcam (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Penicillin, by Cytiva (1 mentions)
Streptomycin, by Cytiva (1 mentions)
4 protocols using horseradish peroxidase hrp labeled goat anti rabbit igg h l
1
Listeriolysin O Expression Analysis
2
Antibody-Based Investigation of Cellular Pathways
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Our study was conducted using the following antibodies: rabbit polyclonal anti-iNOS, anti-GAPDH, anti-COX2, anti-Nur77, anti-LaminB1, anti-Drp1, anti-phospho-Drp1 (Ser637), and anti-Mfn2 purchased from Abcam (Cambridge, UK). We also used Rabbit polyclonal anti-p65, anti-phospho-p65, anti-ERK1/2, anti-phospho-ERK1/2, anti-JNK, anti-phospho-JNK, anti-p38, and anti-phospho-p38 purchased from Cell Signaling Technology Inc. (Danvers, MA, USA).
The chemical agents used in our study included Dulbecco’s modified eagle medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin (Hyclone Laboratories Inc., Logan, UT, USA); Celastrol (MedChemExpress, Monmouth Junction, NJ, USA); LPS (Sigma-Aldrich, St. Louis, MI, USA); horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (H + L) and polyvinylidene fluoride (PVDF) (Beyotime Biotech, Jiangsu, China). The following kits were also used: BeyoECL Plus, Nuclear and Cytoplasmic Protein Extraction, Primary Antibody Dilution Buffer, and Mitochondrial Membrane Potential Assay with JC-1 (Beyotime Biotech, Jiangsu, China).
The chemical agents used in our study included Dulbecco’s modified eagle medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin (Hyclone Laboratories Inc., Logan, UT, USA); Celastrol (MedChemExpress, Monmouth Junction, NJ, USA); LPS (Sigma-Aldrich, St. Louis, MI, USA); horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (H + L) and polyvinylidene fluoride (PVDF) (Beyotime Biotech, Jiangsu, China). The following kits were also used: BeyoECL Plus, Nuclear and Cytoplasmic Protein Extraction, Primary Antibody Dilution Buffer, and Mitochondrial Membrane Potential Assay with JC-1 (Beyotime Biotech, Jiangsu, China).
3
Optimized ER Stress Modulation Assay
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The 4μ8 c (Sigma, SML0949), BiP inducer X (BIX, Sigma, SML1073), salubrinal (SAL, Sigma, SML0951), PERK Inhibitor I (GSK2606414/GSK, Calbiochem, 516535), and PERK activator CCT020312 (CCT, Calbiochem, 324879) were dissolved in dimethyl sulfoxide (DMSO, Sigma, D2650) to make stock solutions (10 mM), and further diluted properly in culture medium before treatment of the cells. Opti-MEM I Reduced Serum Medium (31985088) was purchased from Gibco. Lipofectamine 3000 (L3000015) was obtained from Thermo Fisher Scientific.
The primary antibodies used in our study were specific for phosphor (p)-PERK (Thr980) (Cell Signaling, 3179), total PERK (Cell Signaling, 3192), p-eIF2α (S51) (Bioworld, BS4787), total eIF2α (Bioworld, BS3651), GRP78 (Santa Cruz, sc-13968), ATG5 (Novus Biologicals, NB110-53818), SQSTM1/p62 (Cell Signaling, 39749), LC3B (Cell Signaling, 2775), CSFV-E2 (MEDIAN/JBT, 9011), Tubulin (Beyotime, AT819), and CSFV-Npro (kindly donated by professor Xinglong Yu, Hunan Agricultural University, China). The secondary antibodies including Alexa Fluor 488-labeled goat anti-mouse IgG(H + L) (A0428), 7-Amino-4-methylcoumarin-3-acetic acid-NHS ester (AMCA)-labeled goat anti-mouse IgG (H + L) (A0413), horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG(H + L) (A0208) and goat anti-mouse IgG(H + L) (A0216) were products obtained from Beyotime Biotechnology.
The primary antibodies used in our study were specific for phosphor (p)-PERK (Thr980) (Cell Signaling, 3179), total PERK (Cell Signaling, 3192), p-eIF2α (S51) (Bioworld, BS4787), total eIF2α (Bioworld, BS3651), GRP78 (Santa Cruz, sc-13968), ATG5 (Novus Biologicals, NB110-53818), SQSTM1/p62 (Cell Signaling, 39749), LC3B (Cell Signaling, 2775), CSFV-E2 (MEDIAN/JBT, 9011), Tubulin (Beyotime, AT819), and CSFV-Npro (kindly donated by professor Xinglong Yu, Hunan Agricultural University, China). The secondary antibodies including Alexa Fluor 488-labeled goat anti-mouse IgG(H + L) (A0428), 7-Amino-4-methylcoumarin-3-acetic acid-NHS ester (AMCA)-labeled goat anti-mouse IgG (H + L) (A0413), horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG(H + L) (A0208) and goat anti-mouse IgG(H + L) (A0216) were products obtained from Beyotime Biotechnology.
4
Protein Extraction and Quantification
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For protein extraction and quantification, cells were washed with PBS (Solarbio, Beijing, China) and lysed in RIPA buffer containing 1% phenylmethylsulfonyl fluoride (PMSF) and 1% phosphatase inhibitor (Beyotime, Beijing, China). Samples were centrifuged at 13,000 × g for 15 min at 4 °C and supernatants collected for Western blot analysis. Protein concentration was measured by BCA protein assay (Beyotime, Beijing, China) and 30 μg proteins separated by SDS-PAGE and transferred to a 0.45 μm PVDF membrane (Millipore, MA, USA). After blocking with 5% bovine serum albumin (BSA), the membrane was incubated with the primary antibody at 4 °C overnight. The membrane was washed and incubated with horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (H + L) or HRP-labeled goat anti-mouse IgG (H + L) (1:2000; Beyotime, Beijing, China) for 1 h at room temperature. Immunoblots were analyzed by enhanced ECL and images acquired with an Amersham Imager (General Electric, Boston, MA, USA) and quantified by densitometry (Image J 1.8.0 software). Bands were normalized to the loading control, β-actin.
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