The virulent CSFV strain Shimen was maintained in our laboratory and the virus titers were determined using methods previously described (Pei et al., 2014 (link)). Primary antibodies used in the study included mouse monoclonal anti-CSFV E2 (JBT, 9011), mouse polyclonal anti-CSFV Npro (kindly provided by Dr. Xinglong Yu, Veterinary Department, Hunan Agricultural University, China), rabbit polyclonal anti-caspase-1 p10 (sc-514; Santa Cruz Biotechnology), mouse monoclonal anti-IL-1β (MP425; Thermo Fisher Scientific), rabbit polyclonal anti-IL-1β (ASC0912; Invitrogen), mouse monoclonal anti-GAPDH (AG019; Beyotime), mouse monoclonal anti-tubulin (AT819; Beyotime), rabbit monoclona anti-NLRP3 antibody (bs-23723R; Bioss), rabbit monoclona anti-ASC(bs-6741R; Bioss), rabbit polyclonal anti-Gasdermin D/DFNA5L(bs-14287R; Bioss), rabbit monoclonal anti-RIG-I antibody (D33H10; Cell Signaling Technology), and rabbit monoclonal anti- Caspase-1 antibody (D7F10; Cell Signaling Technology).The secondary antibodies included horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (BS12478; Bioworld), HRP-conjugated goat anti-rabbit IgG (BS13278; Bioworld), Dylight 488 goat anti-mouse IgG (E032210; EarthOx Life Sciences).
Hrp conjugated goat anti mouse igg
Manufactured by Bioworld Technology
Sourced in United States
HRP-conjugated goat anti-mouse IgG is a secondary antibody that specifically binds to mouse immunoglobulin G (IgG) and is conjugated with horseradish peroxidase (HRP). This reagent is commonly used in immunoassays and other applications that require the detection of mouse IgG.
Automatically generated - may contain errors
Lab products found in correlation
Hrp conjugated goat anti rabbit igg, by Bioworld Technology (6 mentions)
Dylight 488 goat anti mouse igg, by EarthOx (2 mentions)
Anti becn1, by ABclonal (1 mentions)
Anti atg5, by ABclonal (1 mentions)
Anti caspase 3, by ABclonal (1 mentions)
Anti parp, by Beyotime (1 mentions)
Anti p62 sqstm1, by Merck Group (1 mentions)
Anti caspase 8, by Beyotime (1 mentions)
Anti caspase 9, by Beyotime (1 mentions)
Anti lc3b, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Beyotime (1 mentions)
Dylight 488 goat anti rabbit igg, by EarthOx (1 mentions)
Goat anti rabbit igg, by Bioworld Technology (1 mentions)
Rabbit monoclonal anti caspase 3 antibody, by Cell Signaling Technology (1 mentions)
Hrp dab kit, by Zhongshan Golden Bridge Biotechnology (1 mentions)
Monoclonal rabbit anti nlrp3 antibody, by Cell Signaling Technology (1 mentions)
Ab209384, by Abcam (1 mentions)
Ab187091, by Abcam (1 mentions)
Caspase 8, by Beyotime (1 mentions)
Necrostatin 1, by Selleck Chemicals (1 mentions)
7 protocols using hrp conjugated goat anti mouse igg
1
CSFV Strain Shimen Characterization
2
Antibody Utilization for Apoptosis and Autophagy Detection
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The following primary antibodies were used in the study: rabbit polyclonal anti-LC3B (Cell Signaling Technology, 2775), rabbit polyclonal anti-SQSTM1/p62 (Sigma, SAB2104334), rabbit polyclonal anti-BECN1 (ABclonal, A7353), rabbit polyclonal anti-ATG5 (ABclonal, A0203), mouse monoclonal anti-GAPDH (Beyotime, AG019), rabbit polyclonal anti-caspase-8 (Beyotime, AC056), rabbit polyclonal anti-caspase-9 (Beyotime, AC062), rabbit polyclonal anti-Caspase-3 (ABclonal, A2156), rabbit polyclonal anti-PARP (Beyotime, AP102) and mouse monoclonal anti-CSFV E2 (WH303) (JBT, 9011). Mouse polyclonal anti-CSFV Npro was kindly provided by Dr. Xinglong Yu (Veterinary Department, Hunan Agricultural University, China). The secondary antibodies used for immunoblotting analysis were HRP-conjugated goat anti-mouse IgG (Bioworld, BS12478), HRP-conjugated goat anti-rabbit IgG (Bioworld, BS13278). The secondary antibodies used for immunofluorescence including Dylight 488 goat anti-mouse IgG (EarthOx, E032210), Dylight 488 goat anti-rabbit IgG (EarthOx, E032220), Dylight 549 goat anti-mouse IgG (EarthOx, E032310) and Dylight 549 goat anti-rabbit IgG (EarthOx, E032320).
3
Recombinant HIV-1 Protein Characterization
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The recombinant HIV-1 protein p24 and glycoprotein gp41 were purchased from Guangzhou Wondfo Biotech Co., Ltd (Guangzhou, Guangdong, China). Six peptides derived from HIV-1 p24 and gp41 (S1 Table ) were synthesized and purified by high performance liquid chromatography (HPLC) at Sangon Biotech Co., Ltd (Shanghai, China). The sources and characteristics of anti-HIV p24 antibodies are listed in the S2 Table . Horseradish peroxidase (HRP) conjugated goat anti-human immunoglobulin G (IgG), mouse anti-human IgG and HRP conjugated goat anti-human λ light chain antibodies were purchased from Abcam (Cambridge, UK). HRP conjugated goat anti-human κ light chain antibody was purchased from Thermo Fisher (Rockford, IL, USA). HRP conjugated goat anti-mouse IgG and goat anti-rabbit IgG were purchased from Bioworld (Minneapolis, MN, USA). HIV-1 Limiting antigen avidity EIA kits were purchased from Maxim Biotech (Rockville, MD, USA) and KingHawk Pharmaceuticals Inc. (Beijing, China) and are called Maxim LAg Avidity EIA Kit and KingHawk LAg Avidity EIA Kit, respectively, in our study.
4
Western Blot Analysis of Autophagy and Inflammasome Markers
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Protein lysate samples were separated by 12% SDS-PAGE gel and transferred to the NC membrane for 40 min (100 mM of Tris-HCl, 150 Mm of NaCl, 0.05% Tween 20, and pH 7.2). The NC membrane was blotted with 5% non-fat milk in TBST for 1 h, followed by incubation with a primary antibody diluted with TBST for 2 h at 37°C. The membrane was washed with TBST 3 times and incubated with a secondary antibody at 37°C for 2 h. After washing with PBS 3 times, the NC membrane was stained using a HRP-DAB kit (Zhongshan Golden Bridge, Beijing, China).
The primary antibodies that we used are: mouse anti-p62 monoclonal antibody (Abcam, UK), rabbit anti-LC3A/B monoclonal antibody, rabbit anti-NLRP3 monoclonal antibody, rabbit anti-Caspase-3 monoclonal antibody (Cell Signaling Technology, USA), rabbit anti-caspase-1 p10 monoclonal antibody, and rabbit anti-β-actin monoclonal antibody (Jackson ImmunoResearch, USA). Secondary antibodies were Cy3-conjugated donkey anti-rabbit monoclonal antibody (Jackson ImmunoResearch, USA), HRP-conjugated goat anti-rabbit IgG, and HRP-conjugated goat anti-mouse IgG (Bioworld, USA).
The primary antibodies that we used are: mouse anti-p62 monoclonal antibody (Abcam, UK), rabbit anti-LC3A/B monoclonal antibody, rabbit anti-NLRP3 monoclonal antibody, rabbit anti-Caspase-3 monoclonal antibody (Cell Signaling Technology, USA), rabbit anti-caspase-1 p10 monoclonal antibody, and rabbit anti-β-actin monoclonal antibody (Jackson ImmunoResearch, USA). Secondary antibodies were Cy3-conjugated donkey anti-rabbit monoclonal antibody (Jackson ImmunoResearch, USA), HRP-conjugated goat anti-rabbit IgG, and HRP-conjugated goat anti-mouse IgG (Bioworld, USA).
5
Molecular Mechanisms of Cell Death Pathways
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The pan-caspase inhibitor Z-VAD-FMK (S7023) and RIPK1 inhibitor necrostatin-1 (S8037) were purchased from Selleck Chemicals. TNF-α (RP0080S) was the product of Kingfisher Biotech. Staurosporine (S1882) and SM-164 (SC0114) were obtained from Beyotime. The primary antibodies used in this study were as follows: rabbit polyclonal Caspase-3 (AC030; Beyotime), rabbit polyclonal Caspase-8 (AC056; Beyotime), rabbit polyclonal RIPK3 (AF7893; Beyotime), rabbit polyclonal MLKL (A5579; ABclonal), rabbit polyclonal TNF-α (A0277; ABclonal), rabbit polyclonal FasL (A0234; ABclonal), rabbit polyclonal TraiL (A12312; ABclonal), mouse monoclonal GAPDH (AG019; Beyotime), rabbit monoclonal (EPR9627) RIP3 (phospho S227) (ab209384; Abcam), rabbit monoclonal (EPR9514) to MLKL (phospho S358) (ab187091; Abcam), and mouse monoclonal anti-PRV gE (kindly provided by Dr. Gaiping Zhang, College of Veterinary Medicine, Henan Agricultural University, China). The secondary antibodies used for Western blot were HRP-conjugated goat anti-mouse IgG (BS12478; Bioworld Technology) and HRP-conjugated goat anti-rabbit IgG (BS13278; Bioworld Technology).
6
Antibody Immunoblotting with Poly I:C
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The chemical reagent poly I:C used in this study was purchased from Sigma–Aldrich (CAS Number: P1530). The primary antibodies used in this study were as follows: Mouse monoclonal anti-Tubulin (Beyotime, Haimen, China, AT819), rabbit monoclonal anti-Flag (Cell Signaling Technology, Danforth, MA, USA, 14793), mouse monoclonal anti-Myc (Beyotime, AF2864), rabbit polyclonal anti-stat-1 α/β (Beyotime, AF0288), mouse monoclonal anti-IRF3 (Beyotime, AG2321), rabbit monoclonal anti-phospho-IRF3 (Ser386) (Beyotime, AF1594), rabbit monoclonal anti-RIG-I (D33H10) (Cell Signaling Technology, 4200), and rabbit monoclonal TRIM25 (D9T7G) (Cell Signaling Technology, 13773). The secondary antibodies used for immunoblot analysis were HRP-conjugated goat anti-mouse IgG (Bioworld Technology, St Louis Park, MN, USA, BS12478) and HRP-conjugated goat anti-rabbit IgG (Bioworld Technology, BS13278).
7
Western Blot Protein Extraction and Analysis
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The PSM cells 48 h after transfection were harvested to extract the proteins using the lysis buffer (KeyGen Biotech, China) complement with protease and protein phosphatase inhibitor on ice. Protein concentration was determined using BCA Protein Assay Kit (Beyotime, China) and 30 μg protein per sample was loaded and separated using a 5% stacking gel and a 12% separating gel. Separated proteins were transferred to PVDF membrane, which was then blocked in 5% BSA for 2 h at room temperature and incubated with primary antibodies at 4 °C overnight. After washing thrice (10 min once) in TBST, membranes were incubated with HRP conjugated goat anti-rabbit IgG or HRP conjugated goat anti-mouse IgG (Bioworld Technology, USA, 1:2000) 2 h at room temperature.
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