In order to construct EGFP fusion expression vectors of bovine CTLA-4, CD80, CD86, and CD28, we designed the primers to amplify the ORF region that did not have a stop codon. The amplified region fragment of bovine CTLA-4, CD80, CD86, and CD28 was inserted into the cloning site of a pEGFP-N2 vector (Clontech, Palo Alto, CA, USA). The complete vectors, pEGFP-N2-CD80, pEGFP-N2-CD86, and pEGFP-N2-CD28 were transfected into Cos-7 cells by Lipofectamine 2000 reagent (Thermo Fisher Scientific) and cultured for 48 h. The complete vector, pEGFP-N2-CTLA-4 was transfected into CHO-DG44 cells (provided by Dr. Suzuki, Hokkaido University, Japan) with the Lipofectamine LTX reagent (Thermo Fisher Scientific). CTLA-4-EGFP expressing CHO-DG44 cells were cloned and established CTLA-4-EGFP highly expressing CHO-DG44 cells. The binding of CTLA-4-Ig to the CD80 or CD86 expressing cells or the binding of CD80-Ig or CD86-Ig to CTLA-4 expressing cells was confirmed using flow cytometry with FACSVerse (BD Biosciences, San Jose, CA, USA) and FCS Express 4 (De Novo Software, Glendale, CA, USA) as previously described with some modifications [22 (link)]. Rabbit IgG (Southern Biotech, Birmingham, AL, USA) was used as a control Ig.
Rabbit igg
Manufactured by Southern Biotech
Sourced in United States
Rabbit IgG is a type of immunoglobulin G (IgG) antibody purified from rabbit serum. IgG antibodies are the most abundant class of antibodies in the body and play a crucial role in the adaptive immune response.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions)
Fcs express 4, by De Novo Software (1 mentions)
Pegfp n2 vector, by Takara Bio (1 mentions)
Pegfp n2, by Thermo Fisher Scientific (1 mentions)
Lipofectamine ltx reagent, by Thermo Fisher Scientific (1 mentions)
Facsverse, by BD (1 mentions)
Tubulin, by Merck Group (1 mentions)
Cisplatin, by Selleck Chemicals (1 mentions)
Mouse igg, by Southern Biotech (1 mentions)
Caspase 8, by Cell Signaling Technology (1 mentions)
C flip, by Cell Signaling Technology (1 mentions)
Rna pol 2, by BioLegend (1 mentions)
C myc, by Cell Signaling Technology (1 mentions)
Mcl 1, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
Caspase 9, by Abcam (1 mentions)
Ultravision protein block, by Thermo Fisher Scientific (1 mentions)
Ultravision hydrogen peroxide block, by Thermo Fisher Scientific (1 mentions)
10 protocols using rabbit igg
1
Constructing EGFP Fusion Vectors of Bovine Immune Regulators
2
Molecular Mechanisms of Cell Death
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The following inhibitors and drugs were used at the indicated concentration unless otherwise specified in the Figure or Figure legends: dinaciclib 50 nM (Selleck-Chemicals, Houston, TX, USA, cat# S2768), NVP-2 50 nM (Selleck-Chemicals Cat# S8981), etoposide (Absource diagnostic, Munich, Germany, cat# S1225-0100), cisplatin (Selleck-Chemicals, cat# S1166), Propidium Iodide (Sigma-Aldrich, Cat# P4864). Recombinant iz-huTRAIL was provided by H. Walczak. Antibodies against the following antigens were used: RNA pol II RBP1 pSer2 (Biolegend, San Diego, CA, USA, cat# 920204), RNA pol II (Biolegend, cat# 904001), MCL-1 (Cell Signalling, Danvers, MA, USA, cat# 5453), cFLIP (Cell Signalling, cat# 56343), C-Myc (Cell Signalling, cat# 5605), PARP (BD, Franklin Lakes, NJ, USA, cat# 556362), caspase 9 (Abcam, Cambridge, UK, cat# 202068), caspase 8 (Cell Signalling, cat# 9746), cleaved caspase 3 (Cell Signalling, cat# 9664), β-Actin (Sigma-Aldrich, cat# A1978), Tubulin (Sigma-Aldrich, cat# T9026), Rabbit IgG (SouthernBiotech, Birmingham, AL, USA, cat# 4050-05), Mouse IgG (SouthernBiotech, cat# 1031-05). VC-1 was produced and provided by Vichem Chemie Research Ltd.
3
Immunohistochemical Analysis of B4GALNT3 in Colorectal Cancer
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Human colorectal cancer tissue sections were de-paraffinized in xylene and re-hydrated in a series of graded alcohols. After rinsing twice with PBS, the sections were then quenched the activity of endogenous peroxidase with Ultravision Hydrogen Peroxide Block (Thermo scientific, Barrington, IL) for 10 min and incubated with Ultravision Protein Block (Thermo scientific, Barrington, IL) for 10 min to reduce non-specific binding. Sections were incubated with an rabbit anti-B4GALNT3 polyclonal antibody (Sigma, St. Louis, MO, 1:100) diluted with 1% bovine serum albumin (MDBio, Inc., Taipei, Taiwan)/PBS for 16 h at 4 °C. After rinsing twice with PBS, the sections were processed using the Ultravision Quanta Detection System (Thermo scientific, Barrington, IL). Specific immuno-staining was visualized with DAB Quanto (Thermo scientific, Barrington, IL). All sections were counterstained with hematoxylin for 1 min and mounted with UltraKitt (J.T. Baker, Deventer, Holland). Negative controls were performed by replacing primary antibodies with rabbit IgG (SouthernBiotech, Birmingham, AL) at the same concentration.
4
NK Cell Depletion in Ischemia-Reperfusion Injury
5
Comprehensive Antibody Characterization Protocol
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The following commercially available antibodies used were: anti-PAI-1 (clone 41/PAI-1; BD Biosciences), anti-p53 (DO-1; Calbiochem), anti-β-actin (AC-15; Sigma), anti-phospho-Smad2 (Ser465/467) (138D4; Cell Signaling Technology), anti-Smad2/3 (clone 18/Smad2/3; BD Bioscience), anti-CBP (A-22; Santa Cruz Biotechnology), anti-acetyl-Histone H3 (catalog no. 06–599; EMD Millipore), anti-Myc (4A6; EMD Millipore), anti-FLAG (M2; Sigma), anti-HA (Y-11; Santa Cruz Biotechnology), and anti-GFP (B-2; Santa Cruz Biotechnology). Mouse immunoglobulin G1 (IgG1) (MB002; R & D Systems) and rabbit IgG (Southern Biotech) were used as controls.
6
Feline PD-1 and PD-L1/PD-L2 Binding Assay
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To examine whether feline PD-1 binds to PD-L1/PD-L2, 2 × 105 fePD-1–EGFP- and fePD-L1–EGFP-expressing cells were incubated for 30 min with 10 μg/mL of either fePD-1–Ig, fePD-L1–Ig, or fePD-L2–Ig at room temperature (RT), followed by another 30 min incubation with Alexa Fluor 647-conjugated F(ab′)2-goat anti-Rabbit IgG (H+L) secondary antibody (Thermo Fisher Scientific). Rabbit IgG (Southern Biotech) was used as a control protein. Cell fluorescence was analyzed using BD FACSLyric system (BD Biosciences, Franklin Lakes, NJ, USA).
To examine PD-L1 expression on cell lines, 2 × 105 cells were incubated for 30 min with 10 μg/mL CL1Mab-7 or mouse IgG1κ isotype-matched control antibody (15H6; Southern Biotech) at RT, followed by another 30 min incubation with Alexa Fluor 647-conjugated F(ab′)2-goat anti-mouse IgG (H+L) secondary antibody (Thermo Fisher Scientific). Cell fluorescence was analyzed using BD FACSLyric system (BD Biosciences). For fePD-1–EGFP- and fePD-L1–EGFP-expressing cells, only EGFP-positive cells were gated and subjected to further analysis.
To examine PD-L1 expression on cell lines, 2 × 105 cells were incubated for 30 min with 10 μg/mL CL1Mab-7 or mouse IgG1κ isotype-matched control antibody (15H6; Southern Biotech) at RT, followed by another 30 min incubation with Alexa Fluor 647-conjugated F(ab′)2-goat anti-mouse IgG (H+L) secondary antibody (Thermo Fisher Scientific). Cell fluorescence was analyzed using BD FACSLyric system (BD Biosciences). For fePD-1–EGFP- and fePD-L1–EGFP-expressing cells, only EGFP-positive cells were gated and subjected to further analysis.
7
Depletion of NK Cells in Mice
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For systemic depletion of NK cells, we chose treatment with anti-asialo GM1 antibody, which has been used as a precise tool for the specific elimination of NK cells.59 (link) C57 BL/6 mice were given intraperitoneal injections of anti-asialo GM1 antibody (10 µL diluted in 100 µL PBS/mouse, Wako Pure Chemicals, Osaka, Japan) or control (rabbit IgG, Southern Biotech, Birmingham, AL, USA) on days -3, 0, 3, 6, 9 and 12 relative to the injection of B16F0 cells.
8
TGF-α Neutralization in Organoid Culture
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Tspan6 wild-type and Tspan6−/− organoids were cultured in mouse intestinal organoid media without EGF in the presence of TGF-α neutralizing antibody (SI Appendix, Table S1 ) in serial dilutions (2.5, 5, 10, and 20 µg/mL) or control antibody (Rabbit IgG, Southern Biotech). Organoids were cultured for 7 d, and images were acquired using the Zeiss Axiovert 25 microscope equipped with Scion Corporation Greyscale camera, and images were analyzed using ImageJ.
9
Nephrotoxic Serum-Induced Glomerulonephritis
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Nephrotoxic serum nephritis was induced as described previously 21,22. Briefly, mouse glomeruli were isolated from female BALB/c mice and sonicated, and this antigenic preparation was used for immunization of rabbits 27. Rabbit serum collected after sufficient antibody titres were achieved was used as nephrotoxic serum (NTS). Wild-type B6 and B7x -/-mice were primed intraperitoneally (i.p.) with 250 μg of rabbit IgG (Southern Biotech, Birmingham, AL, USA) emulsified with complete Freund's adjuvant (CFA) (Sigma-Aldrich, St Louis, MO, USA) on day 0. On day 5, mice received an intravenous injection of either nephrotoxic serum or phosphatebuffered saline (PBS). Blood and urine were collected at baseline (day 0) and at day 12. Mice were killed on day 12, and kidney tissues were harvested for analysis by histopathology, immunohistochemistry, immunofluorescence and mRNA expression levels. The spleens were obtained for analysis of macrophages, T cells and B cells by flow cytometry.
10
Modulating Immune Responses in Melanoma
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To deplete NK cells, mice received anti-Asialo GM 1 antibodies (1 mg/ml, EBio-Science) or isotype antibodies as control (Rabbit IgG, 0.5 mg/ml, Southern Biotech) every second day from 1 week before B16 inoculation. Five days after tumor cell inoculation, NK cells content was evaluated. Propranolol/Epinephrine Study Eight-week-old C57BL/6 mice were randomized to drinking water containing 0.5 g/l propranolol or nothing. Propranolol administration started 1 week before B16 inoculation. An additional group received daily (Monday to Friday) injections of epinephrine (0.5 mg/kg, 200 ml i.p.) from 1 week before B16 inoculation. For the acute effect of epinephrine, mice were injected with 0.5 mg/kg or 2 mg/kg epinephrine, and sacrified after 30 min, where blood, spleen, and muscle tissue were collected. Anti-IL-6/IL-6 Study Eight-week-old C57BL/6 mice were randomized to groups, receiving i.p. injections of anti-IL-6 antibodies (100 mg/mouse, R&D systems, #AB-406-NA) or vehicle injections twice a week from 1 week before B16 inoculation. An additional group received daily (Monday to Friday) injections of IL-6 (100 ng/mouse, R&D systems, #406-ML-025/CF) from 1 week before B16 inoculation.
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