Mouse macrophage cell line Raw264.7 and mouse aorta vascular smooth cell line (MOVAS) were purchased from ATCC and cultured in Dulbecco Modified Eagle's Medium/High Glucose (DMEM/H) medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin. CHO‐K1 stably expressing AT1R was purchased from PerkinElmer (Shanghai, China) and cultured in DMEM/F12 medium containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin. All these cells are maintained at 37°C in a humidified 5% CO2 incubator. For stimulation, MOVAS cells were pretreated with the anti‐ATR‐001 antibody (10 μg/mL), telmisartan (1×10−6 mol/L), or the normal mouse immunoglobulin G (Santa Cruz Biotechnology, sc‐2025) for 2 hours, respectively, subsequently stimulated by Ang II (1×10−5 mol/L) for 72 hours. And Raw264.7 cells were pretreated with the anti‐ATR‐001 antibody (10 μg/mL), telmisartan (1×10−6 mol/L), or the normal mouse immunoglobulin G for 2 hours, respectively, subsequently stimulated by Ang II (1×10−5 mol/L) for 24 hours.
Normal mouse immunoglobulin g
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Normal mouse immunoglobulin G is a type of antibody purified from the serum of normal, healthy mice. It is a complex of proteins that plays a crucial role in the adaptive immune response by recognizing and binding to specific antigens.
Automatically generated - may contain errors
Lab products found in correlation
Raw264, by American Type Culture Collection (1 mentions)
Movas, by American Type Culture Collection (1 mentions)
Telmisartan, by Santa Cruz Biotechnology (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Magna chip g kit, by Merck Group (1 mentions)
Protein a g plus agarose bead, by Santa Cruz Biotechnology (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
4 protocols using normal mouse immunoglobulin g
1
Ang II-Induced Macrophage and Vascular Smooth Cell Response
2
Blocking TRAIL-Induced Apoptosis in MDA-MB-231 Cells
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The MDA-MB-231 cells (1 × 105 cells) were seeded in 35-mm cell culture dishes and cultured for 24 h. For blocking TRAIL-mediated interaction, anti-DR4 antibody (AF347, R&D systems, Minneapolis, MN, USA; 200 ng/mL) or anti-DR5 antibody (CDM234, Cellsciences, Newburyport, MA, USA; 5 µg/mL) were pretreated to cells for 1 h in 37 ℃, and TRAIL (2.5 nM) or TRAIL-ATNC (0.3125 nM) were added and incubated for 3 h in 37 ℃. As a control, normal goat immunoglobulin G (Santa cruz, Dallas, TX, USA; 200 ng/mL) and normal mouse immunoglobulin G (Santa cruz, Dallas, TX, USA; 5 µg/mL) were used. To evaluate blocking effect of antibodies, the apoptotic cell percentage was measured as above.
3
ChIP Analysis of Histone Acetylation in Pancreatic Cells
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ChIP analysis was performed using a Magna ChIP G Kit (Millipore). In brief, isolated islets and MIN6 cells were fixed with 1% formaldehyde for 30 min at room temperature and then subjected to ultrasonic disruption in a solution containing a protease inhibitor cocktail. The lysates were centrifuged to remove debris, diluted 1:10 with a solution containing 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), and 150 mM NaCl, and incubated for 2 h at 4°C with protein G–Sepharose beads. The beads were removed by centrifugation, and the supernatants were subjected to immunoprecipitation by incubation at 4°C overnight with antibodies to acetylated H3K9/K14 (Cell Signaling) or with normal mouse immunoglobulin G (Santa Cruz Biotechnology) followed by 1 h incubation with protein G–Sepharose. The precipitates were washed and then subjected to extraction for 4 h at 65°C with 1% SDS in 100 mM NaHCO3. Proteins were digested with proteinase K, and the remaining DNA was purified using a QIAquick PCR Purification Kit (QIAGEN) and subjected to PCR with Irs2-specific primers (sense, 5′-CTATTACATCCAGAACAGGCG-3′; antisense, 5′-ATGGCAGCTCGGTGCCTTTT-3′ ).
4
Immunoprecipitation of α1 Subunit
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For immunoprecipitation using cell lysates (500 μg) and the mouse brain homogenates (1 mg), they were precleared with 30 μl of protein A/G plus-agarose beads (Santa Cruz; catalog no.: sc-2003) and 1.0 μg of normal mouse immunoglobulin G (Santa Cruz; catalog no.: sc-2025) for 1 h at 4 °C to remove nonspecific binding proteins. The precleared cell lysates were incubated with 2.0 μg of mouse anti-α1 antibody for 1 h at 4 °C and then with 30 μl of protein A/G plus agarose beads overnight at 4 °C. Afterward, the beads were collected by centrifugation at 8000g for 30 s and washed three times with lysis buffer. The complex was eluted by incubation with 40 μl of 2× Laemmli sample buffer (Bio-Rad; catalog no.: 1610737) in the presence of DTT. The immunopurified eluents were separated in 8% SDS-PAGE gel, and Western blot analysis was performed using appropriate antibodies.
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