The RAW 264.7 murine macrophage cells and HL-60 human promyelocytic leukemia cells were purchased from the Korean Cell Line Bank. RAW 264.7 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, Gibco BRL, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS, Gibco BRL). HL-60 cells were incubated under the same conditions using RPMI 1640 medium instead of DMEM. In order to induce differentiation into the neutrophil-like cells, HL-60 cells were incubated with 1.25% v/v DMSO for 5 days without media change. Lipopolysaccharide, gelatin, fibronectin, type I collagen, laminin, Sudan black, neutral red, and Griess reagent were purchased from Sigma-Aldrich (St. Louis, MO, USA). Phaseolin was purchased from Molport (CAS: 13401-40-6, Beacon, NY, USA). Polyclonal antibodies against inducible nitric oxide synthase (iNOS), tubulin, nuclear factor kappa B (NF-κB), inhibitor kappa B (I-κB), glyceraldehyde 3-phosphate dehydrogenas (GAPDH), and lamin A were purchased from Santa Cruz Biotechnology. Polyclonal antibodies against endogenous Ninj1 were obtained from Dr. Kyu-Won Kim (Seoul National University, Seoul, Korea).
Polyclonal antibodies against
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Polyclonal antibodies are a mixture of immunoglobulins produced by different B-cells in response to an antigen. They recognize multiple epitopes on the target antigen and can bind to it with varying affinities. Polyclonal antibodies are commonly used in research, diagnostic, and therapeutic applications.
Automatically generated - may contain errors
Lab products found in correlation
β actin monoclonal antibody, by Merck Group (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Neutral red, by Merck Group (1 mentions)
Laminin, by Merck Group (1 mentions)
Collagen type 1, by Merck Group (1 mentions)
Raw 264.7 murine macrophage cells, by Korean Cell Line Bank (1 mentions)
Lamin a, by Santa Cruz Biotechnology (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Sudan black, by Merck Group (1 mentions)
Raw264.7 cells, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Gelatin, by Merck Group (1 mentions)
Lipopolysaccharide, by Merck Group (1 mentions)
Griess reagent, by Merck Group (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Easysep cell isolation kit, by STEMCELL (1 mentions)
Phospho stat3 y705 antibody, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Sodium orthovanadate, by Merck Group (1 mentions)
12 protocols using polyclonal antibodies against
1
Macrophage and Promyelocyte Differentiation
2
Western Blotting of STAT3 and S1PR1
3
Protein Expression Analysis by Western Blot
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Equal amounts of protein were used for total extracts with specific antibodies followed by electrophoresis on 8–15% SDS polyacrylamide gels under reducing conditions, transferred to nitrocellulose membranes, stained with appropriate primary antibodies and infrared secondary antibodies. The membranes were visualized with chemiluminescence using an ECL Plus (GE-Healthcare). Quantitative analyses of the optical intensities of the protein bands were determined with Un-Scan-It Gel 6.1 (Silk Scientific Inc.). Monoclonal antibodies against P70S6K (sc-8418) and polyclonal antibodies against AKT1/2/3 (sc-8312), GAPDH (sc-32233), p-AKT1/2/3 (sc-33437), and p-P70S6K (sc-7984) were purchased from Santa Cruz Biotechnology. ERK1/2 [(pT185/Y187); 44-680G] and ERK1/2 (44-654G) from Invitrogen and pHCK (Y410; ab61055) and HCK (ab75839) from Abcam.
4
Immunoblot Analysis of Cell Lysates
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Whole cell lysates were prepared by direct lysis in SDS loading buffer. For immunoblot analysis, lysates were separated by 7.5% SDS-PAGE and transferred to a PVDF membrane for probing with antibody. Polyclonal antibodies against arginase 1, IRF8, p38, and Shp2 were from Santa Cruz Biotechnology. pY-Stat6 antibody was from Cell Signaling Technology.
5
Pygo2, JNK, c-Jun, Bcl-2 Western Blot
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Western blotting was performed as described previously [24 (link)]. Briefly, the total protein from lysates of U-87MG and U251 cells was extracted by re-suspending the cell pellets in RIPA buffer. Western blot analyses were performed with polyclonal antibodies against Pygo2, JNK, c-Jun and Bcl-2 (Santa Cruz Biotechnology, USA); monoclonal β-actin antibody was used as a control (Sigma, USA).
6
Western Blot Antibody Validation
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GR monoclonal antibody and polyclonal antibodies against PEPCK and the p65 subunit of NF-κB were purchased from Santa Cruz Biotechnology (Inc., Europe, Heidelberg, Germany). Procaspase-3 polyclonal antibodies specific and monoclonal antibodies against procaspase-9 were obtained from Cell Signaling Technology (Leiden, The Netherlands). Monoclonal antibody against β-actin was obtained from Sigma Aldrich (Sigma Aldrich, St. Louis, MO, USA).
7
Antibody Acquisition for Cell Signaling
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Polyclonal antibodies against phospho-AMPK-T172, phospho-MST1/2-T180/183, MST1, PAK1, PAK3, and ARK5 were purchased from Cell Signaling (Boston, MA, USA). Polyclonal antibody against phospho-ACC-S79 was purchased from Millipore (Charlottesville, VA, USA). Polyclonal antibodies against γ-tubulin, NLP, and survivin as well as monoclonal anti-LKB1 antibody (Ley 37D/G6) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Polyclonal antibodies against PLK1-T210 and pericentrin, and an LKB1 monoclonal antibody were obtained from Abcam (Cambridge, MA, USA). GAPDH antibody was obtained from Bethyl Laboratories (Montgomery, TX, USA). NEK2 (monoclonal) antibody was purchased from BD Biosciences (San Jose, CA, USA). PP1C-γ, PLK1, and phospho-CDC2-T161 antibodies were purchased from Novus Biologicals (Littleton, CO, USA). Polyclonal antibody for α-tubulin and monoclonal antibodies against γ-tubulin and α-tubulin were purchased from Sigma-Aldrich.
8
Western Blot Analysis of Subcellular Fractions
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Nuclear and cytoplasmic fractions were prepared using nuclear extraction kit (Active Motif) according to the manufacture’s instruction. Fractionated proteins were assessed using a BCA (bicinchoninic acid) kit (Pierce), separated on a 4–12% sodium dodecyl sulfate—polyacrylamide gel (Novex), and transferred to a polyvinylidene fluoride (PVDF) membrane (Invitrogen). Membrane was immersed for 1 h in blocking solution (5% milk powder (Roth) in PBS containing 0.1% Tween 20 (PBS-T)) and probed with a molecular specific antibody in blocking solution overnight at 4°C. The membranes were washed extensively in PBS-T and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody in blocking solution at room temperature for 1h. Blots were washed and visualized with enhanced chemiluminescence (ECL; Amersham). Polyclonal antibodies against PAK2 were purchased from Santa Cruz. The Laminin A/C polyclonal antibody (Cell Signaling) and actin monoclonal antibody (Sigma) were used as loading controls for nuclear and cytoplasmic fractions, respectively.
9
Investigating Apoptosis-Inducing Agents
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All-trans retinoic acid, nitroblue tetrazolium (NBT) and dimethylsulfoxide (DMSO) were purchased from Sigma-Aldrich (MO, USA). Arsenic trioxide was obtained from Tongji Hospital (Wuhan, China). Cremophor® EL was purchased from Aladdin Chemicals (Shanghai, China). The cell lysis buffer, BCA Protein Assay Kit and ACK buffer were purchased from Beyotime Biotechnology (Shanghai, China). Matrigel was purchased from BD Biosciences (CA, USA). The Wright-Giemsa Staining Kit was purchased from Jiancheng Bioengineering Institute (Nanjing, China). Z-VAD-FMK, 5-fluorouracil and cisplatin were purchased from Selleck Chemicals (Shanghai, China). Doxorubicin, idarubicin, chloroquine and MG-132 were purchased from MedChemExpress (NJ, USA). Antibodies against PARP, cleaved-caspase-3, Bcl-2, Bax, Bak, γH2AX, β-actin, GAPDH and the appropriate secondary antibodies were purchased from Cell Signaling Technology (MA, USA). Polyclonal antibodies against RARα (C-20, sc-551) and PKCδ (C20, sc-937) were purchased from Santa Cruz Biotechnology (CA, USA).
10
Doxorubicin and Flavonoid Treatment Protocol
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