Sodium chloride, potassium chloride, sodium fluoride, acetylcholine, galangin, U46619 and phorbol 12,13-dibutyrate were obtained from Sigma (St. Louis, MO, USA). DTT, TCA and acetone were purchased from Fisher Scientific (Hampton, NH, USA). Enhanced chemiluminescence (ECL) kits were purchased from Pierce (Rockford, IL, USA). Antibodies against phospho-myosin phosphatase targeting subunit 1 (phospho-MYPT1) at Thr855 (1:5,000), MYPT1, phospho-phosphorylation-dependent inhibitory protein of myosin phosphatase (phospho-CPI-17) at Thr38 (1:1,000), CPI-17, adducin or phospho-adducin at Ser662, ERK or phosphoERK at Thr202/Tyr204 (Upstate Biotechnology, Lake Placid, NY, USA or Cell Signaling Technology, Danvers, MA, USA) were used to determine levels of RhoA/ROCK activity (Kitazawa et al., 2000 (link); Wooldridge et al., 2004 (link); Wilson et al., 2005 (link)) or MEK activity. Anti-rabbit IgG (goat) and anti-mouse IgM (goat) conjugated with horseradish peroxidase were used as secondary antibodies (1:2,000 dilutions for both, Upstate B.). A specific MLC20 antibody (1:1,500, Sigma) and anti-mouse IgG (goat) conjugated with horseradish peroxidase (1:2,000, Upstate B.) were used to determine the level of myosin light chain (LC20) phosphorylation. galangin was prepared in dimethyl sulfoxide (DMSO) as a 0.1 M stock solution and frozen at –20°C for later use.
Phorbol 12 13 dibutyrate
Manufactured by Merck Group
Sourced in United States
Phorbol 12,13-dibutyrate is a chemical compound used as a laboratory reagent. It is a phorbol ester that serves as a potent activator of protein kinase C.
Automatically generated - may contain errors
Lab products found in correlation
Dithiothreitol (dtt), by Thermo Fisher Scientific (6 mentions)
Potassium chloride (kcl), by Merck Group (6 mentions)
Acetone, by Thermo Fisher Scientific (6 mentions)
Sodium fluoride, by Merck Group (6 mentions)
Acetylcholine, by Merck Group (6 mentions)
U46619, by Merck Group (5 mentions)
Ionomycin, by Merck Group (4 mentions)
Mlc20 antibody, by Merck Group (3 mentions)
Phenylephrine, by Merck Group (3 mentions)
Phospho mypt1, by Cell Signaling Technology (3 mentions)
Brefeldin a, by Merck Group (2 mentions)
Brefeldin a, by Thermo Fisher Scientific (2 mentions)
Monensin, by Thermo Fisher Scientific (2 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Galangin, by Merck Group (1 mentions)
Efluor 780, by Thermo Fisher Scientific (1 mentions)
Cytofix, by BD (1 mentions)
Anti mouse cd16 32 clone 93, by BioLegend (1 mentions)
Trustain fcx fc receptor blocking solution, by BioLegend (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
12 protocols using phorbol 12 13 dibutyrate
1
Quantification of RhoA/ROCK and MEK Activity
2
Multiparameter Flow Cytometry Analysis
3
Comprehensive Immune Cell Profiling
4
Differentiation of HT22 cells into neurons
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HT22 cells were purchased from the National Laboratory of Molecular Oncology, Cancer Institute and Hospital (Chinese Academy of Medical Sciences and the Peking Union Medical College, Beijing, China) and were cultured in Dulbecco’s modified Eagle’s medium (Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (Gibco), 100 U/mL penicillin (Gibco), and 100 µg/mL streptomycin (Gibco), in a humidified atmosphere of 5% CO2/95% air at 37°C.
A 24-hour incubation with modified DMEM containing 100 mM dibutyryl cyclic adenosine monophosphate (Sigma, St. Louis, MO, USA), 100 mM phorbol 12,13-dibutyrate (Sigma), 50 ng/mL nerve growth factor-beta (Sigma), and 1× N2 supplement (Gibco) was used to induce the differentiation of HT22 cells into cells with neuronal characteristics and functions (Zhao et al., 2016).
A 24-hour incubation with modified DMEM containing 100 mM dibutyryl cyclic adenosine monophosphate (Sigma, St. Louis, MO, USA), 100 mM phorbol 12,13-dibutyrate (Sigma), 50 ng/mL nerve growth factor-beta (Sigma), and 1× N2 supplement (Gibco) was used to induce the differentiation of HT22 cells into cells with neuronal characteristics and functions (Zhao et al., 2016).
5
Assessing RhoA/Rho-kinase and MEK Activity
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Sodium fluoride, KCl, phenylephrine, acetylcholine, U46619, Y-27632, and phorbol 12,13-dibutyrate were purchased from Sigma (St. Louis, MO, USA). DTT, TCA, and acetone were obtained from Fisher Scientific (Hampton, NH, USA). Enhanced chemiluminescence (ECL) kits were from Pierce (Rockford, IL, USA). Antibodies against phospho-myosin phosphatase targeting subunit protein 1 (phospho-MYPT1) at Thr855 (1:5,000), MYPT1, ERK, or phosphoERK at Thr202/Tyr204 (Cell Signaling Technology, Danvers, MA, USA or Upstate Biotechnology, Lake Placid, NY, USA) were used to determine levels of RhoA/Rho-kinase activity (Wooldridge et al., 2004 (link); Wilson et al., 2005 (link)) or MEK activity. Anti-mouse IgM (goat) and anti-rabbit IgG (goat) conjugated with horseradish peroxidase were used as secondary antibodies (1:2,000 dilutions for both, Upstate). A specific MLC20 antibody (1:1500, Sigma) and anti-mouse IgG (goat) conjugated to horseradish peroxidase (1:2000, Upstate) were used to determine the level of LC20 phosphorylation.
6
Multiparametric Phenotyping of Immune Cells
7
Quantifying RhoA/Rho-kinase Signaling
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Drugs and chemicals were obtained from the following sources. Sodium fluoride, KCl, acetylcholine, cardamonin, phenylephrine, and phorbol 12,13-dibutyrate were purchased from Sigma (St. Louis, MO, USA). DTT, TCA, and acetone were obtained from Fisher Scientific (Hampton, NH, USA). Enhanced chemiluminescence (ECL) kits were from Pierce (Rockford, IL, USA). Antibodies against phospho-myosin phosphatase-targeting subunit protein 1 (phospho-MYPT1) at Thr855 (1:5,000), MYPT1, ERK, or phosphoERK at Thr202/Tyr204 were purchased from Cell Signaling Technology (Danvers, MA, USA) or Upstate Biotechnology (Lake Placid, NY, USA) to determine levels of RhoA/Rho-kinase activity [13 (link)14 (link)] or MEK activity. Anti-mouse IgM (goat) and anti-rabbit IgG (goat) conjugated with horseradish peroxidase were used as secondary antibodies (1:2,000 and 1:2,000, respectively, Upstate). A specific MLC20 antibody (1:1500, Sigma) and anti-mouse IgG (Goat) conjugated with horseradish peroxidase (1:2000, Upstate) were used to determine the level of LC20 phosphorylation. cardamonin was prepared in dimethyl sulfoxide (DMSO) as a 100 mM stock solution and frozen at –20℃ for later use. DMSO alone had no observable effect at the concentrations used (data not shown).
8
Rho-kinase Pathway Regulation Assay
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Drugs and chemicals were obtained from the following sources. Sodium fluoride, KCl, acetylcholine, apigenin, U46619 and phorbol 12,13-dibutyrate were purchased from Sigma (St. Louis, MO, USA). DTT, TCA and acetone were obtained from Fisher Scientific (Hampton, NH, USA). Enhanced chemiluminescence (ECL) kits were from Pierce (Rockford, IL, USA). Antibodies against phospho-myosin phosphatase targeting subunit protein 1 (phospho-MYPT1) at Thr855 (1:5,000), MYPT1, ERK or phosphoERK at Thr202/Tyr204 were purchased from Cell Signaling Technology (Danvers, MA, USA) or Upstate Biotechnology (Lake Placid, NY, USA) to determine levels of RhoA/Rho-kinase activity (Wilson et al., 2005 (link); Wooldridge et al., 2004 (link)) or MEK activity. Anti-mouse IgM (goat) and anti-rabbit IgG (goat), conjugated with horseradish peroxidase, were used as secondary antibodies (1:2,000 and 1:2,000, respectively, Upstate, Lake Placid, NY). apigenin was prepared in dimethyl sulfoxide (DMSO) as a 100 mM stock solution and frozen at −20°C for later use. DMSO alone had no observable effect at concentrations used (data not shown).
9
Calcium Signaling Assay Reagents
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Thapsigargin, phorbol 12,13-dibutyrate, C2-ceramide and C6-ceramide and C2-dihydroceramide were purchased from Sigma-Aldrich. FuGENE HD was from Promega (Madison, USA), and TransIT/Jurkat was from Mirus Corporation (Madison, USA). mAb OKT3 and mAb 4G10 were from Thermo Fisher Scientific and A488-conjugated goat anti-mouse γ2b was from Invitrogen. The genetically encoded Ca2+ indicator RGECO-1 (Zhao et al., 2011 (link)) was purchased from Addgene (plasmid #32444; Cambridge, USA). A488-CTxB was from Invitrogen and TX-100 Surfact-Amps was from Thermo Fisher Scientific.
10
Biochemical Markers of RhoA/ROCK Activity
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Potassium chloride, sodium chloride, sodium fluoride, acetylcholine, luteolin, phenylephrine, phorbol 12,13-dibutyrate and U-46619 were obtained from Sigma-Aldrich (St. Louis, MO, USA). Acetone, TCA and DTT were purchased from Fisher Scientific (Hampton, NH, USA) and enhanced chemiluminescence (ECL) kits were purchased from Pierce (Rockford, IL, USA). Antibodies against phospho-CPI-17 at Thr38 (1:1,000), CPI-17, phospho-MYPT1 at Thr855 (1:5,000), MYPT1, adducin or phospho-adducin at Ser662, β-actin, ERK or phospho-ERK at Thr202/Tyr204 (Upstate Biotechnology, Lake Placid, NY, USA or Cell Signaling Technology, Danvers, MA, USA) were used to determine levels of RhoA/ROCK activity (Kitazawa et al., 2000 (link); Wooldridge et al., 2004 (link); Wilson et al., 2005 (link)) or MEK activity. Anti-rabbit IgG (Goat) and anti-mouse IgM (Goat) conjugated with horseradish peroxidase were used as secondary antibodies (1:2,000 dilutions, Upstate Biotechnology). A specific antibody against MLC20 (1:1,500, Sigma-Aldrich) and anti-mouse IgG (Goat) conjugated with horseradish peroxidase (1:2,000, Upstate Biotechnology) were used to verify the level of LC20 phosphorylation. luteolin was prepared in dimethyl sulfoxide (DMSO) as a 0.1 M stock solution and frozen at –20°C for later use.
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