Microcystin-LR, BSA, PMSF, Tris base, EGTA, leupeptin and pepstatin were purchased from Sigma-Aldrich. The ATM inhibitor (KU55933) was from Tocris. The PLK1 inhibitor (BI2536), the DNA-PK inhibitor (NU7441) and the Aurora A inhibitor (Aurora A Inhibitor-1) were purchased from Selleck Chemicals. Antibodies to PP6c, SAPS1, SAPS2 and SAPS3 were from Bethyl Laboratories. Antibodies to DNA-PKcs and CEP55 were from Abcam. Phosphospecific antibodies to Ser3205 and Thr3950 of DNA-PKcs and antibodies to total DNA-PKcs were as in [15 (link),18 (link)]. Antibodies to actin, α -tubulin and FITC-conjugated α-tubulin were from Sigma-Aldrich. Antibodies to TPX2 (targeting protein for Xklp2) and Chk2 total were from Novus, whereas the phosphospecific antibody to phospho-Thr68 of Chk2 was from Cell Signalling. Antibodies to cyclin B and lamin A/C were from Santa Cruz. The phosphospecific antibody to phospho-Thr210 of PLK1 was from BD Pharmingen. The antibody to Ku 70 was as in [31 (link)]. Antibodies to total PLK1, Aurora A and Aurora A phospho-Thr288 were purchased from Abcam, Serotec and Cell Signalling, respectively.
Microcystin lr
Manufactured by Merck Group
Sourced in United States
Microcystin-LR is a laboratory reference standard used for the detection and quantification of microcystin-LR, a toxic substance produced by certain cyanobacteria. It serves as a calibration and verification tool for analytical methods employed in environmental monitoring and research.
Automatically generated - may contain errors
Lab products found in correlation
Leupeptin, by Merck Group (2 mentions)
2 chloroacetamide, by Merck Group (2 mentions)
Pepstatin, by Merck Group (1 mentions)
Atm inhibitor ku55933, by Bio-Techne (1 mentions)
Ethylene glycol tetraacetic acid (egta), by Merck Group (1 mentions)
Nu7441, by Selleck Chemicals (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
Tris base, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Recombinant wnt5a, by R&D Systems (1 mentions)
Birb 0796, by Cell Signaling Technology (1 mentions)
Pd184352, by Bio-Techne (1 mentions)
U0126, by Cell Signaling Technology (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Glycerol, by Vetec (1 mentions)
Isradipine, by Enzo Life Sciences (1 mentions)
Tween 20, by Thermo Fisher Scientific (1 mentions)
Ici 118 551, by Merck Group (1 mentions)
13 protocols using microcystin lr
1
Mitotic Kinase Inhibitor Protocol
2
Immortalized mouse preadipocyte models
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White preadipocytes from wild type mice (Wt) and knockout (KO) mice lacking p38α (p38α−/−), p38β (p38β−/−), p38γ (p38γ−/−), p38δ (p38δ−/−), MKK3 (MKK3−/−) and MKK6 (MKK6−/−) kinases were immortalized by infection with SV40TpBABE-neo virus as previously described (Matesanz et al., 2017 (link)). The validity of these cell culture model has been previously supported (Matesanz et al., 2017 (link), 2018 (link)). In any case, we have reconfirmed the KO of MKK3 and MKK6 by western blot and those of p38 by qPCR (Supplementary Figure S1 ).
Cells were grown in Dulbecco’s Modified Eagle Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco),l -glutamine (2 mM, Gibco), streptomycin (100 μg/ml, Gibco) and penicillin (100 U/ml, Gibco) and incubated at 37°C under a 5% CO2/95% air atmosphere. Confluent cells were trypsinized and seeded in tissue dishes at a density of 6 × 105 cells/ml. After 8 h, the medium was aspirated and replaced with fresh medium without sera. After 4–5 h, the medium was replaced with fresh medium containing DMSO (control cells) or the indicated concentrations of BIRB 0796 (Cell Signaling), Box5 (EMD Millipore), JNK-IN-8 (Calbiochem), U0126 (Cell Signaling), PD184352 (Tocris), Microcystin L-R (Sigma) or recombinant Wnt5a (R&D Systems) and the incubation was continued for a further 8 h.
Cells were grown in Dulbecco’s Modified Eagle Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco),
3
Antimicrobial Potential of Microcystis
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The antimicrobial activity of Microcystis aeruginosa extract and microcystin were evaluated against Mycobacterium tuberculosis H37Rv (ATCC 27294) pan-susceptible strain and against two clinical isolate mono-resistant to isoniazid and rifampicin with katG S315T and rpoB S531L respectively. Furthermore, M. aeruginosa extract, [D-Leu1] MC-LR, and microcystin-LR (Sigma, USA) toxins were tested against the nontuberculous mycobacteria: M. terrae (ATCC15755), M. chelonae (ATCC 946) and M. kansasii (ATCC12478).
The bacterial suspensions obtained of culture in Ogawa Kudoh medium for about 14 days were prepared in sterile water containing pearls of glass of 3 mm. The suspension was homogenized by vortex agitation and the turbidity was adjusted in agreement with tube one of the scale of McFarland (3.2 × 107 cfu/mL). The inoculum was prepared diluting the bacterial suspension in the proportion of 1:25 in medium 7H9 broth [4.7 g of Middlebrook 7H9 broth base (BD Difco, USA) 2 mL of glycerol (Vetec, Brazil) in 900 mL of water] enriched with 10 % oleic acid-albumin-dextrose-catalase (OADC – BBL, Media Additives, USA) [27 (link)].
The bacterial suspensions obtained of culture in Ogawa Kudoh medium for about 14 days were prepared in sterile water containing pearls of glass of 3 mm. The suspension was homogenized by vortex agitation and the turbidity was adjusted in agreement with tube one of the scale of McFarland (3.2 × 107 cfu/mL). The inoculum was prepared diluting the bacterial suspension in the proportion of 1:25 in medium 7H9 broth [4.7 g of Middlebrook 7H9 broth base (BD Difco, USA) 2 mL of glycerol (Vetec, Brazil) in 900 mL of water] enriched with 10 % oleic acid-albumin-dextrose-catalase (OADC – BBL, Media Additives, USA) [27 (link)].
4
Calcium Channel Protein Immunoblotting
5
Fabrication of Metal-Organic Framework
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Iron(iii ) chloride hexahydrate (Fe3Cl·6H2O), dopamine hydrochloride, 1,3,5-benzenetricarboxylic acid and N,N-dimethyformamide (DMF) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Copper(ii ) acetate (Cu(Ac)2), ethanol, sodium acetate (NaAc) and glycol were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Microcystin-LR, tris(hydroxymethyl)aminomethane (Tris) and α-cyano-4-hydroxycinnamic acid (CHCA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Trifluoroacetic acid (TFA) and acetonitrile (ACN) were purchased from Merck (Darmstadt, Germany). Distilled water was purified by a Milli-Q system (Milford, MA, USA). Other chemicals were of analytical grade and were commercially available.
6
Bisphenol A Detection Using Antibody
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N-(4-Maleimidobutyryloxy) succinimide (GMBS), bovine serum albumin (BSA), 3-mercaptopropyl-trimethoxysilane (MTS), 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC), BPA and humic acid were purchased from Sigma–Aldrich (Germany). The atrazine, microcystin-LR (MC-LR) and 2,4-dichlorophenoxyacetic acid (2,4-D), which are the commonly existed organic pollutants in the water bodies, and four phenolic compounds of 4,4-bis (4-hydroxyphenyl) valeric acid (BVA), nonylphenol, phenol and resorcinol, used for cross-reactivity tests were also purchased from Sigma–Aldrich. All other reagents, if not specified, were supplied by the Beijing Chemical Agents; they were all at analytical grade and used without further purification. Distilled deionized water was used throughout the experiments.
1 mg/mL BPA stock solution was prepared in mixture of 4:6 (v/v) methanol and deionized water and stored at 4°C. Series of BPA standard solutions at different concentrations were freshly prepared by serial dilutions of stock solution using 0.01 M PBS buffer (pH = 7.4). Monoclonal anti-BPA antibody (BPA-MAb, 4D11) was produced by our research group and labeled by Cy5.5 (GE Healthcare Life Sciences) as previously described by Mujumdar et al.32 . The hapten conjugate of BPA and carrier protein was synthesized according to the procedure proposed by Moorhead et al.33 (link).
1 mg/mL BPA stock solution was prepared in mixture of 4:6 (v/v) methanol and deionized water and stored at 4°C. Series of BPA standard solutions at different concentrations were freshly prepared by serial dilutions of stock solution using 0.01 M PBS buffer (pH = 7.4). Monoclonal anti-BPA antibody (BPA-MAb, 4D11) was produced by our research group and labeled by Cy5.5 (GE Healthcare Life Sciences) as previously described by Mujumdar et al.32 . The hapten conjugate of BPA and carrier protein was synthesized according to the procedure proposed by Moorhead et al.33 (link).
7
LRRK2 Kinase Inhibition and Microtubule Binding
8
Western Blot Analysis of 37LRP/67LR
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Cells were lysed in PBS (0.08 M NaCl, 0.002 M KCl, 0.0115 M Na2HPO4, 0.002 M KH2PO4, pH 7.2) containing 1% Triton X-100, in the presence of a protease inhibitor cocktail containing AEBSF, Aprotinin, Bestatin, E-64, Leupeptin and Pepstatin A (Sigma-Aldrich, St. Louis, MO, USA) and a phosphatase inhibitor cocktail containing microcystin LR, cantharidin, and bromotetramisole (Sigma-Aldrich). Protein concentration of lysates was determined using a colorimetric assay (Biorad, Richmond, CA, USA). Equal amounts of protein were subjected to SDS-polyacrylamide gel electrophoresis, transferred to poly-vinyl difluoride (PVDF) filters (Millipore, Windsor, MA, USA), then subjected to Western blot with a polyclonal anti-37LRP/67LR antibody or with non-immune immunoglobulins (Igs) (Jackson ImmunoResearch, Suffolk, England), as decribed [23 (link), 60 (link)].
9
Synaptosomal Fractionation from Thalamic and Cortical Tissue
10
Microcystin-LR Phosphatase Inhibition Assay
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Microcystin-LR was purchased from Sigma (Saint-Quentin Fallavier, France). PP2A was obtained from New England Biolabs Inc. Na2S2O3, MgCl2, MnCl2, HCl, Ca(ClO)2, high-purity CO2, p-Nitrophenyldisodium orthophorphate (p-NPP), tris(hydroxymethyl)aminomethane (Tris), bovine serum albumin (BSA), dithiothreitol (DTT), neoprene rubber, sodium nitrobenzene disodium, and ascorbic acid were purchased from Sinopharm (Shanghai, China). HCOOH, CH3OH, CF3COOH, and HPLC acetonitrile were purchased from Merck (Darmstadt, Germany).
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