All reagents used in this work were of analytical grade. Citric acid, FeCl3, and K4[Fe(CN)6] were purchased from Shanghai Jingchun Biological Technology Co., Ltd. (Shanghai, China). PLGA (lactide: glycolide = 50:50, PLGA 12,000 Da Mw) was obtained from Shandong Daigang Biology Engineer Corp. (China). Imiquimod (R837), docetaxel (DTX), Poly (vinyl alcohol) (PVA 25,000 Mw), calcein-AM (CAM), propidium iodide (PI), 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide (DiR) and fluorescence dyes 1,1′-dioctadecyl-3,3,3′,3′ tetra-methylindocarbocyanine perchlorate (DiI) were purchased from Sigma-Aldrich (Shanghai, China). Membrane protein extraction kits, phenylmethanesulfonyl fluoride (PMSF), penicillin–streptomycin solution, and trypsin were purchased from Beyotime (Shanghai, China). Trichloromethane (CHCl3) was purchased from Chongqing Chuandong Chemical Corp. (Chongqing, China). Cell Counting Kit-8 (CCK-8) was obtained from MedChemExpress (Monmouth Junction, NJ, USA). Enzyme-linked immunosorbent assay (ELISA) kits including mouse IL-6, L-12, TNF-α and IL-10 were purchased from Meimian Industrial Co., Ltd. (Jiangsu, China).
Phenylmethanesulfonyl fluoride pmsf
Manufactured by Beyotime
Sourced in China
Phenylmethanesulfonyl fluoride (PMSF) is a lab equipment product that functions as a serine protease inhibitor. It is commonly used in biochemical research and applications to prevent the degradation of proteins by inhibiting the activity of serine proteases.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Beyotime (8 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyl tetrazolium bromide mtt, by Roche (4 mentions)
Bicinchoninic acid bca protein assay kit, by Beyotime (4 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Trypsin, by Beyotime (2 mentions)
Penicillin streptomycin solution, by Beyotime (2 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (2 mentions)
Cell lysis buffer, by Beyotime (2 mentions)
Glutathione peroxidase gsh px detection kits, by Nanjing Jiancheng (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Duolink in situ pla probe anti mouse minus, by Merck Group (2 mentions)
Duolink in situ pla probe anti rabbit plus, by Merck Group (2 mentions)
Protein a g plus agarose, by Santa Cruz Biotechnology (2 mentions)
Duolink in situ detection reagents red, by Merck Group (2 mentions)
Phosphatase inhibitor, by Beyotime (2 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
Cell counting kit 8 cck 8, by Beyotime (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Cell lysis buffer for western and ip, by Beyotime (2 mentions)
Membrane protein extraction kit, by Beyotime (2 mentions)
31 protocols using phenylmethanesulfonyl fluoride pmsf
1
Polymeric Nanocarrier-Mediated Drug Delivery
2
Western Blot Analysis of PHEV-infected Mouse Brains
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PHEV-infected or mock-infected mouse brains were collected, homogenized, and prepared as 10% (wt/vol) suspensions in PBS (pH 7.4). After centrifugation, the supernatants were collected and stored at −80°C. The brain extracts or cell samples were lysed with radioimmunoprecipitation assay (RIPA) lysis buffer (catalog number p0013B; Beyotime, China) with 1 mM phenylmethanesulfonyl fluoride (PMSF) (catalog number ST506; Beyotime, China) on ice for 30 min. The lysates were then subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Separated proteins were transferred to polyvinylidene difluoride (PVDF) membranes, blocked with 5% skim milk, and then incubated with primary antibodies at 4°C overnight. After extensive washing with PBST (PBS, 0.1% Tween 20), the membranes were incubated with 1:5,000-diluted secondary antibodies for 1 h at 37°C. The membranes were detected using a Tanon 5200 automatic chemiluminescence imaging analysis system (Tanon, China). The intensity of each band was measured by ImageJ software (version 1.51j8).
3
Protein Extraction and Analysis
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Cells were lysed in Radioimmunoprecipitation assay (RIPA) buffer buffer containing the protease inhibitor Phenylmethanesulfonyl fluoride (PMSF) (Beyotime, Shanghai, China), and, then, nucleic and cytoplasmic protein extraction kits were applied (Beyotime, Shanghai, China). Protein concentrations were quantitated with a Bicinchoninic Acid Assay (BCA) Protein Assay kit (Pierce, Waltham, MA, USA). Samples were run by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the membrane was blocked with 5% skim milk for 1 h and then incubated with the primary antibodies and secondary antibodies. Protein was determined with the Ultra High Sensitivity ECL Kit. All antibodies are listed in Table S2
4
Synthesis and Characterization of Nanomaterials
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Sodium hydrosulfide (NaHS), chloroauric acid (HAuCl4), hemoglobin (Hb), hydrochloric acid (HCl) and acetone were bought from Innochem. Ethylene glycol (EG), silver nitrate (AgNO3) and 3,3’-Dioctadecyloxacarbocyanine perchlorate (DiO) were obtained from Aladdin. Poly(viny pyrrolidone) (PVP, Mw ≈ 55,000) was purchased from Sigma-Aldrich (St. Louis). Perfluorohexane (PFO) was bought from Energy Chemical. Silver trifluoroacetate (CF3COOAg) was obtained from Adamas. Indocyanine green (ICG) was purchased from ARK Pharm, Inc. Hoechst 33342, Cytosol Protein Extraction Kit and phenylmethanesulfonyl fluoride (PMSF) were purchased from Beyotime Biotechnology. All reagents for cell culture were bought from Gibco. The purified deionized water was prepared by the Milli-Q plus system (Millipore, USA).
5
Regulation of CD73 by Rapamycin and Chloroquine
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MDA-MB-468 cells were centrifuged for 3 min at 500 g after trypsinization, and the medium was removed. Next, cells were resuspended and washed by PBS, and then these cells in a medium with 10% FBS were plated in 6-well plates, grown to approximately 80% confluent at 24 h. Rapamycin (20 nM) and CQ (50 μM) were added into 6-well plates. After 24 h or 48 h, the cells were harvested and shaken vigorously with RIPA (Beyotime) and 1 mM phenylmethanesulfonyl fluoride (PMSF) (Beyotime); the proteins were analyzed using a 15% or 12% separation gel. For SDS-PAGE, the CD73 protein was visualized by Coomassie Brilliant Blue. For the Western blot analysis, the total proteins were transferred to an isopropyl β-D-thiogalactoside (PVDF) membrane. After incubation with the anti-LC3B antibody (1:1000), anti-p62 antibody (1:1000), or anti-CD73 antibody (1D7,1:1000), the proteins on the membrane were washed by TBSTand incubated with the HRP-conjugated antibody for 45 min. The proteins were detected using ChemiDoc XRS+ imaging systems (Bio-Rad, Hercules, CA, USA).
6
Dioscin-Based Hepatoprotective Assay
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Dioscin (purity > 98%) was obtained from Dioscorea nipponica Makino (Yin et al., 2010 (link)), which was dissolved in 0.5% carboxymethyl cellulose sodium (CMC-Na) for in vivo experiments and in 0.1% dimethyl sulfoxide (DMSO) for in vitro tests. The alanine transaminase (ALT), aspartate transaminase (AST), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), and glutathione peroxidase (GSH-Px) kits were from Nanjing Jiancheng Institute of Biotechnology (Nanjing, China). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was provided by Roche Diagnostics (Basel, Switzerland). The bicinchoninic acid (BCA) protein assay kit, cell lysis buffer, and phenylmethanesulfonyl fluoride (PMSF) were obtained from Beyotime Institute of Biotechnology (Jiangsu, China). Dox and were purchased from Sigma-Aldrich (St. Louis, MO, USA).
7
Assessing Pgp Expression in MDCK-MDR1 Cells
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The effect of BS20 on Pgp expression in MDCK-MDR1 cells was determined using Western blotting assay. After treatment with BS20 (5 or 10 µM) for 3 or 24 h, the cells were harvested, and proteins were extracted with radio-immunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology, Shanghai, China) containing a protease inhibitor cocktail (Thermo Fisher Scientific Inc), phenylmethanesulfonyl fluoride (PMSF) (Beyotime Biotechnology) for 30 min on ice. The sample was centrifuged at 20,800 ×g for 15 min at 4°C. The supernatant was collected, and the protein content was measured using the bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific Inc). Equal amounts of individual protein samples were separated by 8% SDS–PAGE gel and then electro-transferred onto the polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories Inc, Hercules, CA, USA). Membranes were blocked for 30 min with 5% non-fat dry milk (Bio-Rad) in TBST buffer (composed of 50 mM Tris (pH 7.6), 150 mM NaCl, and 0.1% Tween-20) and incubated with the antibodies against Pgp (1:1,000) and GAPDH (1:1,000) overnight at 4°C. After incubation with secondary antibody (1:1,000), the specific protein bands were visualized using a chemiluminescence kit (Thermo Fisher Scientific), and the chemiluminescent signals were quantified using ChemiDoc MP Gel Imaging System (Bio-Rad, Hercules, CA, USA).
8
Moxa Cone-Induced Inflammatory Response
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The following reagents and equipment were used: Freund’s adjuvant (Sigma, St Louis, MO, United States), refined moxa wool (Nanyang Hanyi Moxa Co. Ltd., He’nan, China), Fugui formula 1 (Huaji Pharmaceutical Co. Ltd., Shanghai, China), hematoxylin and eosin (HE) staining kit (Nanjing Jiancheng Technology Co. Ltd., Nanjing, China), self-made cone-like copper moxa-cone mold, self-made columnar copper herbal cake mold, biotin label-based rat antibody array (90 rat proteins, RayBiotech, Norcross, GA, United States), ELISA kits for rat Fas/TNFRSF6, FasL/TNFSF6, IL-1R6 (IL-1Rrp2) and IL-1β (Bio-Techne, Minneapolis, MN, United States), phosphatase/protease inhibitor cocktail (Roche, Basel, Switzerland), RIPA lysis buffer, phenylmethanesulfonyl fluoride (PMSF) (Beyotime, Jiangsu, China), microplate reader (Thermo Fisher Scientific, Waltham, MA, United States), scanner (Qinghua Ziguang, Beijing, China), ScanAlyze image analysis software (Stanford University, CA, United States), pathological analysis system (Leica, Wetzlar, Germany), and light microscope and imaging system (Olympus, Tokyo, Japan).
9
Cellular Stress and Antioxidant Assays
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DOX was purchased from Sigma (Santa Clara, CA, USA). Tissue Protein Extraction Kit was obtained from KEYGEN Biotech. Co., Ltd. (Nanjing, China). The bicinchoninic acid (BCA) protein assay kit, ROS assay kit, cell lysis buffer and phenylmethanesulfonyl fluoride (PMSF) were obtained from Beyotime Institute of Biotechnology (Jiangsu, China). Creatine kinase (CK), lactate dehydrogenase (LDH), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH) and glutathione peroxidase (GSH-Px) detection kits were purchased from Nanjing Jiancheng Institute of Biotechnology (Nanjing, China). Sodium dodecyl sulfate (SDS), hydroxymethyl aminomethane (Tris) and 4′,6′-Diamidino-2-phenylindole (DAPI) were purchased from Sigma (St. Louis, MO, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was provided by Roche Diagnostics (Basel, Switzerland). Lipofectamin2000, TransZolTM, TransScript® All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal), TransStart® Top Green qPCR SuperMix were purchased from Beijing TransGen Biotech Co., Ltd. (Beijing, China). SanPrep Column MicroRNA Mini-Preps Kit, MicroRNA First Strand cDNA Synthesis Kit and MicroRNAs Quantitation PCR Kit were purchased from Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China).
10
Molecular Mechanisms of Endothelial Activation
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Lipopolysaccharide LPS L2880 was obtained from Sigma-Aldrich St. Louis, MO, USA. A myeloperoxidase (MPO) assay kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Anti-CD31 (AF3628) was purchased from R&D Systems (Minneapolis, MN, USA). Anti-MYH9 (11128–1-AP) and anti-IgG (B900610) were purchased from Proteintech Group (Chicago, IL, USA). Anti-Wnt5a (sc-365,370), Anti-β-catenin (sc-7963), Anti-TLR4 (sc293072), and Protein A/G PLUS-Agarose (sc-2003) were obtained from Santa Cruz Biotechnology Inc. (Texas, CA, USA). Anti-VE-cadherin (ab33168) was provided by Abcam (Cambridge, MA, USA). Duolink®In Situ PLA® Probe Anti-Rabbit PLUS (DUO92002), Duolink® In Situ PLA® Probe Anti-Mouse MINUS (DUO92004), and Duolink®In Situ Detection Reagents Red (DUO92008) were obtained from Sigma–Aldrich (St. Louis, MO, USA).
The bicinchoninic acid (BCA) protein assay kit and phenylmethanesulfonyl fluoride (PMSF) were procured from Beyotime Biotechnology (Shanghai, China). Alexa Fluor 488- and 594-conjugated secondary antibodies and Dynabeads™ Sheep Anti-Rat IgG (11035) were from Thermo Fisher Scientific (Waltham, MA, USA).
The bicinchoninic acid (BCA) protein assay kit and phenylmethanesulfonyl fluoride (PMSF) were procured from Beyotime Biotechnology (Shanghai, China). Alexa Fluor 488- and 594-conjugated secondary antibodies and Dynabeads™ Sheep Anti-Rat IgG (11035) were from Thermo Fisher Scientific (Waltham, MA, USA).
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