Various inhibitors were used to study the transport mechanisms of Aβ and its isoforms. The contribution of RAGE to the transport of Aβ across the endothelial monolayer was assessed using the antagonist of this receptor FPS-ZM1 (Sigma) at a concentration of 20 μM (to obtain a stock solution, FPS-ZM1 was dissolved in DMSO to a concentration of 305 mM). To study the caveolin-dependent transport of Aβ isoforms, the inhibitor filipin (Sigma) was used at a concentration of 3 μg/ml (to obtain a stock solution, filipin was dissolved in DMSO to a concentration of 5 mg/ml). An equivalent amount of DMSO was added to the control samples. The contribution of clathrin-dependent endocytosis was assessed using chlorpromazine (Merck) at a concentration of 5 μg/ml (to obtain a stock solution, chlorpromazine was dissolved in DMEM). These concentrations were selected based on literature data and tested for toxicity to bEnd.3 cells using MTT (filipin) and WST (FPS-ZM1 and chlorpromazine) assays according to the manufacturer's protocol (Supplementary Figure 2 ). Before experiments, cells were preincubated with inhibitors added to the upper transwell compartment for 1 h, after which they were filled with solutions containing the inhibitor and 1 μM Aβ, and samples were taken from the lower compartment after 2, 6 and 24 h.
Fps zm1
Manufactured by Merck Group
Sourced in United States, Italy, Germany
The FPS-ZM1 is a laboratory equipment product from Merck Group. It is designed for specific laboratory functions, but a detailed description cannot be provided while maintaining an unbiased and factual approach. Additional information about the core function and intended use of this product is not available.
Automatically generated - may contain errors
Lab products found in correlation
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Tak 242, by Merck Group (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Chlorpromazine, by Merck Group (1 mentions)
Filipin, by Merck Group (1 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
Insulin, by Thermo Fisher Scientific (1 mentions)
Ly294002, by Merck Group (1 mentions)
Peroxynitrite, by Merck Group (1 mentions)
Apocynin, by Merck Group (1 mentions)
Iec 6, by American Type Culture Collection (1 mentions)
Peroxynitrite, by Cayman Chemical (1 mentions)
Mitotracker red cmxros, by Thermo Fisher Scientific (1 mentions)
Viper, by Novus Biologicals (1 mentions)
Hmgb1, by Merck Group (1 mentions)
Sch772984, by MedChemExpress (1 mentions)
Sh 4 54, by MedChemExpress (1 mentions)
31 protocols using fps zm1
1
Inhibiting Aβ Transport Mechanisms
2
Evaluating Inflammatory Pathways in Rat IEC-6 Cells
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Immortalized rat intestinal epithelial cells (IEC-6) were purchased from ATCC (CRL-1592) was maintained in high glucose Dulbelcos modified eagles medium (DMEM), Corning (Tewksbury, MA) supplemented with 10% fetal bovine serum (FBS), Atlanta biologicals (Norcross, GA) suplimented with 2 mM glutamine, 0.1 U/ml Insulin, 100 U/ml Penicillin, and 100 μg/ml streptomycin; Gibco (Grand Island, NY) at 37 °C in a humidified atmosphere of 5% CO2. The cells were then treated with recombinant rat HMGB1 (Mybiosource. San Diego, CA) 30 ng/ml a cytokine mediator of inflammation released by activated macrophages able to bind to and activate RAGE & TLR4, Apocynin (Sigma-Aldrich) 100 µM an inhibitor of NADPH oxidase activity and thus preventing production of superoxide, RAGE antagonist (FPS-ZM1, Milipore, MA) 200 nM V-domain ligand binding RAGE inhibitor, Peroxynitrite (Cayman Chem, Ann Arbor, MI) 300 µM a highly reactive structural isomer of nitrate, Phenyl Boronic Acid (FBA) (Sigma Aldrich) 100 µM was used as a scavenger for Peroxynitrite, PI3K inhibitor (LY294002, Sigma Aldrich) 10 µM as an inhibitor for AKT phosphorylation as required.
3
Evaluating Autophagy Modulators in Cell Cultures
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MG, 3-methyladenine (3-MA), N-acetyl-L-cysteine (NAC), bafilomycin A1 (Baf A), albumin from bovine serum (BSA), thiazolyl blue tetrazolium bromide (MTT), propidium iodide (PI), fluorescein isothiocyanate (FITC)-dextran, and other chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA). MitoTracker Red CMXRos was purchased from Invitrogen (Burlington, ONT, Canada). FPS-ZM1, a specific antagonist for RAGE, was from Millipore (#553030; Temecula, CA, USA).
4
5-FU Cytotoxicity Regulation in MCS2 Cells
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In brief, 5 × 105 MCS2 cells were incubated with 80-nM 5-FU (Sigma) for 24 h. For blocking experiments, 5 × 105 MCS2 cells were pretreated with the appropriate reagent for 30 min at 37°C. TLR4 was specifically inhibited by 10-µM VIPER (Novus) (32 (link)), RAGE by 100-nM FPS-ZM1 (Millipore) (33 (link)), and ERK1/2 by 50-nM SCH772984 (ChemCatch).
5
Astrocytic Inflammatory Response Regulation
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Astrocytes were plated at 1.7×105 cells/well in 48-wells culture plates and cultured for 18h-24h before experiment. Then they were stimulated with HMGB1 (1-2μg/mL, Sigma, St. Louis, MO, USA) or interstitial fluid (100μg/mL) with/without the combination of antibody (HMGB1 Ab or IgG: 5 μg/mL) for 24 h. The culture supernatants were collected for ELISA while the remaining cells were performed for the following RT-PCR or Western blot assay.
Blocking agents for TLR4 (100nM TAK-242, Milipore, Temecula, CA, USA) or RAGE (148nM FPS-ZM1, Milipore, Temecula, CA, USA) in 10% FBS-DMEM/F12 medium were incubated with astrocytes for 2h at 37°C, 5%CO2 incubator. Inhibitor agents for p38 (3μM SB203580, MedChemExpress, Monmouth Junction, NJ,USA), ERK (2μM SCH772984, MedChemExpress, Monmouth Junction, NJ,USA), JNK (5μM SP600125, MedChemExpress, Monmouth Junction, NJ,USA) and stat3 (10μM SH-4-54, MedChemExpress, Monmouth Junction, NJ,USA) in 10% FBS-DMEM/F12 medium were incubated with astrocytes for appointed time at 37°C, 5%CO2 incubator. Subsequently appropriate HMGB1 were used to reach to the final concentration 1-2μg/mL. The remaining steps are the same as before.
Blocking agents for TLR4 (100nM TAK-242, Milipore, Temecula, CA, USA) or RAGE (148nM FPS-ZM1, Milipore, Temecula, CA, USA) in 10% FBS-DMEM/F12 medium were incubated with astrocytes for 2h at 37°C, 5%CO2 incubator. Inhibitor agents for p38 (3μM SB203580, MedChemExpress, Monmouth Junction, NJ,USA), ERK (2μM SCH772984, MedChemExpress, Monmouth Junction, NJ,USA), JNK (5μM SP600125, MedChemExpress, Monmouth Junction, NJ,USA) and stat3 (10μM SH-4-54, MedChemExpress, Monmouth Junction, NJ,USA) in 10% FBS-DMEM/F12 medium were incubated with astrocytes for appointed time at 37°C, 5%CO2 incubator. Subsequently appropriate HMGB1 were used to reach to the final concentration 1-2μg/mL. The remaining steps are the same as before.
6
Albumin and Glyc-alb Effects on TEER
7
Isolation and Culture of Jejunum Intestinal Organoids
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Jejunum intestinal organoids were obtained as previously described [21 (link)]. Briefly, jejunum intestinal organoids from wild-type (WT) mice were dissected and incubated for 30 min at 4 °C in PBS with 2 mM EDTA. After shaking, dissociated fragments were passed through 70 µm filter and crypts were counted and centrifuged for seeding. The pellet was resuspended in Corning-Matrigel® (Fisher Scientific, Madrid, Spain) and IntestiCultTM (StemCell, Grenoble, France) with a 1:1 ratio. Domes were cultured in 24 well plates with IntestiCultTM supplemented with penicillin–streptomycin (Sigma-Aldrich). Intestinal organoids were stimulated with 1% (v/v) of fecal water overnight and then processed for RNA extraction. For experiments with inhibitors, 10 µM of BAY11-7082, 10 µM FPS-ZM1 or 1 µM wortmannin (Sigma Aldrich) were added for 24 h and then RNA was extracted as previously described [22 (link),23 (link)].
8
Insulin-like Growth Factor Signaling Inhibition
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Insulin-like growth factor 1 (IGF-1) was from Prepotech. FPS-ZM-1 was purchased from Sigma-Aldrich (distributed by Biogenerica); AG490 was from Calbiochem. Linsitinib (OSI-906), PD0325901, and LY294002 were purchased from Selleck Chemicals. Human recombinant (hr) S100A7 protein was from R&D systems. All compounds were solubilized in DMSO, except for IGF-1 and S100A7, which were dissolved in 10 mM Acetic Acid and PBS, respectively.
9
Investigating Neuro-2a Cell Response to S100B
10
Tanshinone IIA Modulates HMGB1-Mediated Responses
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Special reagents and antibodies included recombinant HMGB1 (rHMGB1, ProSpec, Rehovot, Israel), IL-6 (PeproTech, NJ, USA), FPS-ZM1 (Sigma–Aldrich, MO, USA), anti-HMGB1 antibody (Abcam, Cambridge, UK), anti-AQP4 antibody (Abcam), anti-NF-κB antibody (Cell Signaling Technology, MA, USA), anti-IL-6 antibody (Millipore, MA, USA), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (Beyotime, Shanghai, China), anti-histone H3 antibody (Beyotime), and anti-Cadherin antibody (Beyotime). Tanshinone IIA was sourced from IMAM International Pharmaceuticals, Ltd. (Tianjin, China).
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