Mouse ileal epithelial cells were collected by scraping from mouse ileum as previously described.65 (link) Briefly, mouse epithelial cells were lysed in lysis buffer (1% Triton X-100 (Sigma-Aldrich, X100), 150 mM NaCl ( J.T.Baker 3624-19), 10 mM Tris ( Fisher Scientific, BP152-5) pH 7.4, 1 mM EDTA(Fisher Scientific, BP120-1), 1 mM EGTA(Sigma-Aldrich, E3889) pH 8.0, 0.2 mM sodium ortho-vanadate (Sigma-Aldrich, S6508), and protease inhibitor cocktail (Roche Diagnostics, 118367001)). Cultured cells were rinsed twice in ice-cold HBSS (Sigma-Aldrich, H1387), lysed in protein loading buffer (50 mM Tris, pH 6.8, 100 mM dithiothreitol (Amresco, 0281), 2% SDS (Sigma-Aldrich, L3771), 0.1% bromophenol blue (IBI Scientific, IB74040), and 10% glycerol (Sigma-Aldrich, G5516)), and sonicated (Branson Sonifier, 250). Equal amount of protein was separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose (Bio-rad, 162-0112), and immunoblotted with primary antibodies: VDR (Santa Cruz, sc13133), Beclin-1(Santa Cruz, sc10086), Villin (Santa Cruz, sc7672), LC3B (Cell Signal Technology Inc., 2775), ATG16L1 (Abgent, AP18176), p62 (Abgent, AP2183B) or β-actin (Sigma-Aldrich,A1978) antibodies and visualized by ECL chemiluminescence (Thermo Scientific, 32106).
E3889
Manufactured by Merck Group
Sourced in United States
The E3889 is a laboratory equipment manufactured by Merck Group. It serves as a core function for scientific research and analysis. The detailed specifications and intended use of this product are not available in this response.
Automatically generated - may contain errors
Lab products found in correlation
G5516, by Merck Group (4 mentions)
Nitrocellulose, by Bio-Rad (3 mentions)
Bp152 5, by Thermo Fisher Scientific (3 mentions)
Bp120 1, by Merck Group (3 mentions)
S6508, by Roche (3 mentions)
L3771, by IBI Scientific (3 mentions)
Ib74040, by Merck Group (3 mentions)
Ecl chemiluminescence, by Thermo Fisher Scientific (3 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
Triton x 100, by Merck Group (3 mentions)
Dithiothreitol (dtt), by Avantor (2 mentions)
Villin, by Santa Cruz Biotechnology (2 mentions)
S7653, by Merck Group (2 mentions)
P2850, by Merck Group (2 mentions)
S7920, by Merck Group (2 mentions)
P8340, by Merck Group (2 mentions)
Res3098t b7, by Merck Group (2 mentions)
Sodium chloride (nacl), by Merck Group (2 mentions)
H1387, by Merck Group (2 mentions)
Image lab 4, by Bio-Rad (2 mentions)
20 protocols using e3889
1
Isolation and Analysis of Mouse Ileal Epithelial Cells
2
Cell Culture and Inhibitor Treatment
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Unless stated otherwise, for all cell experiments, cells were seeded at 3 × 10^5 cells in 1.5 ml of media onto 10 µg/ml fibronectin (Sigma-Aldrich, F1141) coated MatTek dishes (MatTek Corporation, P35G-1.5-14-C) and left to incubate 16–24 h before treatments, fixation, or live imaging. For inhibitor treatments, cells were treated with the following: DMSO (Life technologies, D12345) at 0.5%, LatA (EMD Millipore, 428021) at a 5 µM dilution from a 1 mM stock in DMSO, Noc (Sigma-Aldrich, M1404) at a 50 µM dilution from a 33 mM stock in DMSO, CK666 (Sigma-Aldrich, SML0006) at a 100 or 200 µM dilution from a 20 mM stock in DMSO, and cytochalasin D (Sigma-Aldrich, C8273) at a 1 µM dilution from a 2 mM stock in DMSO. For EGTA treatment, cells were incubated for 3 h with 3 mM EGTA (Sigma-Aldrich, E3889) in media from a 0.95 M stock. Trypan blue (Bio-Rad, 145-0013) exclusion assay showed that cell viability was at least 93% for all inhibitor treatment conditions, conducted at the longest time point.
3
Protein Extraction and Western Blot Analysis
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For protein extraction, cells were washed twice with ice‐cold PBS and lysed on ice in golden lysis buffer (20 mmol/L Tris‐HCl (Sigma Aldrich RES3098T‐B7, St. Louis, MI), pH 8.0, 137 mmol/L NaCl (Sigma Aldrich S7653, St. Louis, MI), 5.95 mmol/L EDTA (Sigma Aldrich 1233508, St. Louis, MI), 5 mmol/L EGTA (Sigma Aldrich E3889, St. Louis, MI), 10 mmol/L NaF (Sigma Aldrich S7920, St. Louis, MI), 1% Triton X‐100 (Sigma Aldrich X100, St. Louis, MI), and 10% glycerol (Sigma Aldrich G5516, St. Louis, MI)) supplemented with protease inhibitors (Sigma Aldrich P8340, St. Louis, MI) and phosphatase inhibitors (Sigma Aldrich P2850, St. Louis, MI). The proteins were separated via 12% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred to PVDF membranes (Thermo Fisher Scientific LC2002, CA). HAS3 (Sigma Aldrich SAB2108148, St. Louis, MI), GFAP (GeneTex GTX108711, CA), β‐tubulin (GeneTex GTX11307, CA), TUBB3 (GeneTex GTX631836, CA) and α‐actin (GeneTex GTX109639, CA) antibodies were diluted 1:2000 in TBST, and the membranes were incubated for 2 hours at room temperature. Horseradish peroxidase (HRP)‐conjugated anti‐mouse and anti‐rabbit IgG (Santa Cruz Biotechnology SC‐2005, CA) secondary antibodies were diluted 1:4000, and the membranes were incubated for 1 hour at room temperature. α‐Actin was used as the protein loading control.
4
Mouse Intestinal Mucosal Cell Isolation and Analysis
5
Decellularization of Raw264.7 Macrophages
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The decellularization of Raw264.7 cells was carried out as reported elsewhere 16 (link). The TGF-β1-stimulated Raw264.7 were shaken for 120 min at 4 °C in calcium-free phosphate-buffered saline (PBS) containing EGTA (0.5 mM, pH 7.4) (#E3889; Sigma-Aldrich). We repeated the treatment 3-4 times until all cells are removed from the extracellular matrix. The ECM prepared from macrophages were washed with PBS and stored in a 4°C refrigerator for subsequent experiments.
6
Integrin Expression in Caco-2 Cells
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Integrin subunit expression was determined in ELISA assays in 21-day-old Caco-2 monolayers after treatment with ethylene glycol-bis-(β-aminoethyl ether)-N, N, N′, N′-tetraacetic acid (EGTA) (E3889, Sigma-Aldrich) at a 10 μM concentration in Dulbecco's phosphate buffered saline without calcium and magnesium (DPBS; Euroclone S.p.A., Milan, Italy) for 15 min. Briefly, EGTA-treated cell monolayers were washed with DPBS and fixed with 3.7% formaldehyde for 15 min at room temperature (RT). Subsequently, enterocytes were washed and blocked with 2% bovine serum albumin (BSA) in DPBS for 2 h at RT with gentle agitation. All antibodies against integrin subunit were diluted 1:1,000 in 1% BSA and incubated with enterocytes for 1 h. Removal of excess of anti-integrin antibodies with several washes was followed by an additional 1 h of incubation with AP-conjugated secondary antibodies diluted 1:10,000 in 1% BSA. After the addition of para-nitrophenyl phosphate (pNPP), absorbance at 405 nm was determined using a plate reader. Data concerning integrin expression in EGTA-treated cell monolayers were expressed as fold changes compared to untreated monolayers.
7
Mouse Brain Protein Fractionation
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Mouse brain tissues were homogenized in 0.2 ml of ice-cold buffer (0.1 M 3-[N-morpholino]propanesulfonic acid, pH 7.0, 1 mM ethylenediaminetetraacetic acid, 0.5 mM MgSO4, 1 M sucrose [Panreac AppliChem, A2211]) containing 1 mM NaF, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride and 10 μg/ml each of aprotinin (Sigma-Aldrich, A1153) and 1 μg/ml leupeptin (Sigma-Aldrich, L8511). The homogenates were cleared by centrifugation at 50,000 × g for 20 min at 4°C, and the supernatants were collected as soluble fractions. To prepare the sarkosyl-insoluble fraction, the pellets were resuspended in lysis buffer (0.1 M 3-[N-morpholino]propanesulfonic acid, pH 7.0, 10% sucrose, 2 mM ethylene glycol tetraacetic acid [Sigma-Aldrich, E3889], 0.5 mM MgSO4, 500 mM NaCl, 1 mM MgCl2, 10 mM NaH2PO4, 20 mM NaF) containing 1% N-lauroylsarcosine (sarkosyl; Sigma-Aldrich, L9150) with protease inhibitors (Sigma-Aldrich, L8511, A1153 and P7626), vortexed for 1 min at room temperature, incubated at 4°C for 16 h, and then centrifuged at 200,000 × g for 30 min at room temperature. The supernatant fractions were collected as sarkosyl-soluble fractions, and the pellets, sarkosyl-insoluble fractions, were resuspended in sodium dodecyl sulfate protein loading buffer and incubated at 95°C for 5 min.
8
Kinetic Analysis of PDGF-CC Antibodies
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Biosensor analysis was performed on a BIAcore 2000 biosensor (GE Healthcare) using an NTA sensor chip as described in detail [33 (link)]. Briefly, a NTA sensor chip (GE Healthcare, BR1004-07) was loaded with Ni2+ and used to immobilize histidine tagged PDGF-CC ligand. The PDGF-CC antibodies were passed over the chip at varying concentrations in order to determine apparent affinity (KD). Chips were regenerated with ethylene glycol tetraacetic acid (EDTA, Sigma, E3889). The chip was re-equilibrated by washing with HBS (containing no EDTA) before further analysis. Using BIAevaluation software, a 1:1 Langmuir binding analysis model was used to estimate the apparent association (ka) and disassociation (kd) rate constants for each antibody generated (49).
9
Isolation of Mitochondria from Heart
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Mitochondria were isolated from the whole heart according to the method described in [28 (link)]. The heart was purified from blood vessels, crushed and homogenized. The homogenate was suspended in the isolated medium containing 75 mM sucrose (Sigma S7903, Saint-Louis, MO, USA), 10 mM Tris-HCl (pH 7.4), 225 mM mannitol (Sigma M4125, Saint-Louis, MO, USA), 0.5 mM EDTA (Sigma E9884, Saint-Louis, MO, USA), 0.5 mM EGTA (Sigma E3889, Saint-Louis, MO, USA), and 0.1% BSA (Sigma A6003, Saint-Louis, MO, USA). Mitochondria were isolated by differential centrifugation. At first, the homogenate was centrifuged at 1000 × g for 10 min to precipitate blood and destroyed mitochondria. The supernatant was precipitated at 8500 × g for 10 min. The precipitated RHM were washed and in isolation medium without EDTA or BSA (8500× g, 10 min). All procedures were conducted at 4 °C. The concentration of protein RHM was determined using the Bradford assay (30–35 mg/mL).
10
Investigating Cellular Responses to Mechanical Stimuli
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For experiments involving inhibitors, cells were exposed to the inhibitor for 0.5 h, unless stated otherwise, in the presence or absence of compression. For inhibiting the function of mechanically sensitive ion channels, cells were treated with gadolinium chloride (Gd3+, 5 μM, # 203289, Sigma) or GsMTx4 (5 μM, #ab141871; Abcam, Cambridge, MA). To remove calcium ions from the DMEM, EGTA (2 mM, # E3889; Sigma) was added to the medium. To disrupt caveolae in the membrane, cells were treated with 5 mM of methyl-β-cyclodextrin (MβCD, # SLBP3372V, Sigma). To evaluate the impact of HIF-1a, cells were treated with inhibitor CAY10585 (10 μM, # ab144422, Abcam). For inhibiting the activity of Src, cells were treated with PP2 (10 μM, Calbiotech, Spring Valley, CA). For inhibiting the activity of MMP, cells were treated with GM-6001 (a broad-spectrum MMP inhibitor, 15 μM, #CC1000; Sigma).
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