Nuclear extracts of peritoneal cells were prepared based on an adapted version of Rong and Baudry [33 (link)]. Briefly, cells were lysed in cold phosphate buffered saline (PBS) [supplemented with 2 μg/mL leupeptin, 2 μg/mL antipain, and 0.5 mM PMSF (all obtained from Sigma Aldrich)] and centrifuged at 4°C for 2 min at 12,000 g. Pellets were resuspended in lysis buffer [10 mM HEPES pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.1 mM EDTA, 0.5 mM PMSF, 2 μg/mL leupeptin, 2 μg/mL antipain, 3 mM sodium orthovanadate, 30 mM sodium fluoride, and 20 mM sodium pyrophosphate (all obtained from Sigma Aldrich)] and incubated on ice for 15 min. After that, NP-40 (Sigma Aldrich) (final concentration of 0.5%) was added, and samples were vigorously mixed and centrifuged for 30 s at 12,000 g. Pellets were then resuspended in extraction buffer [20 mM HEPES, pH 7.9, 25% glycerol, 1.5 mM MgCl2, 300 mM NaCl, 0.25 mM EDTA, 0.5 mM PMSF, 2 μg/mL leupeptin, and 2 μg/mL antipain (all obtained from Sigma Aldrich)], incubated for 20 min on ice, and centrifuged for 20 min at 13,000 g at 4°C. The resulting supernatants containing nuclear proteins were stored at −80°C. Protein concentration was determined using the Bio-Rad (Richmond, CA, USA) colorimetric assay [34 (link)].
Leupeptin
Manufactured by Merck Group
Sourced in United States, Germany, Italy, China, Japan, France, Israel, Switzerland, Canada, Sao Tome and Principe, United Kingdom, Australia, Ireland
Leupeptin is a protease inhibitor that can be used in laboratory settings to inhibit the activity of certain proteases. It is a tripeptide compound that binds to and inhibits the catalytic sites of proteases.
Automatically generated - may contain errors
Lab products found in correlation
Aprotinin, by Merck Group (423 mentions)
Pepstatin, by Merck Group (149 mentions)
Pepstatin a, by Merck Group (144 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (125 mentions)
Phenylmethylsulfonyl fluoride, by Merck Group (99 mentions)
Triton x 100, by Merck Group (57 mentions)
Sodium orthovanadate, by Merck Group (54 mentions)
Dithiothreitol (dtt), by Merck Group (52 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (47 mentions)
Bestatin, by Merck Group (41 mentions)
Bovine serum albumin (bsa), by Merck Group (39 mentions)
Mg132, by Merck Group (35 mentions)
Antipain, by Merck Group (34 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (33 mentions)
Protease inhibitor cocktail, by Merck Group (31 mentions)
Na3vo4, by Merck Group (29 mentions)
Chymostatin, by Merck Group (26 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (25 mentions)
Streptomycin, by Thermo Fisher Scientific (25 mentions)
Sodium fluoride, by Merck Group (24 mentions)
889 protocols using leupeptin
1
Nuclear Protein Extraction from Peritoneal Cells
2
Muscle Fiber Preparation and Solutions
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The dissecting solution was composed of the following (in mM): K‐proprionate (250), Imidazole (40), EGTA (10), MgCl2·6H2O (4), Na2H2ATP (2), H2O. The storage solution was composed of K‐proprionate (250), Imidazole (40), EGTA (10), MgCl2·6H2O (4), Na2H2ATP (2), glycerol (50% of total volume after transfer to 50:50 dissecting:glycerol solution), as well as leupeptin (Sigma) protease inhibitors. The skinning solution with Brij 58 was composed of K‐proprionate (250), Imidazole (40), EGTA (10), MgCl2·6H2O (4), 1 g of Brij 58 (0.5% w/v). The relaxing solution: Imidazole (59.4), K.MSA (86), Ca(MSA)2 (0.13), Mg(MSA)2 (10.8), K3EGTA (5.5), KH2PO4 (1), H2O, leupeptin (0.05), Na2ATP (5.1), as well as leupeptin (Sigma) protease inhibitors. The pre‐activating solution: KPr (185), MOPS (20), Mg(CH3COOH)2 (2.5), ATP (2.5). Activating solution: Ca2+ (15.11), Mg (6.93), EGTA (15), MOPS (80), ATP (5), CP (15), K (43.27), Na (13.09), H2O. All solutions were adjusted to a pH of 7.0 with the appropriate acid (HCl) or base (Tris).
3
Neuronal Starvation and Leupeptin
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Seven days after plating, neurons were incubated either in EBSS alone (Invitrogen), 20 μM leupeptin (Sigma) diluted in water or EBSS and 20 μM leupeptin for 3–9 hrs. Control cells were incubated in complete Neurobasal medium for 3–9 hrs. Then, the medium was harvested and the cells lysed as described below.
4
Cell Culture Drug Preparation Protocol
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Drugs for cell culture experiments were obtained as follows: Bafilomycin A (SML1661; Sigma-Aldrich), copanlisib (S2802; SelleckChem), leupeptin (L2884; Sigma-Aldrich), and ammonium chloride (A4514; Sigma-Aldrich).
Bafilomycin A was used at a concentration of 160 nM for all experiments. All other drug doses and incubation times are as indicated in the figure legends or directly on the figures. Bafilomycin A was dissolved in DMSO (D2650; Sigma-Aldrich). copanlisib, which is not DMSO soluble, was initially solubilized in a 10% trifluoroacetic acid (T6508; Sigma-Aldrich)/90% water solution to a concentration of 2 mM and then diluted with water to appropriate working stock concentrations. leupeptin (L2884; Sigma-Aldrich) was solubilized in sterile PBS.
For all drug treatments, the cells were maintained in a complete growth medium with serum, according to the recipes specified above.
Bafilomycin A was used at a concentration of 160 nM for all experiments. All other drug doses and incubation times are as indicated in the figure legends or directly on the figures. Bafilomycin A was dissolved in DMSO (D2650; Sigma-Aldrich). copanlisib, which is not DMSO soluble, was initially solubilized in a 10% trifluoroacetic acid (T6508; Sigma-Aldrich)/90% water solution to a concentration of 2 mM and then diluted with water to appropriate working stock concentrations. leupeptin (L2884; Sigma-Aldrich) was solubilized in sterile PBS.
For all drug treatments, the cells were maintained in a complete growth medium with serum, according to the recipes specified above.
5
Autophagic Flux Inhibition in Cardiomyocytes
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Autophagic flux in Atg5+/+ animals was blocked using leupeptin-based inhibition of LC3-II (microtubule-associated protein 1 light chain 3-II) turnover, as described previously.22 (link) Atg5+/+ mice received intraperitoneal injections of leupeptin (40 mg/kg body weight, Sigma–Aldrich, Vienna, Austria) or vehicle (sterile 0.9% NaCl solution), and were sacrificed 50 min after the injection. Isolated cardiomyocytes were subjected to subcellular Ca2+ imaging and immunoblot analysis of LC3 and p62/SQSTM1.
6
Isolation and Lysis of Mitochondrial Fraction
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Cells (2 × 106 (link)) were plated in 100 mm dishes. Following treatment the cells were scraped off the plate using a rubber policeman and centrifuged at 700 × g for 10 min at 4°C. The cell pellets were resuspended in 1 ml of 20 mm HEPES-KOH, pH 7.5, 10 mm KCl, 1.5 mm MgCl2, 1 mm EDTA, 1 mm EGTA, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 250 mm sucrose (Sigma-Aldrich, S9378). The cells were broken open with 6 passages through a 26-gauge needle applied to a 1-ml syringe. The homogenate was centrifuged at 1000 g for 5 min at 4°C. The supernatant fraction was transferred to a high-speed centrifuge tube and centrifuged at 10,000 g for 25 min at 4°C. The pellet (mitochondrial) fraction was lysed in 50 mM Tris (tris[hydroxymethyl]aminomethane)–HCl, 150 mM NaCl, 1% Triton X-100, 0,1% Nonidet P-40 (Sigma-Aldrich, 74388), 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin. Protein concentration of the fraction was determined by the Bradford assay (Bio-Rad). The purity as well as equal loading of mitochondrial fraction was determined by measuring levels of mitochondrial protein COX4I1.
7
Porcine RPE Phagocytosis Assay
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Monolayers of first-passage primary porcine RPE cells seeded onto polycarbonate Transwell® membrane inserts were challenged for 1 h at 37°C with POS in DMEM with 10% FBS (5×107 POS/ml), after POS were subjected to a 10-min sonication step in an ultrasonic bath. After the pulse, cells were washed three times with warm DMEM to remove the unbound POS and chased for 2 h and 4 h. Cells were then washed three times with warm PBS and fixed with 4% paraformaldehyde (PFA) and 0.1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 3 h at room temperature and processed as described below. For the leupeptin assay, cells were pre-incubated with 5 µg/ml leupeptin (Sigma-Aldrich, St Louis, MO) in DMEM with 10% FBS for 2 h, and the inhibitor was maintained throughout the whole course of the experiment. For the phagocytosis assay with BSA–gold, porcine RPE cells were incubated in the lower chamber with 5-nm BSA–gold diluted in DMEM containing 0.5% BSA for 3 h at 37°C before adding POS to the upper chamber. BSA–gold was maintained throughout the experiment. Cells were chased for 2 h and processed for cryo-immuno-electron microscopy, as described below. The same method was used for the phagocytic assay with mouse macrophages J774A.1.
8
Protease Inhibitor Cocktail for In Vitro and In Vivo Studies
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4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochlorine (AEBSF, A8456), aprotinin (A1153), bestatin (Sigma B8385), pepstatin A (P5318), N-(trans-Epoxysuccinyl)-L-leucine 4-guanidinobutylamide (E64, E3132), leupeptin (L2884), N-ethylmaleimide (NEM, E3876), calpeptin (C8999), calpain inhibitor-I (A6185), -II (A6060), -III (M6690), pan-caspase inhibitor (V116), BAPTA-AM (A1076), ionomycin (I0634) were supplied by Sigma. Calpistatin peptide (208902), negative control peptide (208904), elastase inhibitor IV (324759) and cathepsin G inhibitor I (219372) were supplied by Calbiochem. Tryptase inhibitor nafamostat mesylate (NM, 3081) was supplied by Tocris. AEBSF, aprotinin, bestatin, pepstatin A, E64, leupeptin and NEM were used in vitro at concentrations according to manufacturer’s instructions (Sigma Inhib1). calpeptin, calpain, NE, CG, caspase and tryptase inhibitors, BAPTA-AM were used at 100 μM during in vitro studies. Calpistatin and control peptide were used at 10 μM during in vitro studies. Doses of inhibitors used in vivo and details of all other reagents used are described in other method sections.
9
Cytochrome c Detection in Cells
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Cells were treated for 24 h, fractioned and processed for Cytochrome c detection as previously reported [26 (link)]. Briefly, cells were harvested by centrifugation at 2500 rpm for 10 min at 4°C. Pellets were resuspended in 50 μl of sucrose buffer (250 mM sucrose; 10 mM Hepes; 10 mM KCl; 1.5 mM MgCl2; 1 mM EDTA; 1 mM EGTA) (all from Sigma-Aldrich, Milano, Italy) containing 20 μg/ml aprotinin, 20 μg/ml leupeptin, 1 mM PMSF and 0.05% digitonine (Sigma-Aldrich). Cells were incubated for 20 min at 4°C and then centrifuged at 13,000 rpm for 15 min at 4°C. Supernatants containing cytosolic protein fraction were transferred to new tubes and the resulting mitochondrial pellets were resuspended in 50 μl of lysis buffer (1% Triton X-100; 1 mM EDTA; 1 mM EGTA; 10 mM Tris-HCl, pH 7.4) (all from Sigma-Aldrich) containing 20 μg/ml aprotinin, 20 μg/ml leupeptin, 1 mM PMSF (Sigma-Aldrich) and then centrifuged at 13,000 rpm for 10 min at 4°C. Equal amounts of proteins were resolved by 11% SDS/polyacrylamide gel as indicated in the Western blot analysis paragraph.
10
Subcellular Fractionation of Chromatin-Bound Proteins
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For detergent/salt soluble extracts, HeLa S3 and U-2-OS cells were washed with cold PBS, scraped and incubated for 15 mins on ice in HS buffer supplemented with trichostatin A (TSA) and protease and phosphatase inhibitors (5 mM sodium fluoride, 10 mM β-glycerolphosphate, 0.2 mM sodium vanadate, 10 μg/ml leupeptin, 10 μg/ml pepstatin, 0.1 mM PMSF, Sigma). After centrifugation at 16.000 g for 15 min at 4°C, the supernatant was collected. To analyse chromatin-bound complexes, cells were washed twice in cold PBS, scraped and centrifuged at 1.500 g for 10 min at 4°C. The pellet was incubated on ice for 10 mins in CSK buffer (10 mM PIPES pH 7, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2)/0.5% Triton X-100, supplemented with TSA and protease and phosphatase inhibitors (5 mM sodium fluoride, 10 mM β-glycerolphosphate, 0.2 mM sodium vanadate, 10 μg/ml leupeptin, 10 μg/ml pepstatin, 0.1 mM PMSF, Sigma) and subsequently centrifuged at 1.500 g for 10 min to collect soluble proteins. For DNaseI or Benzonase release of chromatin material, the remaining pellet was resuspended in CSK/0.1% Triton X-100 containing DNase I (1000 U/mL, Roche), or Benzonase (2500 U/mL, Millipore), and incubated at 30°C for 30 mins. Solubilized chromatin was then collected by centrifugation at 16.000 g for 10 mins.
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