Harvested Huh7 cells (2 × 106) were washed in ice-cold PBS and incubated for 25 min on ice with 300 μL of ice cold buffer A (250 mmol/L Sucrose, 20 mmol/L (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) HEPES-KOH (pH 7.4), 10 mmol/L KCl, 1 mmol/L Na-EGTA, 1 mmol/L Na-EDTA, 1.5 mmol/L MgCl2, 1 mmol/L dithiothreitol (DTT), 0.1 mmol/L phenylmethylsulfonyl fluoride (PMSF), and cocktail of protease inhibitors [Sigma-Aldrich Co. LLC.]). Cells were then lysed with 30 strokes using a syringe fitted with a needle (22 gauges and 30 mm) and centrifuged at 4°C (600 g). The supernatant was centrifuged at 21,000g for 10 min. The supernatant (cytosol) was saved at −20°C and the pellet (mitochondria) was incubated for 20 min on ice in 100 μL of buffer B (50 mmol/L HEPES (pH 7.4), 1% Nonidet P-40 (NP-40), 10% (v/v) glycerol, 1 mmol/L Na-EDTA, 2 mmol/L DTT, 0.1 mmol/L PMSF, and cocktail of protease inhibitors [Sigma-Aldrich Co. LLC.]). Samples were centrifuged at 21,000g for 15 min, the supernatant containing mitochondrial proteins was stored at −20°C. 40 μg protein of each fraction were electrophoresed in 4–10% precasted SDS-PAGE gel (Thermo Scientific) prior to immunoblotting (see previous section).
Cocktail of protease inhibitor
Manufactured by Merck Group
Sourced in United States, Germany, China
The Cocktail of Protease Inhibitors is a laboratory product containing a mixture of chemical compounds that inhibit the activity of proteases, which are enzymes involved in the breakdown of proteins. The core function of this product is to provide a means to study and control proteolytic processes in various experimental settings.
Automatically generated - may contain errors
Lab products found in correlation
Bradford assay, by Bio-Rad (3 mentions)
Ripa buffer, by Cell Signaling Technology (3 mentions)
Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (3 mentions)
Quantity one, by Bio-Rad (3 mentions)
Chemidoc xr, by Bio-Rad (3 mentions)
Nitrocellulose membrane, by Merck Group (3 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
β actin, by Abcam (2 mentions)
α tubulin mouse antibody, by Merck Group (2 mentions)
Ampkα rabbit monoclonal antibody, by Cell Signaling Technology (2 mentions)
Pampkα thr172 rabbit monoclonal antibody, by Cell Signaling Technology (2 mentions)
Secondary antibody against rabbit or mouse igg, by Promega (2 mentions)
β actin, by Merck Group (2 mentions)
Ecl reagent, by Merck Group (2 mentions)
Gapdh, by Merck Group (2 mentions)
Anti β actin, by Abcam (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Enhanced chemi luminescence (ecl), by Cytiva (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Torin1, by Bio-Techne (2 mentions)
84 protocols using cocktail of protease inhibitor
1
Isolation and Fractionation of Mitochondria from Huh7 Cells
2
Western Blot Analysis of SLC7A11 Expression
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HGC27 and SGC7901 cells at logarithmic growth phase were digested and placed in six-well plates at a density of 5 × 105 cells per well, and transfected with miR-489-3p mimics, miR-489-3p inhibitor or NC. The cells were lyzed by RIPA lysis buffer (Sigma, China) added with the cocktail of protease inhibitors (Sigma). An equivalent amount of protein (30 μg) was separated in an SDS-PAGE and shifted onto the polyvinylidene fluoride (PVDF) membranes. The membranes were soaked in a fast-blocking reagent (Thermo) for 15 min, followed by incubation with primary antibodies against SLC7A11 (1: 1,000, Abcam, United States) and β-actin (1: 1,000, Abcam) at 4°C with gentle rocking all night. Next day, wash the membranes three times in TBST and incubate them in appropriate secondary antibodies (1: 1,000, Abcam). The membranes were visualized by a Enhanced chemiluminescence (ECL) reagent, and captured in a gel imaging system (BD Biosciences, United States).
3
Hypoxia-Induced HIF-1α Quantification
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A549 and RAW 264.7 cells were seeded for 24 h in 6-well plates (106 cells/well). After 6 and 24 h under hypoxia (1% O2) or normoxia, cells were washed three times with PBS, harvested using a cell scraper, homogenized in radioimmunoprecipitation assay (RIPA) buffer supplemented with 1 mM phenylmethylsulfonyl fluoride and a 10% cocktail of protease inhibitors (Sigma, Spain), and centrifuged at 13,000 × g and 4°C for 20 min. The supernatant was removed, and the amount of proteins was determined using the bicinchoninic acid (BCA) assay (Promega, Spain). The samples were stored at −80°C. Forty micrograms of proteins from each sample was used to measure HIF-1α levels with an enzyme-linked immunosorbent assay (ELISA) kit (Thermo Fisher Scientific, Spain).
4
AMPK and IKK-β Protein Analysis
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The hypothalamus was homogenized in the lysis buffer (10 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 12% glycerol, 0.5 mM DTT, 0.1 mM EGTA) with a cocktail of protease inhibitors (Sigma Aldrich). Proteins (20 or 40 μg/lane) were separated on 12% SDS-PAGE and transferred to nitrocellulose membranes. The blots were incubated with AMPKα rabbit monoclonal antibody (Cell Signaling Technology; dil 1:1000), pAMPKα (Thr172) rabbit monoclonal antibody (Cell Signaling Technology; dil 1:1000), IKK-β rabbit monoclonal antibody (Abcam; dil 1:500) or α-tubulin mouse antibody (Sigma Aldrich; dil 1:1000) overnight at 4°C and then with secondary antibody against rabbit or mouse IgG (Promega; dil 1:2500) for 1 h at RT. The signals were visualized with the ECL system (Pierce). The expression level of α-tubulin was used to normalize the data.
5
Western Blot Analysis of EMT Markers
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Tissue samples and cells were collected and washed three times with cold PBS and then homogenized in RIPA lysis buffer (1% NP-40, 0.5% deoxycholic acid, 50 mM Tris-HCl [pH 8.0], 0.1% sodium dodecyl sulfate (SDS), 150 mM NaCl) containing a cocktail of protease inhibitors (Sigma-Aldrich, St Louis, MO, USA). Following sonication and centrifugation (13,000× g, 15 min, 4°C), the supernatant was collected. The BCA protein assay kit (Pierce, Waltham, MA, USA) was used to determine the protein concentration according to our protocol. Samples were denatured in loading buffer, separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. Anti-GATA-3 (Abcam, Cambridge, UK; diluted 1:200), anti-ZEB1 (Abcam; diluted 1:200), EMT kit (CST, Danvers, MA, USA; diluted 1:1000) and β-actin (Sigma-Aldrich; diluted 1:5000) were incubated at 4°C overnight followed by incubation with HRP-conjugated secondary antibody (Abcam; diluted 1:5000). Specific blots were visualized using the ECL kit (Thermo Fisher Scientific).
6
Western Blot Protein Analysis Protocol
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Subconfluent cells were washed with PBS, and lysed with RIPA lysis buffer (150 mmol/L NaCl, 50 mmol/L Tris–HCl (pH 7.4), 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) with 1 mmol/L sodium orthovanadate, 1 mmol/L PMSF, and 1% cocktail of protease inhibitors (Sigma, St. Louis, MO). The lysates were clarified by centrifugation at 12000 g for 20 min at 4°C and separated by SDS-PAGE followed by transfer onto nitrocellulose membranes. The following antibodies were used: mouse anti-E-cadherin antibody (BD Biosciences, San Jose, CA), goat anti-biotin antibody (Sigma), mouse anti-GAPDH antibody (KangChen Bio-tech, Shanghai, China), rabbit anti-GFP antibody (Cell Signaling Technology, Beverly, MA), rabbit anti-Arf6 antibody (Abcam, Cambridge, MA). Protein bands were detected by incubating with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) and visualized with ECL reagent (Millipore). Digital images of immunoblots were obtained with a Chemidoc XRS and analyzed using the image analysis program Quantity One (Bio-Rad, Hercules, CA).
7
Western Blot Analysis of Cell Signaling Proteins
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Western blotting was performed as described previously31 (link). Tissue samples were lysed in buffer containing 0.1% Triton X100 and a cocktail of protease inhibitors (Sigma-Aldrich). Protein concentration was measured by Bradford assay (Bio-Rad Laboratories). Protein extracts were then separated on 8–12% SDS-PAGE and transferred onto PVDF membrane (GE Healthcare Life Sciences). Membranes were probed with primary antibodies against p16INK4 (ab54210), SIRT1 (ab110304), acetyl-p53Lys381 (ab61241), TGF-β (ab66043), SMAD3 (ab28379), IL-6 (ab9324), TNF-α (ab6671), MMP2 (ab86607) (Abcam); p53 (sc126, Santa Cruz); Bcl-2 (B9804, Sigma-Aldrich); phospho-SMAD3Ser423/425 (9,520), SMAD2/3 (8685S, Cell Signaling Technology); NF-κB p65 (PA5-16,545), acetyl-SMAD2/3 (PA576015, ThermoFisher). Loading conditions were determined with GAPDH (G8795, Sigma-Aldrich). Images were obtained by ChemiDoc-it 500 Imaging System (Bio-Rad Laboratories) and the optical density of the bands was analyzed with Quantity One Analysis Software (Bio-Rad Laboratories).
8
Protein analysis of muscle tissues
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For the protein extracts, myotubes or TA muscles were homogenized in a radioimmunoprecipitation assay (RIPA) buffer with 1 mM of a cocktail of protease inhibitors (Sigma-Aldrich, St. Louis, MO, USA) and 1 mM of phenylmethylsulfonyl fluoride (Sigma-Aldrich, St. Louis, MO, USA). Proteins were subjected to SDS-PAGE, transferred onto polyvinylidene difluoride membranes (Thermo Fisher Scientific, Waltham, MA, USA), and probed with anti-OXPHOS (1:1000; Abcam, Cambridge, MA, USA), anti-PGC-1α (1:1000, Cell Signaling, Danvers, MA, USA), anti-PINK-1 (1:1000, Cell Signaling, Danvers, MA, USA), anti-LC3B (1:1000, Cell Signaling, Danvers, MA, USA), anti-TOM20 (1:1000, Cell Signaling, Danvers, MA, USA), anti-GAPDH (1:2000; Santa Cruz, Dallas, TX, USA) and anti-β-actin (1:1000; Abcam, Cambridge, MA, USA) antibodies. All immunoreactions were visualized by enhanced chemiluminescence (Thermo Scientific, Waltham, MA, USA). Images were acquired using the Fotodyne FOTO/Analyst Luminary Workstation Systems (Fisher Scientific, St. Waltham, MA, USA). Densitometry analysis was determined by scanning immunoreactive bands. Intensity values were obtained for further normalization against the control group using ImageJ software (National Institutes of Health [NIH], Bethesda, MD, USA).
9
BMP9 Signaling Activation Assay
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Cells were stimulated in 0% FCS with recombinant BMP9 (R&D Systems) at the concentration indicated for 1 h after 1 h30 of serum deprivation. Cell extracts were lysed in 50 mmol/L Tris-HCl pH 7.4, 0.5 mol/L NaCl and a cocktail of protease inhibitors (Sigma) by sonication. 20 μg of proteins from cell lysates were separated on a SDS/PAGE, 4–20% (Bio-Rad) and analyzed by immunoblotting with anti-pSmad1/5/9 antibody (Cell Signaling #9511). The same membrane was reprobed with a monoclonal antibody against β-actin (clone AC-15; Sigma) to confirm equal protein loading.
10
Western Blot Analysis of Signaling Proteins
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Subconfluent cells were washed with PBS, and lysed with RIPA lysis buffer (150 mmol/L NaCl, 50 mmol/L Tris–HCl (pH 7.4), 1 % Triton X-100, 1 % sodium deoxycholate, 0.1 % SDS) with 1 mM sodium orthovanadate, 1 mM PMSF, and 1 % cocktail of protease inhibitors (Sigma). The lysates were clarified by centrifugation at 12,000g for 20 min at 4 °C and separated by SDS-PAGE followed by transfer onto nitrocellulose membranes. The following antibodies were used: rabbit anti-P38 antibody, rabbit anti-P-P38 antibody, rabbit anti-P-Akt antibody, mouse anti-HSP27 antibody and rabbit anti-P-HSP27 antibody (Cell Signaling, Danvers, MA, USA), rabbit anti-Akt antibody (Bioworld, Louis Park, USA), rabbit anti-GAPDH antibody (Santa Cruz, CA, USA). Protein bands were detected by incubating with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz) and visualized with ECL reagent (Millipore, Billerica, MA, USA). Digital images of immunoblots were obtained with a Chemidoc XRS and analyzed using the image analysis program Quantity One (Bio-Rad, Hercules, CA, USA).
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