Peripheral blood mononuclear cells were homogenized in ice PBS containing 0.05% Triton X‐100 and protease inhibitor cocktail (Sigma). Protein samples were run on 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (Sigma) and transferred onto PVDF membranes according to standard protocol, and then blocked with 5% dried skimmed milk in TBST for at least 1h. The membranes were then incubated overnight at 4°C, either with a mouse monoclonal anti‐CD64 antibody and anti‐CD32 antibody (1:800; Abcam), a polyclonal rabbit anti‐CD16 antibody (1:800; Abcam), or a polyclonal rabbit anti‐GAPDH (1:1000; Abcam), and then incubated with horseradish peroxidase‐conjugated secondary antibodies (1:1000; Bio‐Rad) at room temperature for 1 hour after washing three times using TBST. Blots were washed again and developed using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech). Immunoblot bands quantification was calculated by means of a Bio‐Rad calibrated densitometer (GS‐800) using the vendor's software (Bio‐Rad Laboratories); GAPDH was used as an internal reference for analyses.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
Manufactured by Merck Group
Sourced in United States
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a laboratory technique used for the separation and analysis of proteins. It involves the use of a polyacrylamide gel matrix and an electric current to separate proteins based on their molecular weight.
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Lab products found in correlation
Ripa lysis buffer, by Santa Cruz Biotechnology (2 mentions)
Polyvinylidene fluoride membrane, by Roche (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Protein assay, by Bio-Rad (2 mentions)
Skim milk, by BD (2 mentions)
Nitrocellulose membrane, by Merck Group (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Enhanced chemiluminescence kit, by Cytiva (1 mentions)
Rabbit anti gapdh, by Abcam (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Bio-Rad (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Quantity one software version 4, by Bio-Rad (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Imagequant las 4000 mini, by GE Healthcare (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
20 protocols using sodium dodecyl sulfate polyacrylamide gel electrophoresis
1
Western Blot Analysis of Immune Cells
2
Liver Protein Expression Analysis
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The liver tissues were treated with TRIzol protein extraction reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), according to the manufacturer's instructions, and the protein concentrations were determined using Lowry's method (23 ). The protein samples were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Sigma-Aldrich) and electroblotted onto polyvinylidenedifluoride membranes (Millipore Corporation, Billerica, MA, USA). Following blocking with 1.5% bovine serum albumin (Sigma-Aldrich), the membranes were incubated overnight at 4°C with primary antibodies against peroxisome proliferator-activated receptor (PPAR)-γ (mouse anti-PPAR-γ; cat. no. SC-7273), α-SMA (mouse anti-α-SMA; cat. no.SC-32251), TGF-β1 (rabbit anti-TGF-β1; cat. no.SC-146), TIMP-1 (rabbit anti-TIMP-1; cat. no.SC-5538) and MMP-2 (rabbit anti-MMP-2; cat. no. SC-10736) obtained from Santa Cruz Biotechnology, Inc. The membranes were further incubated with secondary antibody for 1 h at room temperature. The protein bands were detected using an enhanced chemiluminescence detection system (Pierce Biotechnology, Inc., Rockford, IL, USA) and the protein expression levels were determined using Quantity One software version 4.5 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
3
Western Blot Analysis of Protein Expression
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Cell or tissue lysates were harvested in lysis buffer containing 150 mM NaCl, 50 mM Tris–HCl, and 0.01% NP40 with 1 mM PMSF (Sigma-Aldrich). Total protein (20 μg) was segregated by 8, 10, and 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Sigma-Aldrich) under reducing conditions and transferred to a polyvinylidene difluoride membrane. The membrane (Millipore) was blocked using 5% nonfat dry milk for 1 h at room temperature. The membrane was hybridized with the primary antibody overnight at 4 °C and washed by PBST before being incubated with horseradish peroxidase-conjugated secondary antibody (Invitrogen) for 2 h at room temperature. An enhanced chemiluminescence reagent (Millipore) was used to detect the signal captured by ImageQuant LAS 4000 mini (GE Healthcare Life Sciences, Chicago, Illinois, USA). The antibodies are listed in Additional file 1 : Table S3.
4
Western Blot Analysis of Protein Lysates
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Protein lysates were obtained by scraping and subjecting cells to 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Sigma), and then transferring onto nitrocellulose membranes (Thermo). Membranes were blocked in a TBST solution consisting of 20 mM Tris-HCl (pH 7.5), 137 mM NaCl, and 0.05% TWEEN® 20, containing 5% non-fat milk powder for 1 hour at 25°C, then incubated with antibodies against RIP140 (1:500; Rabbit mAb; Santa Cruz Biotechnology) or phosphorylated ERK1/2 (p-ERK1/2, 1:500; Mouse mAb; Cell Signaling Technology, Danvers, MA, USA) or β-actin (1:1,500; Mouse mAb; Cell Signaling Technology) overnight at 4°C. After TBST washes, membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG antibodies (1:2,000; Cell Signaling Technology) for 1 hour at 25°C. Protein expression was subsequently detected and visualized using an Enhanced Chemilumenescence substrate (Thermo) (Feng et al., 2015). Results were quantified by ImageJ 2× software (National Institutes of Health, Stapleton, NY, USA). All experiments were conducted in triplicate.
5
Protein Expression Analysis by Western Blot
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The sample supernatant was collected using a RIPA lysis solution (Thermo Fisher Scientific), and the protein was electroblotted to a polyvinylidene fluoride (Shanghai Yongke Biotechnology Co.) membrane (0.45 μm) by sodium dodecyl sulfate polyacrylamide gel electrophoresis (Sigma Aldrich). After cutting the membrane, the bands were sealed in 5 % skim milk for 2 h, and then POSTN (Abcam, ab231130), YAP (Abcam, ab205270), IL-6 (Abcam, ab233706), and GAPDH (Abcam, ab8245) primary antibodies (1:3000) were added for a night-long incubation at 4 °C. The next day, the bands were immersed in a HRP secondary antibody (1:5,000, Abcam, ab288151) to incubate at 37 °C for 1.5 h, followed by development with a chemiluminescent imager. Finally, we determined the gray values of each group of protein bands with an Image J image analysis software.
6
Protein Expression Analysis in Lung Tissue
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A mixture of RIPA buffer (Beyotime Institute of Biotechnology) with 1% phenylmethylsulfonyl fluoride was added into right-lung tissue or 16HBE cells to extract the total protein. The concentration of total protein was measured using a BCA kit. Samples of total protein from each group were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Sigma-Aldrich) and transferred onto polyvinylidene difluoride (PVDF) membranes. The latter were sealed with 5% skimmed milk for 2 h at room temperature and then, respectively, incubated with primary antibodies overnight at 4°C: anti-Nrf2 (1:1,000 dilution), anti-HO-1 (1:2,000), anti-MRP1 (1:50), anti-HDAC2 (1:2,000), or anti-β-actin (1:2,000). The next day, PVDF membranes were rinsed with phosphate-buffered saline and incubated with peroxidase-conjugated secondary antibodies (1:5,000) for 2 h at room temperature on a shaker. Finally, the PVDF membranes were coated with electrochemiluminescence luminescence solution (Solarbio) evenly in a dark room and exposed in a gel imager. β-actin was used as the quantitative internal reference. Gray values for proteins were analyzed with Image J (National Institutes of Health, Bethesda, MD, USA).
7
Protein Expression Analysis Using SDS-PAGE
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We collected whole cell lysates using RIPA lysis buffer (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and calculated the protein concentrations using a BCA Protein Assay Kit (Beyotime, Jiangsu, China). Then, 4–20% sodium dodecyl sulfate polyacrylamide gel electrophoresis (Sigma-Aldrich St. Louis, MO, USA) protein loading buffer was added to the collected 40µg protein samples and heated in a boiling water bath for 3–5 minutes to fully denature the protein. After the protein sample was cooled to room temperature, it was directly loaded into the sample well of SDS-PAGE gel for electrophoresis, which was followed by protein electrophoretic transfer to polyvinylidene fluoride membranes (Roche, Indianapolis, IN, USA). After this, we blocked the membranes in 5% milk, and then incubated them with primary antibodies (Abcam, Cambridge, MA, USA) to evaluate SLC7A5, p-AKT (Abcam), p-p70-S6K, (Abcam), and p-mTOR (Abcam) expression. Subsequent incubations using the secondary antibody were performed, and GAPDH was used as an internal reference.
8
Protein Extraction and Western Blot
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For protein extraction, 1 mL of NP-40 lysis buffer (Biyuntian) was transferred to each well of a six-well plate of microglia on ice for 30 min, and the cells were then centrifuged at 12,000 rpm for 10 min. The soluble protein solutions were then mixed with 5x sample buffer (Biyuntian) and boiled at 90oC for 10 min. Equal amounts of protein (60 μg) from each sample were separated via 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (Sigma) and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked in 5% milk with Tris-buffered saline for 1 h, and then were incubated with the following primary antibodies at 4°C overnight: rabbit anti-iNOS, anti-Arg1 (1:500, Bioss) and rabbit anti-actin (1:1000, Santa Cruz Biotechnology). The membranes were washed three times in Tris-buffered saline with Tween and then incubated with the secondary antibody (peroxidase-conjugated IgG, 1:5000, Santa) at room temperature for 2 h. Binding antibodies were visualized using enhanced chemiluminescence on an imaging system (Amersham Imager 600).
9
Western Blot Analysis of Cell Protein Signaling
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Cell proteins were collected using a mixture of RIPA lysis buffer (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and phenylmethanesulfonyl fluoride (Roche Ltd, Dublin, Ireland). About 30 μg protein was subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (Sigma-Aldrich, St Louis, MO, USA), and the bands were transferred to polyvinylidene fluoride membranes (Hoffman-La Roche Ltd., Basel, Switzerland). The membranes were then blocked in 5% nonfat milk and incubated with primary antibodies against PTEN, AKT, p-AKT, CD44, CD133, EpCAM (1:1,000; Abcam, Cambridge, UK), and subsequently with matched secondary antibodies (Cell Signaling Technology, Danvers, MA, USA). Signals for the expression of each protein were detected with the Bio-Rad Gel imaging system (Bio-Rad, Hercules, CA, USA). PI3K inhibitor LY294002 was purchased from LC Laboratories. GAPDH (G8140; US Biological, Swampscott, MA, USA) was used as a loading control.
10
Western Blot Analysis of β-Catenin
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Cell extracts were harvested from the SP and non-SP cells using RIPA buffer (Sigma-Aldrich) containing protease inhibitor cocktail (Roche Diagnostics Deutschland GmbH, Mannheim, Germany) and protein concentration was determined using a Bradford assay (Sigma-Aldrich) (18 (link)). Protein lysates (40 μg) from each sample were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Sigma-Aldrich). Separated proteins were transferred to a polyvinylidene difluoride membrane (Sigma-Aldrich). The membranes were treated with the primary antibodies against β-catenin (1:2,400; cat. no. ab6302; Abcam, Shanghai, China) and GAPDH (0.7 μg/ml; cat. no. ab37168, Abcam). Subsequently, the membranes were incubated in the secondary antibodies horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit IgG with alkaline phosphatase markers; cat. no. ab97048, Abcam). The protein was detected using chemiluminescence reagents (Amersham Biosciences). Blots were scanned using a Bio-Rad GS-710 densitometer (Bio-Rad Laboratories, Inc.).
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