Primary antibodies included anti-USP7, anti-Ubiquitin, anti-CHK1, anti-γ-H2AX, anti-AGO2, anti-Flag, anti-HA, anti-Myc (Cell Signaling Technology, Beverly, MA, USA), and anti-β-actin (CWBIO, Guagnzhou, China). Cell pellets were lysed with radioimmunoprecipitation assay (RIPA) buffer (Cell Signaling Technology) containing proteinase and phosphatase inhibitor cocktails (Sigma-Aldrich). The resultant proteins were electrophoresed and transferred to a nitrocellulose membrane (Bio-Rad). Membranes were blocked with 5% bovine serum albumin for 1 h before incubation with primary antibodies overnight at 4 °C, followed by incubation with horseradish peroxidase-conjugated secondary antibody and development with an ECL western blotting substrate (Pierce, Rockford, IL, USA).
Proteinase and phosphatase inhibitor cocktail
Manufactured by Merck Group
Sourced in China, United States
Proteinase and phosphatase inhibitor cocktails are multi-component solutions designed to inhibit a broad range of proteases and phosphatases that may be present in biological samples. They are commonly used to preserve the native state of proteins during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Bio-Rad (5 mentions)
Ripa buffer, by Cell Signaling Technology (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Ecl select kit, by GE Healthcare (2 mentions)
β actin, by Abcam (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Lysis buffer, by Beyotime (2 mentions)
Donkey anti rabbit 711 035 152, by Jackson ImmunoResearch (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Magnetic bead, by Thermo Fisher Scientific (2 mentions)
Magna rip rna binding protein immunoprecipitation kit, by Merck Group (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Bca assay, by Thermo Fisher Scientific (2 mentions)
Odyssey imaging system, by LI COR (2 mentions)
Startingblock, by Thermo Fisher Scientific (2 mentions)
Immobilon fl pvdf membrane, by Merck Group (2 mentions)
Anti ubiquitin, by Cell Signaling Technology (1 mentions)
Anti chk1, by Cell Signaling Technology (1 mentions)
Anti ago2, by Cell Signaling Technology (1 mentions)
Anti ha, by Cell Signaling Technology (1 mentions)
28 protocols using proteinase and phosphatase inhibitor cocktail
1
Western Blotting Analysis of Protein Expression
2
Western Blot Analysis Protocol
3
Protein Expression Analysis in Cells
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The proteins of tissues or cells were extracted by using radioimmunoprecipitation assay (RIPA) buffer containing 1% proteinase and phosphatase inhibitor cocktails (Sigma-Aldrich). Protein lysates were separated by 11% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel electrophoresis, transferred onto polyvinylidene fluoride (PVDF) membrane, and separately immunoblotted with primary antibodies including 3-NT (1:1000), iNOS (1:1000), p47phox (1:1000), p67phox (1:1000), p-p65Ser536 (1:1000), p65 (1:1000), p-IKKα/βSer176/180 (1:1000), IKKβ (1:1000), p-IκBαSer32 (1:1000), IκBα (1:1000) and GAPDH (1:2000) following HRP-conjugated secondary antibodies (1:2000). The signals were detected by chemiluminescent ECL Select Kit (GE Healthcare, IL, USA), captured by Gel-Doc system (Bio-Rad, CA, USA) and analyzed by Image Lab software (Bio-Rad, CA, USA).
4
Analyzing EMT Markers by Immunoblotting
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Whole cell lysates were prepared by lysing the cells in a buffer containing 50 mM Tris–HCl (pH 7.5), 0.15 M NaCl, 0.1% sodium dodecylsulfate, 1% sodium deoxycholate, 1% Triton X-100, and proteinase and phosphatase inhibitor cocktails (Sigma-Aldrich). Lysates were centrifuged for 10 min at 4 °C and an equal amount of protein 25–50 μg from the supernatants was used for SDS-PAGE and immunoblotting.
For Western blot analysis, the primary antibodies were as follows: E-cadherin (BD Biosciences, San Jose, CA, USA); N-cadherin, Snail and Slug (Cell Signaling Technology); integrin α5, β1, and β3 (BD Biosciences); GAPDH (EMD Millipore, Bedford, MA, USA) and SDC4 (Sigma-Aldrich). For Dot blot analysis, the conditioned medium from A549 cells was collected after 24 h of TGF-β1 stimulation, and blotted onto PVDF membranes. Then the membrane was probed with SDC4-specific antibody and visualized with ECL detection reagent.
For Western blot analysis, the primary antibodies were as follows: E-cadherin (BD Biosciences, San Jose, CA, USA); N-cadherin, Snail and Slug (Cell Signaling Technology); integrin α5, β1, and β3 (BD Biosciences); GAPDH (EMD Millipore, Bedford, MA, USA) and SDC4 (Sigma-Aldrich). For Dot blot analysis, the conditioned medium from A549 cells was collected after 24 h of TGF-β1 stimulation, and blotted onto PVDF membranes. Then the membrane was probed with SDC4-specific antibody and visualized with ECL detection reagent.
5
Western Blot Analysis of Mouse RVs
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Mouse RVs were homogenized in RIPA buffer (PBS, 1% Ipegal, 0.5% sodium deoxycholate, 0.1% SDS) with proteinase and phosphatase inhibitor cocktails (Sigma-Aldrich, St. Louis, MO, USA). The protein concentration in the samples was determined by using Bradford assay (Pierce Co., Rockford, IL, USA) and was stored at –70°C until use. Primary antibodies used for Western blot included CD36 (3313, Cell Signaling Technologies [CST], Danvers, MA, USA), β-microglobulin (2101, CST), HSP70 (ab8227, Abcam, Cambridge, MA, USA), and β-actin (ab8227, Abcam).
6
Western Blot Analysis of Cellular Proteins
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Cells were lysed using a lysis buffer (Beyotime, Shanghai, China) with proteinase- and phosphatase-inhibitor cocktails (Sigma-Aldrich). Protein concentrations in the samples were measured by use of a BCA protein assay kit (Beyotime). Samples were then used for a conventional Western blot analysis according to the methods introduced in a previous study [16 (link)]. Primary anti-LC3B (ab48394), anti-p62 (ab91526), anti-NDRG2 (ab57429), anti-HIF1-α (ab16066), and anti-GAPDH (ab125247) and HRP-conjugated secondary antibodies were purchased from Abcam (Cambridge, MA, USA). The protein signals were detected using the BeyoECL Plus kit (Beyotime) according to the manufacturer’s instructions.
7
Protein Extraction and Western Blot Analysis
8
Western Blot Analysis of Autophagy Markers
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Cell samples were lysed using a lysis buffer (Beyotime, Shanghai, China) supplemented with proteinase and phosphatase inhibitor cocktails (Sigma-Aldrich). The protein concentration was determined using a BCA protein assay kit (Beyotime). The proteins were separated on 10% SDS PAGE gel and then transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat milk for 1 h and then incubated with primary antibodies overnight at 4°. The primary antibodies used were rabbit anti-LC3B (L7543, 1:1000, Sigma-Aldrich), anti-SQSTM1/p62 (ab91526, 1:1000, Abcam, Cambridge, MA, USA), anti-cleaved PARP (#9546, 1:2000, Cell Signaling, Danvers, MA, USA), anti-HMGB1 (ab77302, 1:1000; Abcam), and anti-β-actin (ab189073, 1:1000; Abcam). After washing 3 times, the membranes were incubated in HRP-conjugated secondary antibodies for another 1 h at room temperature. Then, the protein signals were detected using the ECL Western blotting substrate (Promega, Madison, WI, USA).
9
RNA-Binding Protein Immunoprecipitation Protocol
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RIP experiments were carried out according to the manufacturer’s protocol of the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Bedford, MA). Briefly, cells were lysed with RIPA buffer (Cell Signaling Technology) containing proteinase and phosphatase inhibitor cocktails (Sigma-Aldrich). Magnetic beads (Invitrogen) were pre-incubated with primary antibodies or anti-rabbit IgG (Cell Signaling Technology) for 30 min, and lysates were immunoprecipitated with beads, rotated overnight at 4 °C. RNA was purified from RNA–protein complexes bounded to the beads and then was analyzed by qRT-PCR.
10
Western Blot Analysis of Liver Proteins
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Protein was extracted from liver tissue or cell cultures as described [7 (link)]. Protein was extracted from liver tissue or cell cultures with ice-cold protein lysis buffer (50 mM Tris, 150 mM NaCl, 0.1% sodium dodecyl sulfate, 1% sodium deoxycholate, and 1% Triton-100). The buffer contains 1% proteinase and phosphatase inhibitor cocktails (Sigma-Aldrich, St. Louis, MO). Proteins (30 µg/sample) in SDS-loading buffer (50 mM Tris, pH 7.6, 10% glycerol, and 1% SDS) were subjected to 4–20% SDS–polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane (Bio-Rad). The membrane was blocked with 5% dry milk and 0.1% Tween 20 (USB, Cleveland, OH). The nuclear and cytosolic fractions were prepared with NE-PER Nuclear and Cytoplasmic Extraction Reagents (ThermoFisher Scientific). The Foxo1, p-JNK, JNK, p-Akt, p-β-catenin, β-catenin, Snail, HMGB1, RIPK3, p-MLKL, NLRP3, cleaved caspase-1, Lamin B2, β-actin (Cell Signaling Technology), Gli1 (Santa Cruz Biotechnology), Sonic Hedgehog (Shh), SMO, NEK7, and TCF4 (Abcam) mAbs were used. The membranes were incubated with Abs, and then added Western ECL substrate mixture (Bio-Rad) for imaging with the iBright FL1000 (ThermoFisher Scientific). Relative quantities of protein were determined by comparing to the β-actin expression, using iBright image analysis software (ThermoFisher Scientific).
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