Cell media were aspirated, and cells were washed with PBS before being lysed with lysis buffer. The amount of lysis buffer used depended on culture size. Cell lysates were then incubated on ice for 5 min and transferred to microfuge tubes. Cell lysates were then rotated for 30 min on a Labinoco LD79 Test-tube Rotator (Wolf Laboratories, York, UK) prior to centrifugation at 12,000× g for 15 min at 4 °C. Supernatant (protein lysate) was then transferred into a fresh microfuge tube and either stored at −20 °C ready for protein sample quantification or equal volumes of LaemmLi 2 X Concentrate (Sigma-Aldrich, Gillingham, Dorset, UK) added prior to boiling at 100 °C for 10 min. The Bio-Rad DC™ Protein Assay Kit (Bio-Rad, Hertfordshire, UK) was used for protein sample quantification. SDS-PAGE was undertaken on an acrylamide gel composed of a 10% (v/v) running gel and 5% (v/v) stacking gel in an OmniPAGE VS10DYS Vertical Electrophoresis System (OmniPAGE, Cleaver Scientific Ltd., Rugby, UK). Samples were then transferred from the acrylamide gel to a PVDF Transfer Membrane (Merck Millipore, Sigma-Aldrich, Gillingham, Dorset, UK) using the Mini Trans-Blot® Cell (Bio-Rad, Hertfordshire, UK) wet transfer system. Standard Western blotting was carried out. EZ-ECL Chemiluminescent Detection Kit (Geneflow, Staffordshire, UK) was used for protein visualization.
Pvdf transfer membrane
Manufactured by Merck Group
Sourced in United States, Germany
PVDF transfer membrane is a type of lab equipment used for the transfer and immobilization of proteins and nucleic acids from gels to a solid support. It is made of polyvinylidene fluoride (PVDF) material and is commonly used in Western blotting and other protein analysis techniques.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Beyotime (6 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (4 mentions)
Ripa lysis buffer, by Beyotime (4 mentions)
Ripa buffer, by Beyotime (3 mentions)
Protein assay dye, by Bio-Rad (3 mentions)
Restore western blot stripping buffer, by Thermo Fisher Scientific (3 mentions)
Rabbit igg hrp linked whole antibody, by Merck Group (3 mentions)
Mouse igg hrp linked whole antibody, by Merck Group (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Histone h3, by Cell Signaling Technology (2 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
Anti pstat3 antibody d3a7, by Cell Signaling Technology (2 mentions)
Horseradish peroxidase conjugated anti rabbit igg antibody, by GE Healthcare (2 mentions)
Ecl western blotting detection reagent, by GE Healthcare (2 mentions)
Anti β actin antibody c4, by Santa Cruz Biotechnology (2 mentions)
Ecl western blotting detection reagent, by Thermo Fisher Scientific (2 mentions)
Ecl detection kit, by GE Healthcare (2 mentions)
Versadoc system, by Bio-Rad (2 mentions)
88 protocols using pvdf transfer membrane
1
Protein Extraction and Western Blotting
2
Kinetics of p-AKT and EGFR in iTreg Cells
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To analyze the shorter kinetics of p-AKT and EGFR, 3 days differentiated iTreg cells were transferred into isotype IgG (5 μg ml−1) or 6F01 (5 μg ml−1) coated wells and were spun down for receiving signals from mAbs. The cells were cultured for the indicated times and were harvested. To examine the level of p-AKT, the cells were fixed/permeabilized using FIX & PERM™ Cell Permeabilization Kit (Thermo Fisher Scientific). The fixed cells were stained with anti-p-AKT-APC and were analyzed by Flow cytometry. To examine the level of EGFR, the cells were lysed with RIPA buffer (Thermo Fisher Scientific). Lysates were quantified with a BCA protein assay (Thermo Fisher Scientific), and equal amounts of the proteins were separated on 7.5% SDS polyacrylamide gels and transferred onto a PVDF transfer membrane (EMD Millipore). The membrane was blocked by 4% Bovine Serum Albumin (Sigma-Aldrich) and incubated with an anti-EGFR antibody (Invitrogen) followed by the manufacturer’s protocol. After washing, anti-mouse IgG-conjugated horseradish peroxidase (Abcam) was bound in a dilution of 1: 10,000. Anti-β-actin antibody (Cell Signaling Technology) was used as a control. The signals were visualized by chemiluminescence.
3
Quantification of SP-D and SNO-SP-D
4
HL60 Cells ERK Signaling Pathway
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HL60s were seeded in T25 flasks at 50 × 104 cells/mL and cells were treated with either vehicle [0.5% EtOH (v/v)], 1 –4 , 9 or 52 (5 µM), as well as 1 (10 µM as reported [9 (link)]). After 16 h, cells were collected and washed with PBS. Cell pellets were re-suspended in cold cell lysis buffer (radio-immunoprecipitation assay buffer) supplemented with 1% phosphatase inhibitor cocktail 2 and 3 (Sigma) and 10% protease inhibitor (Roche). Cells were lysed for 30 min on ice. Protein concentration was then determined by BCA assay. Lysates were boiled with Laemmli sample buffer [Tris-HCL 50 mM (pH 6.7), glycerol 10% (w/v), sodium dodecyl sulphate 2% (w/v), bromophenol blue 0.02% (w/v)] containing DTT 50 µM for 10 min at 90 °C. Moreover, 20 µg of lysates were resolved by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF transfer membrane (EMD Millipore). Membranes were blocked with 5% non-fat milk and probed with primary antibodies for ERK and phospho-ERK (Cell Signalling). Anti-GAPDH was used as a loading control (Calbiochem).
5
Quantitative Immunoblotting for Protein Expression Analysis
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Total protein was collected by lysing adherent cells with RIPA buffer (Sigma, cat. #R0278) supplemented with phosphatase and protease inhibitors (Thermo Scientific, cat. #78440). Protein concentration was measured using the Bradford reagent (Thermo Scientific, cat. #23236). An equal amount of protein was loaded for immunoblotting. After electrophoresis, the protein was transferred to the PVDF transfer membrane (Merck, cat. #0000240973), followed by a 5% BSA blocking buffer, and incubated overnight with primary antibodies. After washing with TBST (TBS with .1% Tween), anti‐rabbit second antibodies (Cell Signaling Technology, cat. #7074) conjugated with HRP‐conjugated were used to probe the membranes. Samples were developed by Chemiluminescent Substrate (Thermo Scientific, cat. #34577) and exposed by Tanon 5200 Multi‐system (Tanon Co.). Densitometry was performed using ImageJ software (National Institutes of Health), and samples were normalized to internal controls. Primary antibodies used in this study are CDK12 antibody (Cell Signaling Technology, cat. #11973), Phospho‐Rpb1 CTD (Ser2/Ser5) Rabbit mAb (Cell Signaling Technology, cat. #13546), β‐Actin Rabbit mAb (Cell Signaling Technology, cat. #8457), GAPDH Rabbit mAb (Cell Signaling Technology, cat. #2118), α‐Tubulin Rabbit mAb (Cell Signaling Technology, cat. #2125) and recombinant Anti‐FACL4 antibody (Abcam, cat. #EPR8640).
6
Western Blot Protein Extraction Protocol
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Cells were lysed for protein extraction by incubating for 5 min in RIPA buffer (10X, #9806, Cell Signaling) on ice after washing with PBS twice. Cells were then scraped and sonicated briefly, centrifuged for 10 min at 14,000x g in +4°C. Protein concentrations were measured with Bradford or a Nanodrop 2000 instrument. 20–40µg of protein was run into a 10% or 12% polyacrylamide gel (Bio Rad) at 100V for 10 min and then 150V for 50 min. Semi-dry blotting (Thermo Fisher) was conducted transferring the proteins to a PVDF transfer membrane (Merck Millipore) under 36mA for 65 min. Alternatively, proteins were transferred to a nitrocellulose membrane (AmershamTM ProtanTM, GE Healthcare Life Sciences) by wet blotting under 45 V for 15 min, then 120 V for 45 min. 5% milk/TBST was used for blocking the membrane for 1 h at RT followed by incubation with primary antibodies overnight in +4 °C. On the following day, the membrane was washed with TBST and incubated with secondary antibody for 1 h at RT. After washing with TBST, ECL Prime Detection Reagent (GE Healthcare) or ClarityTM Western ECL Substrate (BioRad) was used to detect the bands for imaging with a Chemidoc XRS+ Molecular Imager (Bio-Rad).
7
Liver Protein Isolation and Western Blot Analysis
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Proteins were isolated from liver tissues or cells using RIPA lysis buffer (Invitrogen) supplemented with protease inhibitors (Thermo Scientific, Waltham, USA). Proteins were separated using tris–glycine gel (APPLYGEN, Beijing, China) and transferred from the gel to a PVDF transfer membrane (Merck Millipore, Boston, USA). After blocking with 10% skim milk (BD), membranes were probed with the following primary antibodies (details can be found in Additional file 1 : Table S1): PGAM5, α-SMA, COL1A1, COL3A1, GPX6, CAT, HO-1, SOD1, nuclear respiratory factor 1 (NRF1), NRF2, phosphor-mTOR (p-mTOR), mTOR, phosphor-insulin receptor β (p-InsRβ), InsRβ, protein kinase B (PKB/AKT), phospho-AKT (p-AKT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin. Horseradish peroxidase (HRP)-linked anti-rabbit, anti-rat and anti-mouse were used as secondary antibodies. The protein bands were visualized by enhanced chemiluminescence detection reagents (APPLYGEN). Pixels were quantified in image J software (NIH, USA). Protein levels were corrected for β-actin or GAPDH levels.
8
Western Blot Profiling of Histone Modifications
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Cells were lysed in 2% SDS, 100 mM tris-HCl (pH 7.5) and equal amounts of protein (≈30 g) were resolved by SDS-polyacrylamide gel electrophoresis (4-12% Bis-Tris) and blotted onto the PVDF transfer membrane (Merck Millipore). Membranes were stained with Ponceau and washed in PBS-Tween 0.01%. Blots were blocked in 5% non-fat milk in PBS-Tween at RT for 30 min, followed by incubation o/n at 4 °C with the primary antibody (SDMA, Cell Signaling Technology, 13222, 1:1000 dilution; SmD3, Sigma, HPA001170, 1:1000 dilution; α-Tubulin, Cell Signaling Technology, 2125, 1:1000 dilution; Prmt5, Santa Cruz Biotechnology, sc-376937, 1:1000 dilution; Tri-Methyl-Histone H3 K27, Cell Signaling Technology, 9733, 1:1000 dilution, Histone H3, Cell Signaling Technology, 4499, 1:2000 dilution; Ezh2, Cell Signaling Technology, 5246, 1:1000 dilution; Tri-Methyl-Histone H3 K4, Cell Signaling Technology, 9725, 1:1000 dilution; all prepared in blocking buffer) and the with a secondary antibody at 4 °C for 5 h (goat α-rabbit IgG-680; A-21109, Invitrogen, 1:5000 dilution; goat α-mouse IgG-680; A21057, Invitrogen, 1:5000 dilution). Blots were imaged with the Odyssey Infrared Imaging System (LI-COR, NE, USA).
9
Quantifying STAT3 Activation by Western Blot
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STAT3 activation was assessed by measuring the increased expression of pSTAT3 by western blotting, as previously described [37 (link)]. Solubilized SBC-3 cells and macrophages were run on a 10% SDS–polyacrylamide gel and transferred to a polyvinylidene fluoride (PVDF) transfer membrane (Millipore, Bedford, MA, USA). To detect pSTAT3, the membranes were incubated with an anti-pSTAT3 antibody (D3A7; Cell Signaling Technology Japan, Tokyo, Japan) and visualized using a horseradish peroxidase-conjugated anti-rabbit IgG antibody with ECL western blotting detection reagent (GE Healthcare Life Sciences, Piscataway, NJ, USA). To detect STAT3, the membranes were incubated with an anti-STAT3 antibody (124H6; Cell Signaling Technology Japan, Tokyo, Japan) and visualized using a horseradish peroxidase-conjugated anti-mouse IgG antibody with an ECL western blotting detection reagent. The membranes were re-blotted with anti-β-actin antibody (C4) (sc-47778; Santa Cruz Biotechnology, Inc.) as an internal calibration control. Quantification of the western blots was performed using ImageJ and the Amersham Imager 680 analysis software.
10
Co-IP of FAP and uPAR Interaction
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For coimmunoprecipitation (Co-IP), fap gene (NM_007986.2) was cloned into pCMV-Tag2B-flag vector, and Plaur gene (NM_011113.3) into PCDNA3.1-HA vector. Primary antibodies against uPAR (R&D Systems), FAP (Abgent), HA (Santa Cruz Biotechnology) were used. The rabbit normal IgG antibodies (Santa Cruz Biotechnology) were added as a control. Anti-FLAG M2 Affinity Gel (Sigma) was used for immunoprecipitation. For Western blotting, Protein concentration was measured by using BCA protein assay kit (Pierce). 20 μg protein samples were used and transferred onto polyvinylidene fluoride (PVDF) transfer membrane (Millipore). The membrane was blocked with 5% blotting grade milk powder in TBST (50 mM Tris-HCl, 0.15 M NaCl, 0.1% Tween-20, pH: 7.4). The Western blotting was performed as previously described [21] (link). The antibodies were shown in Supporting Table S1.
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