Conventionally cultured Capan-1 cells were lysed in lysis buffer containing (mM) 250 TRIS, 1,25 NaCl, 20 NaF, 50 EDTA, 5% Trizol, and 1 × Sigma fast protease inhibitor (Sigma-Aldrich, Germany). The lysates were centrifuged for 15 min at 15000 g at 4 °C, and the supernatant was stored at − 80 °C until use. Protein concentration was determined in the samples by use of a Bradford assay. Ten to 20 μg of protein was loaded on 10% polyacrylamide precast gels (NuPAGE, Bis–Tris, Invitrogen), separated by electrophoresis, and transferred to a PVDF membrane (Invitrogen). Membranes were blocked in a solution with 3% BSA and 2% skim milk in TRIS-buffered saline containing 0.1% Tween (TBST) (60 to 90 min, room temperature (RT)). The membranes were incubated overnight at 4 °C with primary antibodies against KCNQ1 (1:1000 rabbit polyclonal, APC-022, Alomone Labs), TREK-2 (1:600 rabbit polyclonal, APC-055, Alomone Labs), and TASK-2 (1:2000 rabbit polyclonal, LS-C291082, (LifeSpan BioSciences, Inc.)), followed by rinsing in TBST and treatment with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:2000 to 1:2500 goat anti-rabbit, sc-2004 (Santa Cruz) in TBST with 3% BSA and 2% skim milk). Chemiluminescence of the immunoreactive bands was visualized with EZ-ECL (Biological Industries) on the Fusion FX (Vilber Lourmat) imaging system.
Ez ecl
Manufactured by Sartorius
Sourced in Israel, United States
The EZ-ECL is a chemiluminescent detection system for Western blot analysis. It provides a rapid and sensitive method for detecting and quantifying proteins. The EZ-ECL kit contains all the necessary reagents for the chemiluminescent detection of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Fusion fx, by Vilber (9 mentions)
Pvdf membrane, by Thermo Fisher Scientific (5 mentions)
Protease inhibitor cocktail, by Merck Group (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Gapdh, by Merck Group (3 mentions)
Polyacrylamide precast gels, by Thermo Fisher Scientific (3 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Ripa lysis buffer, by Roche (2 mentions)
Mtorc1, by Cell Signaling Technology (2 mentions)
Ab8224, by Abcam (2 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Gapdh, by Proteintech (2 mentions)
Protease inhibitor, by Keygen Biotech (2 mentions)
Ponceau s solution, by Merck Group (2 mentions)
Anti mouse horseradish peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (2 mentions)
Anti securin, by Abcam (2 mentions)
78 protocols using ez ecl
1
Western Blot Analysis of Ion Channels
2
Quantitative Protein Analysis Workflow
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Cells were lysed using RIPA buffer, and proteins were collected and preserved at −80°C. The BCA assay was used to determine protein concentration. About 50 μg of the proteins were loaded on 10% SDS-PAGE for separation and transferred onto PVDF membranes for 1.5 h. The membranes were incubated with a primary antibody (1:1000) at 4°C overnight. Subsequently, the membranes were incubated with horseradish peroxidase (HRP) -conjugated secondary antibody (1:30,000) at 37°C for 1.5 h. EZ-ECL (Biological Industries, Israel) was used to detect the proteins, according to the manufacturer’s instructions. Anti-P53 (ab26), and anti-TERT (ab181830) antibodies were obtained from Abcam (Cambridge, UK), anti-α-TUBULIN (CST#138000) was obtained from Invitrogen (California, USA), and anti-HRP antibodies (7074S, 7076S) was purchased from Cell Signaling Technology (Cambridge, USA).
3
Quantification of Thioredoxin Proteins
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Quantifications of Trx and TrxR proteins were conducted by Western blot analyses as previously described, with minor modifications [32 (link), 43 (link)]. Briefly, equal amounts of protein (5 μg) were separated by SDS-PAGE and transferred to a nitrocellulose membrane at 250 mA for 90 min. Membranes were blocked at 4°C overnight with 5% dry skim milk in 0.05 M Tris-buffered saline pH 7.6, containing 0.05% Tween-20 (TBS-T). Subsequently, the membranes were incubated with Trx or TrxR primary antibodies, which were generously provided by Dr. S.G. Rhee (Ewha Women University, Seoul, Korea). After washing with TBS-T, the membranes were incubated for 1h at room temperature with HRP-labeled goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories Inc. West Grove, PA, USA). Next, the membranes were washed with TBS-T and developed using light sensitive film (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK) and the chemiluminescence detection kit for HRP (EZ-ECL; Biological Industries, Beit-Haemek, Israel) according to manufacturer's instructions. Band intensities were quantified using IMAGE J 1.32 software (NIH, USA).
4
Immunoblotting for IRF3 Activation
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Immunoblotting was performed according to Jiménez et al., with modifications (17 (link)). BM-DCs (4 × 106) were incubated for 2 h at 37°C in polystyrene tubes and were then exposed to rSIP or FLH. The cells were lysed at the indicated time points using RIPA lysis buffer supplemented with protease inhibitor cocktail (5 mg/mL; Roche). Proteins were separated in polyacrylamide gels (SDS−PAGE, 10-15%), electrotransferred to nitrocellulose membranes, and blocked with 5% bovine serum albumin (BSA) in 0.1% (v/v) TBS-Tween 20 (TBST). Primary antibodies were dissolved in 5% BSA and incubated with the blocked membranes overnight at 4°C. After exposure of the membranes to horseradish peroxidase-conjugated anti-rabbit secondary antibodies for 1 h in BSA, bands were visualized using the Odyssey system (Li-Cor Bioscience) detection system, and band intensities were analyzed with LI-COR Image Studio Software. SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher) and EZ-ECL (Biological Industries) were the chemiluminescent substrates used for developing the Western blots. The antibodies used for immunoblotting were a recombinant anti-IRF3 antibody [EPR2418Y] (ab68481), goat anti-rabbit IgG H&L (HRP) (Abcam, ab205718) and phospho-IRF-3 (Ser396) (4D4G) rabbit mAb #4947 (Cell Signaling Technology, Danvers, USA).
5
Protein Expression Analysis of Distal Colon
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Distal colonic samples (2–3.5 cm proximal to the anus) were ground and lysed using a total protein extract kit (Keygen). Each sample (30 μg protein) was separated by SDS-PAGE and transferred to a nitrocellulose membrane (Wang et al., 2016 (link)). The membrane was first incubated with primary antibodies against Caspase 3, PCNA, mTORC1, and GCN2 (Cell Signaling) overnight at 4°C and then incubated with the secondary antibody for 2 h at 25°C. The results were visualized via chemiluminescence with EZ-ECL (Biological Industries).
6
Western Blot Analysis of O-GlcNAc Modifications
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The presence of O-GlcNAc on proteins was detected by Western blotting using a pan-GlcNAc mouse monoclonal antibody (CTD 110.6, Covance). For characterization of cytoplasmic and membrane fractions, we used a mouse monoclonal anti–β-actin antibody (ab8224, Abcam), a rabbit polyclonal anti-calnexin antibody (sc-11397, Santa Cruz Biotechnology), and a mouse monoclonal anti-Na(+)/K(+) adenosine triphosphatase α-1 subunit antibody (a6F, Developmental Studies Hybridoma Bank, Iowa City, IA, USA). Briefly, the membrane was blocked for 1 hour using 3% BSA or 5% milk diluted in tris-buffered saline with Tween 20 (TBST) (0.1% Tween) while shaking. Antibodies, diluted 1:1000 in 3% BSA or 5% milk in TBST, were added to the membrane overnight, followed by several washes with TBST. Next, the membrane was incubated with appropriate horseradish peroxidase (HRP)–conjugated secondary antibody for 1 hour at room temperature while shaking. Blots were developed after thorough TBST washes using an enhanced chemiluminescence system (EZ-ECL, Biological Industries) according to the manufacturer’s manual.
7
Western and Far-Western Blotting Protocols
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Following electrophoresis, the proteins were transferred to a nitrocellulose membrane (110 V, 1 h, 4°C) using the Mini Trans-Blot cell apparatus (Bio-Rad). Nonspecific binding sites were blocked using PBS-Tween 20 (0.05% Tween 20, J.T. Baker) containing 5% non-fat dry milk for 1 h at room temperature. The membrane was then overlaid with mouse anti-His antibody (Bio-Rad), diluted 1:1000 in the blocking solution and incubated overnight at 4°C. Next day, the membrane was washed three times with PBS-Tween 20 and overlaid with peroxidase-conjugated goat anti-mouse antibodies (Jackson ImmunoResearch Laboratories) diluted 1:5000 in blocking solution, for 1 h at room temperature. The membrane was then washed three times with PBS-Tween 20. Before imaging (using the ChemiDoc MP imaging system, Bio-Rad), the membrane was incubated in EZ-ECL (Biological Industries) solution for 5 min.
For far western blotting, the experiment was performed similar to the western blot experiment, but CEACAM1-Ig (2 µg/ml) (Gur et al., 2019b (link)) was used instead of the first antibody and the HRP-conjugated α - human IgG (Jackson ImmunoResearch Laboratories, diluted 1:5,000) was used as a secondary antibody.
For far western blotting, the experiment was performed similar to the western blot experiment, but CEACAM1-Ig (2 µg/ml) (Gur et al., 2019b (link)) was used instead of the first antibody and the HRP-conjugated α - human IgG (Jackson ImmunoResearch Laboratories, diluted 1:5,000) was used as a secondary antibody.
8
Western Blot Analysis of Autophagy Markers
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Cells were lysed in luciferase lysis buffer, as described above. The buffer was supplemented with 10 μl/ml 0.1 M PMSF (Sigma-Aldrich, 93,482) and 1% protease (Sigma-Aldrich, P8340) and phosphatase inhibitor cocktails (Sigma-Aldrich, P0044). Proteins were separated by SDS-PAGE and transferred to PVDF membranes, which were incubated with antibodies to LC3B (Sigma, L7543; 1:1000), ATG12 (Cell Signaling Technology, 2010S; 1:1000), VCL (Sigma-Aldrich, V9131; 1:10,000), GAPDH (Merck, MAB374; 1:1000), SQSTM1/p62 (BD Transduction, 610,832; 1:1000), and CASP3 (Cell Signaling Technology, C-9664S; 1:500). Detection was done with either horseradish peroxidase (HRP)-conjugated goat anti-mouse (Jackson ImmunoResearch, 115–035-003) or anti-rabbit (Jackson ImmunoResearch, 111–165-144) secondary antibodies, followed by enhanced chemiluminescence using EZ-ECL (Biological Industries, 20–500). Protein levels were quantitated using Image Studio Lite software (LI-COR Biosciences) with normalization to loading control protein, as indicated in figures, within the linear range of detection.
9
Western Blot Analysis of FGF2, GPR30, and MAPK Signaling
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Samples were homogenized in RIPA buffer (Beyotime Biotechnology) with protease and phosphatase Inhibitor (Thermo Fisher). Western blot analyses were carried out in accordance with standard protocols, and membranes were incubated with anti-FGF2 (1:200, Proteintech), anti-GPR30 (1:100, Abcam), phospho-Mek (1:1000, Cell Signaling Technology), Mek (1:1000, Cell Signaling Technology), phospho-p44/42 MAPK (p-Erk1/2, 1:1000, Cell Signaling Technology), p44/42 MAPK (Erk1/2, 1:1000, Cell Signaling Technology), p-FGFR1 (1:500, Abcam), and GAPDH (1:5000, Proteintech). The membranes were incubated with HRP-conjugated antibodies goat anti-rabbit (1:10000, Abcam) or goat anti-mouse (1:10000, Abcam). They were imaged on the Image Quant LAS 4000 mini biomolecular imager (GE Healthcare Life Sciences) after being applied with EZ-ECL (Biological Industries). The relative protein levels were measured by Image J software (National Institutes of Health) and normalized over the expression of GAPDH.
10
Western Blot Analysis of Macrophage Proteins
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Protein extracts from equal volumes of peritoneal fluids or equal total protein content of isolated macrophages were subjected to SDS-PAGE using 10% polyacrylamide gels, transferred (1 h, 15 V) to PVDF membranes (Bio-Rad), and blocked for 1 h with 5% BSA in TBST (0.1% Tween 20 in Tris-buffered saline). Then, membranes were immuno-blotted overnight at 4°C with either goat anti-mouse CD11b (M-19, 1:200, SantaCruz Biotechnology), goat anti-mouse arginase-1 (ab60176, 1:20,000, Abcam), rabbit anti-mouse 12/15-LO (160704, 1:1,000, Cayman Chemical), rabbit anti-mouse PROS1 (AB15928, 1:1,000, Merck Millipore), and goat anti-mouse actin (I-19, 1:500, SantaCruz Biotechnology). Then, the membranes were washed three times with TBST and incubated with the appropriate HRP-conjugated secondary antibodies (1:10,000, 1 h, room temperature, Jackson ImmunoResearch). Blots were washed and developed using the EZ-ECL (Biological Industries) chemiluminescence kit and analyzed using the LAS-4000 luminescent image analyzer (Fujifilm) and the TotalLab TL-100 software (Nonlinear Dynamics). Band densitometric intensities among different samples were normalized to actin.
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