The middle lobe of the liver was placed in lysis buffer (containing protease and phosphatase inhibitor) and homogenized by using an ultrasonic cell crusher (scientz-II D, Ningbo Scientz Biotech Co., Ltd., Ningbo, China). Then, the targeted proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then blotted onto a nitrocellulose (NC) membrane. Thereafter, the NC membrane was incubated with the corresponding primary and secondary antibodies. After incubation, proteins were examined by using a VILBER Fusion FX7 imaging system (Vilber Lourmat, Marne-la-Vallée, France) with an enhanced chemiluminescent detection reagent (Millipore, Billerica, MA, USA).
Fusion fx7 imaging system
Manufactured by Vilber
Sourced in France, United States, Germany
The Fusion FX7 is a high-performance imaging system designed for laboratory applications. It provides advanced imaging capabilities for a variety of sample types. The system is equipped with a sensitive camera and advanced imaging software to capture and analyze images with precision.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Pvdf membrane, by Bio-Rad (5 mentions)
β actin, by Merck Group (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Cleaved caspase 3, by Cell Signaling Technology (3 mentions)
Ecl plus, by Thermo Fisher Scientific (3 mentions)
Anti p62, by Cell Signaling Technology (2 mentions)
Anti atg5, by Cell Signaling Technology (2 mentions)
Bio1d software, by Vilber (2 mentions)
Anti lc3b, by Cell Signaling Technology (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Cleaved caspase 8, by BD (2 mentions)
Immunocruz western blotting luminol reagent, by Santa Cruz Biotechnology (2 mentions)
Bio 1d densitometer, by Vilber (2 mentions)
Phosphstop, by Roche (2 mentions)
Tris glycin, by Thermo Fisher Scientific (2 mentions)
Complete protease inhibitor, by Roche (2 mentions)
Westernbright quantum, by Advansta (2 mentions)
Hrp conjugated secondary antibody, by Cell Signaling Technology (2 mentions)
42 protocols using fusion fx7 imaging system
1
Protein Extraction and Western Blot Analysis
2
Western Blot Protein Quantification
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Cells were lysed in RIPA buffer (Cell Signaling, 9806S) supplemented with 0,2% SDS, PMSF and protease/phosphatase inhibitor cocktail (Cell Signaling, 9806S). Twenty μg of whole cell lysates were resolved on NuPAGE 4–12% Bis-Tris Protein Gels (Invitrogen) and transferred onto PVDF membranes (neolab Migge GmbH, IPFL00010). Membranes were blocked in 5% BSA and incubated overnight with primary antibodies (Supplementary Table 3). Anti-mouse or anti-Rabbit IgG, HRP-linked Antibody (Cell signaling, 7076; 7074) were used as secondary antibodies and signals were detected using the Vilber FUSION FX7 imaging system (Vilber Lourmat). Vinculin and β-actin served as loading controls. Band intensities were quantified using the ImageJ software.
3
Western Blot Analysis of Apoptosis Signaling
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The cells were lysed in 300 μL cleavage buffer. The protein extract was loaded on 10% polyacrylamide gel containing sodium dodecyl sulfate and transferred to polyvinylidene fluoride membrane after electrophoresis. After the membrane was sealed with 5% skimmed milk for 1 h, the membrane was washed with washing solution 3 times (10 min/time). Then the cells were incubated with specific first antibody for 2 h at room temperature, and the membrane was washed with membrane washing solution for 4 times (each time for 10 min). Proteins were detected by using an enhanced chemiluminescent reagent (Millipore, Billerica, MA, USA) with a VILBER Fusion FX7 imaging system (Vilber Lourmat, Marne-la-Vallée, France). β-Actin was used to ensure the same load of whole-cell protein.
All tests were performed in triplicate. The antibodies used in this study include P-PI3k, PI3k, P-Akt, Akt, Bax, Bcl-2, caspase-9, caspase-3, PARP and β-actin (1 : 1000). All antibodies were purchased from Thermo Fisher Scientific.
All tests were performed in triplicate. The antibodies used in this study include P-PI3k, PI3k, P-Akt, Akt, Bax, Bcl-2, caspase-9, caspase-3, PARP and β-actin (1 : 1000). All antibodies were purchased from Thermo Fisher Scientific.
4
Western Blot Analysis of Protein Targets
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Proteins were separated on polyacrylamide gels (Minigel system, BioRad) and either transferred onto Immobilon-P Membrane (Millipore) using the semi-dry system (Trans-Blot, BioRad) or processed for silver staining (Roti-Black Kit, Roth). For Western blotting, membranes were blocked with Tris-buffered saline (TBS)-0.05% Tween/4% skim milk for 1 h and probed with the following primary antibodies in TBS-0.05% Tween/4% skim milk overnight: mouse monoclonal anti-V5 (R960, Invitrogen, 1:1000), rat monoclonal anti-Notch3 ECD (clone 3G6, [28 (link)] 1:20), goat polyclonal anti-LAP (AF-246-NA, R&D Systems, 1:500), mouse monoclonal anti-LTBP-1 (MAB388, R&D Systems, 1:500) and mouse monoclonal anti-β-Tubulin (clone TUB 2.1, Sigma, 1:1000). Detection was carried out using HRP-coupled secondary antibodies (DAKO), chemiluminescence development (Immobilon Western HRP Substrate, Millipore) and the Fusion FX7 imaging system (Vilber Lourmat).
5
Histone Extraction and VP22 Quantification
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LMH cells transfected with the mutant VP22 constructs were harvested 24 or 48h post-transfection and salt extraction of histones from chromatin was performed as previously described [18 (link),45 (link)]. The proteins included in the extraction fraction were separated in a 10% SDS-PAGE gel and revealed with colloidal Coomassie blue staining. Images of the gels were captured with the Fusion-FX7 imaging system (Vilber Lourmat, Marne-la-Vallée, France) and the level of VP22 proteins found in the histone extract was estimated using the Bio-profil 1D++ software (ChemiSmart 5000) as a ratio of VP22 signal relative to total protein input (for each lane).
6
Immunoblotting Analysis of Autophagy Markers
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Primary neuronal cells and SK-N-SH cells were lysed in lysis buffer [25 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), pH 7.4, 100 mM NaCl, 1 mM EDTA (ethylene diamine tetra acetic acid), 5 mM MgCl2, 0.1 mM DTT (dithiothreitol), and a protease inhibitor mixture]. Whole cell proteins were electrophoretically resolved on a 10%-15% sodium dodecyl sulfate polyacrylamide gel and transferred to a nitrocellulose membrane. Immunoreactivity was detected through sequential incubation with primary antibodies, horseradish peroxidase-conjugated secondary antibodies, and enhanced chemiluminescence reagents i.e. west save gold detection kit (AbFrontier Inc.). The primary antibodies used for immunoblotting were anti-LC3B (#4108, Cell Signaling Technology), anti-P62 (#5114, Cell Signaling Technology), anti-ATG5 (#2630, Cell Signaling Technology), anti-prion protein (ab52604, Abcam, Cambridge, MA, USA) and anti-β-actin (A5441, Sigma Aldrich). Images were examined using a Fusion FX7 imaging system (Vilber Lourmat, Torcy Z.I. Sud, France). Densitometry of the signal bands was analyzed using the Bio-1D software (Vilber Lourmat, Marne La Vallee, France).
7
Western Blot Analysis of Apoptosis Markers
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The cells were harvested, washed in cold PBS, resuspended in lysis buffer (25 mM HEPES [pH 7.4], 100 mM EDTA, 5 mM MgCl2, 0.1 mM DTT, and protease inhibitor mixture), and sonicated to prepare cell lysates. Proteins (35 μg) present in the cell lysates were separated by performing electrophoresis on 10%–15% SDS polyacrylamide gels and were transferred onto nitrocellulose membranes, and analyzed by western blotting as described previously [42 (link)]. Immunoblotting was performed using antibodies against cleaved caspase-3 (Cell Signaling Technology, Danvers, MA, USA), cleaved caspase-8 (BD Pharmingen, USA), Bax and p53 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), DR4, DR5, and ß-actin (Sigma-Aldrich). Images were obtained using Fusion-FX7 imaging system (Vilber Lourmat, Marne-la-Vallée, France).
8
Protein Separation and Detection
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HFF and HEC-LTT were pelleted and subsequently lysed in a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer at 95 °C for 10 min and sonicated for 1 min. SDS-containing 8% polyacrylamide gels were used to separate proteins. The proteins were transferred to PVDF membranes (Bio-Rad Laboratories, Hercules, CA, USA) and detected by chemoluminescence using a FUSION FX7 imaging system (Vilber Lourmat GmbH, Eberhardzell, Germany).
9
Immunoblot Analysis of TRPV1 and ERK Signaling
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Immunoblot analysis was performed as described previously. Skin and trigeminal ganglia were collected and homogenized in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.5% NP-40) containing a protease inhibitor (Nacalai Tesque) and a phosphatase inhibitor cocktail (PhosSTOP; 04906845001, Roche) for 10 minutes on ice. Supernatants were collected, and the protein concentration was quantified with a Pierce BCA Protein Assay Kit (23225, Thermo Fisher Scientific). Protein extracts were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. The membrane was blocked with 4% BSA for 1 hour at room temperature and then incubated overnight at 4 °C with the following primary antibodies: TRPV1 (rabbit, 1:4000), p44/42 MAPK (Erk1/2 [ERK]; rabbit, 1:4000; 9102; Cell Signaling Technology), pERK (rabbit, 1:4000; 9101; Cell Signaling Technology), TATA-binding protein (rabbit, 1:1000; 8515, Cell Signaling Technology), and glyceraldehyde-3phosphate dehydrogenase protein (GAPDH; rabbit, 1:2000; sc25778, Santa Cruz Biotechnology). Specific bands were visualized using an ECL Prime Western Blotting Detection Reagent (RPN2232; GE Healthcare) and analyzed using a FUSION-FX7 imaging system (Vilber).
10
Immunoblotting analysis of bacterial proteins
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Total bacterial extracts were prepared as previously described [66 ]. Bacterial proteins (an equivalent of 108 bacteria/well) were separated by electrophoresis in 15% (wt/vol) SDS-polyacrylamide gel (PAGE) and transferred onto a nitrocellulose membrane. Immunoblotting were performed using either a mouse anti-Hsp60 monoclonal antibody (loading control; 1:6000; Enzo Life Sciences) or a rabbit anti-PefA polyclonal antibody (1:500) [8 ]. Horseradish peroxidase HRP-conjugated rabbit anti-mouse IgG (1:5000; Dako) or HRP-conjugated goat anti-rabbit IgG (1:5000; Dako) were used as secondary antibodies. Detection was performed by chemiluminescence with Fusion FX7 imaging system (Vilber Lourmat) using the SuperSignal™ West Dura Extended Duration Substrate (Thermo Scientific). Exposure times for PefA detection are indicated on all the blots. For Hsp60 detection, exposure times varied between 100 and 500 ms. At least two independent assays were performed for each experiment.
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