We performed western blot analysis as previously described.19 (link) The anti-Capn4 antibody was purchased from LifeSpan Biosciences (Seattle, WA, USA). The anti-β-catenin antibody was obtained from BD Bioscience (San Jose, CA, USA). Anti-cyclin D1, anti-c-Myc, and anti-β-actin antibodies were products of Santa Cruz Biotechnology (Santa Cruz, CA, USA). Protein bands were detected with enhanced chemiluminescence reagent (Thermo Scientific, Logan, UT, USA) and visualized by a chemiluminescence detector (Bio-Rad Laboratories, Hercules, CA, USA). The densitometric analysis of blots was performed by using Bio-Rad Image Lab 4.0 software (Bio-Rad Laboratories).
Chemiluminescence detector
Manufactured by Bio-Rad
Sourced in United States, France
The Chemiluminescence detector is a laboratory instrument used to measure and quantify the light emitted from a chemiluminescent reaction. It is designed to detect and analyze the light signals generated by various biological and chemical assays that utilize chemiluminescence as the detection method.
Automatically generated - may contain errors
Lab products found in correlation
Ecl lumina forte, by Merck Group (3 mentions)
Trans blot turbo system, by Bio-Rad (3 mentions)
Phosphatase inhibitor cocktail, by Merck Group (2 mentions)
Up95424a, by Interchim (2 mentions)
Up95425a, by Interchim (2 mentions)
Protease inhibitor, by Merck Group (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Enhanced chemiluminescence reagent, by Thermo Fisher Scientific (1 mentions)
Anti capn4 antibody, by LifeSpan BioSciences (1 mentions)
Image lab 4, by Bio-Rad (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Anti c myc, by Santa Cruz Biotechnology (1 mentions)
Anti cyclin d1, by Santa Cruz Biotechnology (1 mentions)
Anti β catenin antibody, by BD (1 mentions)
Antiferritin h, by Cell Signaling Technology (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Hrp conjugated goat anti rabbit secondary antibody, by Abcam (1 mentions)
Pierce bca protein assay reagent kit, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
10 protocols using chemiluminescence detector
1
Quantitative Western Blot Analysis
2
Western Blot of FUS Protein
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Cells were washed in 1x PBS and lysed in RIPA buffer (Tris 50mM, NaCl 150mM, EDTA 1mM, SDS 0.1%, Deoxycholate 0.5%, Triton X-100) containing protease inhibitor (Sigma P8340) and phosphatase inhibitor cocktail (Sigma 8345). After centrifugation at 10,000 rpm, 4°C during 10 min, supernatant was saved and used to dose protein extract by using a BCA Assay (Interchim, UP95424A, UP95425A). Proteins were denatured and SDS PAGE was performed with 25 µg of protein on criterion TGX stain free gel 4-20% (Biorad, 5678094). Proteins were blotted on nitrocellulose membrane using semi-dry Transblot Turbo system (BioRad, France) and blocked with 10% non-fat milk during 1 h. Anti-FUS antibody (Proteintech, 11570-1-AP, 1:1000) was incubated overnight at 4°C in 3% non-fat milk. Washing proceeded with washing buffer (1 M Tris pH 7.4, NaCl 5M, Tween 20 100%) and goat anti-rabbit HRP (Abliance, BI2407, 1/5000) and was incubated 1.5 h at room temperature. After successive washes, proteins were visualized with chemiluminescence using ECL Lumina Forte (Millipore, France) and chemiluminescence detector (Bio-Rad, France). Total proteins were detected with stain free gel capacity (Biorad, 5678094) and used to normalized.
3
Quantifying Osteoblast Membrane Protein Levels
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Protein extracts were prepared from primary osteoblasts as previously described.15 Membranes were blotted with antitransferrin receptor 1 (Tfr1; 1:500; Zymed Laboratories, South San Francisco, CA, USA) and antiferritin‐H (1:1000 in 2% BSA; Cell Signaling Technology, Danvers, MA, USA) following the manufacturer's instructions. Membranes were washed and incubated with antirabbit or antimouse horseradish peroxidase‐conjugated antibody (1:5000; Invitrogen, Carlsbad, CA, USA). Reactions were carried out with Luminata Forte Western HRP Substrate Kit (MilliporeSigma, Burlington, MA, USA). As a loading control, anti‐β‐actin (1:10000; Sigma‐Aldrich, St. Louis, MO, USA) was used. Membranes were washed prior to the addition of substrate and visualized in a chemiluminescence detector (Bio‐Rad Laboratories, Hercules, CA, USA).
4
Western Blot Analysis of Cardiac Proteins
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The mice cardiac tissues (60-80 mg) or rat cardiac myoblast H9c2 cells were lysed in RIPA buffer (Thermo Fisher Scientific, Inc.). The supernatant was quantified with Pierce BCA protein assay reagent kit (Thermo Fisher Scientific, Inc.). Total protein (20 µg per sample) was loaded and separated in 10% or 12% SDS-PAGE, and transferred to a PVDF membrane (MilliporeSigma). After blocking in TBST (0.1% Tween-20) containing 5% fat-free milk at room temperature for 1 h, the membrane was incubated with primary anti-RUNX1 (1:1,000; cat. no. ab229482; Abcam), anti-DRD2 (1:500; cat. no. bs-20730R; BIOSS), anti-phosphorylated (p-)P65 subunit of NF-κB (1:1,000; cat. no. ab76302; Abcam), anti-NF-κB P65 subunit (1:1,000; cat. no. bs-20355R; BIOSS), anti-TNF-α (1:500; cat. no. bs-2081R; BIOSS), anti-IL-6 (1:500; cat. no. ab259341; Abcam) or anti-β-tubulin (1:1,000; cat. no. ab18207; Abcam) antibodies respectively, overnight at 4˚C. After washing three times in TBST, the membranes were incubated with HRP-conjugated goat anti-rabbit secondary antibody (1:10,000; cat. no. ab191866; Abcam) for 1.5 h at room temperature. Protein band signals were detected using ECL program (Thermo Fisher Scientific, Inc.) under chemiluminescence detector (Bio-Rad Laboratories). The expression of target protein was normalized to β-tubulin using Image Lab Software (Bio-Rad Laboratories).
5
Western Blot Analysis of Protein Lysates
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Cells and xenograft tumor tissues were lysed on ice in RIPA lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1% TritonX-100, 5 mM ethylenediaminetetraacetic acid). Cell lysates were then centrifuged at 12 000 × g for 15 min at 4 °C. Supernatant was collected and protein concentration was measured using a BCA Kit (Pierce, IL, USA). Equal amounts of protein (50 μg) were separated by SDS-PAGE and transferred onto PVDF membranes. Membranes were incubated with the corresponding primary antibody at 4 °C overnight. Then, the membranes were washed and incubated with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit secondary antibodies (1:5000–1:10000, BioRad, Hercules, CA, USA) for 2 h at room temperature. Specific protein bands were measured with an enhanced chemiluminescence reagent (Millipore Corporation) and visualized by a chemiluminescence detector (Bio-Rad Laboratories, Inc., Berkeley, CA, USA) that can also assess the relative intensity of the bands.
6
Western Blot of FUS Protein
7
Muscle Proteome Analysis by Western Blot
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Snap-frozen muscle tissue was pulverized in a TissueLyser (Qiagen) for 2 × 20 s under liquid nitrogen using stainless steel beads. Tissue powder was homogenized in RIPA lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate) at 1 ml/100 mg tissue containing 1:100 protease inhibitor cocktail (Calbiochem, USA) and phosphatase inhibitor cocktails 2 and 3 (Sigma-Aldrich, Germany). Determination of protein concentration was carried out using a BCA Assay Reagent Kit (UP95424 Uptima, France). Protein was denaturated by boiling, resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to 2-μm nitrocellulose membranes (Bio-Rad, France) using a semi-dry Transblot Turbo system (Bio-Rad, France). After using a chemiluminescent blocker (Millipore, France), membranes were probed with primary antibodies against Glycogen Synthase Rabbit mAb (Cell Signaling; Cat#3886; 1:500), Phospho-Glycogen Synthase Ser641 (Cell Signaling; Cat#3891; 1:500), PGC-1α (Millipore; Cat#AB3242, 1:1,000), and total actin (Sigma-Aldrich; Cat#A2103, 1:10,000). Primary antibodies were detected with anti-rabbit HRP (P.A.R.I.S; Cat#BI2407, 1:5,000). The protein bands were detected by chemiluminescence using ECL Lumina Forte (Millipore) and a chemiluminescence detector (Bio-Rad, France).
8
LPS-TLR4 Signaling Pathway Analysis
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An immunoblotting assay was used for analyzing the expression level of several associated mediators of LPS-TLR4 signaling; these included TLR4, TNF-α, IL-1β, COX-2, VEGFC, pp65, and p65. In short, the cell lysate, from various treated conditions on LPS-pretreated SW480 cells (at 37 °C for 24 h), was equally loaded onto 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Thereafter, gels were blotted onto polyvinylidene difluoride (PVDF) membranes, which were continuously blocked by 5% skimmed milk powder in PBS-T (0.05% Tween-20 in PBS). Afterward, membranes were incubated with diluted specific primary antibodies against TLR4, TNF-α, IL-1β, COX-2, pp65, p65, VEGFC, and β-actin (Santa Cruz, Rio Grande, TX, USA and Merck Millipore, Burlington, MA, USA) at 4 °C overnight. For detection, membranes were soaked with diluted HRP-conjugated secondary antibody (Merck Millipore, Burlington, MA, USA) at room temperature and further reacted with the enhanced chemiluminescence (ECL) substrate (Bio-Rad® Laboratories, Hercules, CA, USA). The bands were visualized and captured using a chemiluminescence detector (Bio-Rad® Laboratories, Hercules, CA, USA). Finally, the intensity of each band was analyzed and normalized with its β-actin band.
9
Protein Extraction and Western Blot Analysis
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Protein extracts were prepared from flash-frozen tissue after homogenization in RIPA lysis buffer (Incomplete RIPA buffer, 7× protease inhibitor cocktail, 200mM sodium orthovandate, 1M sodium fluoride, 100mM PMSF) as previously described [15 (link)]. Total proteins (30–50 µg) were subjected to Western blot analysis with the following antibodies; anti-pMek1/2, anti-Mek1/2, anti-pErk1/2, anti-Erk1/2, anti-pp90Rsk, anti-pRsk1/2, anti-pStat3, and anti-Stat3 (all rabbit, Cell Signaling Technology, MA, USA; 1:1000 concentration). Mouse anti-vinculin (Santa Cruz, CA, USA; 1:2000) and mouse anti-β-actin (Sigma Aldrich, Missouri, USA; 1:10,000) were used as loading controls. Furthermore, membranes were washed and incubated with anti-rabbit or anti-mouse (Invitrogen, CA, USA; 1:5000) horseradish peroxidase-conjugated antibody. Western blot images were acquired using EMD Millipore Luminata HRP chemiluminescence substrate (Millipore, MA, USA) and signal acquired in Bio-Rad chemiluminescence detector (Bio-Rad Laboratories, CA, USA). The signals were semiquantified using image J (ImageJ; www://rsb.info.nih.gov/ij/ ).
10
Western Blot Analysis of Protein Expression
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For protein expression analysis by Western blot, cells were lysed using lysis buffer consisting of 10 mM tris-HCL pH 7.5, 50 mM KCl, 2 mM MgCl2, 1% Triton X-100, 1 mM DTT, 1 mM PMSF, and protease inhibitor cocktail (Roche, Basel, Switzerland). Samples were incubated for 20 min on ice with periodic mixing followed by 10 min centrifugation at 14,000× g at 4 °C. Proteins were mixed with 4× Laemmli-buffer-containing β-mercaptoethanol and then boiled at 95 °C for 5 min. Isolated proteins were separated by SDS-PAGE and then transferred to a PVDF membrane using semi-dry transfer. Membranes were blocked followed by overnight incubation at 4°C with primary antibodies (HPV16 E6 E6-6F4 (Euromedex, Souffelweyersheim, France), HPV16 E7 NM2 (in-house produced), p21 Waf1/Cip1 (F-5), P16INK4A (DCS-50)sc-65476, p53 (DO-1)sc-126 (Santa Cruz Biotechnology, Dallas, Texas), β-actin AC-74 (Sigma-Aldrich), GAPDH (GT239) (Biozol, Eching, Germany). HRP-coupled secondary antibodies (IgG anti-Mouse IgG (H+L)-HRPO (Dianova, Hamburg, Germany) were then incubated with the membrane for 1 h. After adding the chromogenic substrate ECL™ Prime Western Blotting detection reagents (Cytiva Europe GmbH, Freiburg im Breisgau, Germany), a chemiluminescence signal was observed in a Biorad Chemiluminescence Detector (BioRad ChemiDoc, Hercules, CA, USA).
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