For detection of sCLU, cytoplasmic components were isolated using a LysoPure Nuclear and Cytoplasmic Extractor kit (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan), and nuclear components were discarded. Cytoplasmic lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE), and the proteins were transferred onto polyvinylidene fluoride membranes. Blots were incubated with respective primary antibodies overnight at 4°C, followed by incubation with corresponding secondary antibodies for 1 hour. Proteins were visualized by enhanced chemiluminescence using EzWestLumi Plus and Ez‐Capture MG (ATTO Corp., Tokyo, Japan). Anti‐CLU (sc‐5289) and anti‐GAPDH (sc‐32233) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX). Anti‐p‐Akt (4060), anti‐pan Akt (2920), anti‐p‐Erk (1/2) (9106), and anti–phosphorylated mammalian target of rapamycin (p‐mTOR) (5536) antibodies were purchased from Cell Signaling Technology (Danvers, MA). Anti‐Erk (1/2) antibody (MAB1576) was purchased from R&D Systems (Minneapolis, MN). Cleaved Caspase‐3 antibody (#9661) was purchased from Cell Signaling Technology (Danvers, MA).
Ez capture mg
Manufactured by ATTO Corporation
Sourced in Japan
The Ez-Capture MG is a lab equipment product designed for image capture and analysis. It features a high-resolution camera and software for acquiring and processing digital images. The core function of the Ez-Capture MG is to enable users to capture, view, and analyze various samples or specimens within a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Enhanced chemiluminescence detection kit, by Cytiva (2 mentions)
Ap00664pu n, by OriGene (2 mentions)
Hybond p, by Cytiva (2 mentions)
Pvdf membrane, by Cytiva (2 mentions)
P stat3, by Cell Signaling Technology (2 mentions)
Pro prep reagent, by iNtRON Biotechnology (2 mentions)
Anti gapdh sc 32233, by Santa Cruz Biotechnology (1 mentions)
Lysopure nuclear and cytoplasmic extractor kit, by Fujifilm (1 mentions)
Cleaved caspase 3 antibody 9661, by Cell Signaling Technology (1 mentions)
Anti clu sc 5289, by Santa Cruz Biotechnology (1 mentions)
Ezwestlumi plus, by ATTO Corporation (1 mentions)
Anti goat igg conjugated to horseradish peroxidase, by Jackson ImmunoResearch (1 mentions)
Human angiogenesis array kit, by R&D Systems (1 mentions)
Proteome profiler antibody array, by R&D Systems (1 mentions)
Human adipokine array kit, by R&D Systems (1 mentions)
Human soluble receptor array kit, by R&D Systems (1 mentions)
Anti human bcl 2, by Abcam (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
10 protocols using ez capture mg
1
Western Blot Analysis of sCLU
2
Western Blot Analysis of Adenovirus Proteins
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Each fraction was solubilized in an equal volume of 2× sodium dodecyl sulfate (SDS) gel-loading buffer [90 mM Tris–HCl (pH 6.8), 10% mercaptoethanol, 2% SDS, 0.02% bromophenol blue, and 20% glycerol] and boiled for 5 min. Proteins were then resolved by SDS–polyacrylamide gel electrophoresis (PAGE) using an 8% gel before being electroblotted onto a polyvinylidene difluoride (PVDF) membrane (Hybond-P; Amersham-Pharmacia Biotech, Piscataway, NJ) for 60 min at 15 V. Blots were treated with 5% skimmed milk for 1 h at room temperature and then incubated with a goat anti-adenovirus polyclonal antibody (AP00664PU-N, Acris) in PBS containing 0.1% Tween 20 (PBS-T) and 0.5% skimmed milk for 1 h at room temperature. After three washes with PBS-T, the membrane was incubated in horseradish peroxidase (HRP)-conjugated anti-goat IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) in PBS-T and 0.5% skimmed milk for 1 h at room temperature. After three washes with PBS-T, the probed proteins were detected using an enhanced chemiluminescence detection kit (Amersham-Pharmacia Biotech). The signal of chemiluminescence was detected by using Ez-Capture MG (ATTO Corp., Tokyo, Japan). Band intensity was analysed by densitometric analysis with ImageJ software (National Institutes of Health).
3
Adenovirus Protein Detection by Western Blot
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Each fraction was solubilized in an equal volume of 2× sodium dodecyl sulfate (SDS) gel-loading buffer (90 mM Tris–HCl [pH 6.8], 10% mercaptoethanol, 2% SDS, 0.02% bromophenol blue, and 20% glycerol), boiled for 5 minutes, and then resolved by SDS–polyacrylamide gel electrophoresis (PAGE) using an 8% gel. The bands were then electroblotted onto a polyvinylidene difluoride (PVDF) membrane (Hybond-P; Amersham-Pharmacia Biotech, Piscataway, NJ, USA) for 60 minutes at 15 V. The PVDF membrane was blocked with 5% skimmed milk for 1 hour at room temperature and then incubated with goat anti-adenovirus polyclonal antibody (AP00664PU-N, Acris) in PBS containing 0.1% Tween 20 (PBS-T) and 0.5% skimmed milk for 1 hour at room temperature. After three washes with PBS-T, the membrane was incubated in PBS-T and 0.5% skimmed milk supplemented with anti-goat IgG conjugated to horseradish peroxidase (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) for 1 hour at room temperature with gentle shaking. The membrane was then washed three times with PBS-T, and protein bands that reacted with the antibodies were visualized using an enhanced chemiluminescence detection kit (Amersham-Pharmacia Biotech). Chemiluminescence was detected using EzCaptureMG (ATTO Corp., Tokyo, Japan). Band intensity was analyzed by densitometric analysis with ImageJ software (National Institutes of Health).
4
Serum Proteomic Profiling of Angiogenesis, Receptors, and Adipokines
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Proteomic analyses of serum proteins were carried out using Proteome Profiler antibody arrays (R&D Systems, Minneapolis, MN), namely the Human Angiogenesis Array kit (ARY007), Human Soluble Receptor Array kit (ARY012), and Human Adipokine Array kit (ARY024). Each examination was performed according to the respective protocol. Briefly, serum samples for use with each kit were applied to nitrocellulose membranes with bound capture antibodies for target proteins and incubated for 24 hours in a refrigerated room followed by removing serum from the membrane and assaying immediately. Samples were analyzed using a LAS system (Ez‐Capture MG; ATTO Corp., Tokyo, Japan). Measured signals are presented as percentage of the negative control (0%) and positive control (100%). We selected proteins in an unbiased manner that met the following criteria: (1) significant difference (P < 0.05) in rate of change in value between the R group and NR group; (2) exhibit >1.5‐fold difference between before and 1 month after SFN treatment initiation; and (3) measured value >5%.
5
Western Blot Analysis of Cell Signaling
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SDS-PAGE and Western blot analysis were performed according to the protocol described previously [16 (link)]. Briefly, cells were seeded in a 6-well plate, and after 24 h, treated with or without PEF-III (20 and 30 μg/mL) and incubated till 70–80% confluence. Lysates were prepared using lysis buffer (50 mM TrisCl, pH 7.8, 150 mM NaCl, 1% NP 40, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride). The lysates were then resolved on SDS-PAGE and transferred into polyvinylidene difluoride (PVDF) membranes. Next, the membranes were blocked in skim milk (4%) and Tris-buffered solution (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween-20) for 1 h and incubated overnight at 4 °C with specific primary antibodies including anti-human Bcl-2 (Abcam, Tokyo, Japan), Bax (Cell Signaling Technology, Tokyo, Japan), p53 (Cell Signaling Technology), and β-actin (Cell Signaling Technology). After that, the membranes were incubated with horseradish peroxidase conjugated secondary antibodies for 50 min and then washed with Milli-Q (3–4 times), after which bound antibodies were detected by chemiluminescence reaction using Immuno Star Zeta (Wako) and EZ capture MG (ATTO Corporation, Tokyo, Japan) according to the manufacturers’ protocols.
6
Western Blot Protein Analysis Protocol
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Cells were harvested and lysed using a Cell Lysis Buffer (Cell Signaling Technology, Inc., Danvers, MA, USA) containing a Protease Inhibitor Cocktail (Sigma-Aldrich, St. Louis, MO, USA). The cell lysates were incubated on ice for 30 min, then centrifuged for 30 min (15,000× g, 4 °C), and the supernatants were collected. The protein concentration was determined using the Bradford method. Protein samples (20 µg) were separated using 10% SDS-PAGE and then transferred to PVDF membranes. After blocking with 10% skimmed milk in TBS, the membranes were incubated overnight at 4 °C with primary antibodies and then with horseradish peroxidase-labeled secondary antibodies for one hour at room temperature. Immunolabelling bands were visualized using the Clarity Max Western ECL Substrate (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and analyzed using Ez-Capture MG (Atto Corporation, Tokyo, Japan). The primary and secondary antibodies used in the experiments are listed in Table 1 . β-actin was used as an endogenous control. Densitometric analysis of band intensity to compare protein expression levels was performed using the ImageJ software program (version 1.53 t).
7
Western Blot Analysis of Protein Expression
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Lysates from both the treated and the control cells were prepared using TNE buffer, and the protein concentration was quantified using a protein DC quantification kit. Equal amounts of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA), and the membranes were blocked in 4% skim milk for 40 minutes. After blocking, the membranes were incubated with primary antibodies (antibody information provided in Supplementary Table 1 ) at 4°C overnight and washed with tris-buffered saline with Tween (TBST). Horseradish peroxidase (HRP)-conjugated secondary antibodies were added and incubated at 37°C for 40 minutes, the membranes were then washed with TBST, and the protein band was detected by use of the chemiluminescence kit Immuno Star Zeta (Wako, Japan) and EZ capture MG (ATTO Corporation, Japan). The normalized protein expression was calculated to that of the reference protein β-actin by use of ImageJ software.
8
Immunoblotting Analysis of Protein Expression
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The protein cell lysates were isolated by Pro-Prep™ reagent (iNtRON Biotechnology, Seongnam, Korea). Total proteins were separated by electrophoresis on gradient SDS-PAGE gels (8% to 15%) and transferred onto a PVDF membrane (Amersham Biosciences, Bukres, UK). Antibodies against GAPDH, NF-κB, NDRG2, p-IKKα/β, Lamin A/C, and α-actinin were purchased from Santa Cruz Biotechnology. Anti-human PD-L1, p-STAT3, STAT3, α-tubulin, p-IκBα, IκBα, p-NF-κB (p-p65), and PD-1 antibodies were purchased from Cell Signaling (Beverly, MA, USA). Anti-mouse PD-L1 antibody was purchased from R&D Systems (Minneapolis, MN, USA). The blots were visualized by Ez-Capture MG (ATTO Corporation, Tokyo, Japan).
9
Whole Cell Lysate Preparation and Protein Analysis
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For the preparation of whole cell lysates, the cells were lysed on ice in the PRO-PREP™ reagent (iNtRON Biotechnology, Korea) for 20 min. Nuclear and cytoplasmic extracts were prepared using the NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology, USA), according to the manufacturer’s instructions. The proteins were separated in an SDS-polyacrylamide gel and transferred to a PVDF membrane (Amersham Biosciences, UK), which was then blocked with Tris-buffered saline plus 0.05% Tween-20 (TBST) containing 5% skim milk. The membranes were incubated with specific antibodies overnight at 4°C and washed with TBST. The antibody recognizing COX-2 was purchased from Cayman Chemical (USA), and the antibodies against NDRG2, p-IKKα/β (Ser176), IKKβ, NF-κB p65, Akt1/2, Lamin A/C, and actin were purchased from Santa Cruz Biotechnology. The antibodies for p-IκBα (Ser32/36), IκBα, p-Erk1/2 (Thr202/Tyr204), Erk1/2, and p-Akt (Ser473) were purchased from Cell Signaling Technology Inc. (USA). After incubating the membrane with the appropriate secondary antibodies coupled to horseradish peroxidase followed by enhanced chemiluminescence, the blots were visualized using the Ez-Capture MG (ATTO Corporation, Japan).
10
Western Blot Analysis of Cellular Proteins
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The cells were washed using DPBS, and the total protein was isolated by a protein extraction solution (iNtRON Biotechnology, Seongnam, Korea). The supernatant fractions were obtained by centrifugation at 12,000 rpm for 15 min at 4°C. The protein was mixed with a 5X sample buffer and separated on 9% or 12% SDS-polyacrylamide gel. Then, gel was transferred to a PVDF blotting membrane (GE healthcare Life Science, Wauwatosa, WI, USA), and the membrane was blocked with Tris-buffered saline plus 0.1% Tween-20 (TBS-T) containing 5% skim milk (BD Biosciences) or BSA (Gibco). The membrane was incubated with specific antibodies overnight at 4°C and washed by TBS-T. The antibodies against actin, α-actinin, NDRG2 and ICAM1 were purchased from Santa Cruz Biotechnology and the antibodies against NFATc1, MITF, STAT3, p-STAT3, JNK, p-JNK, ERK, p-ERK, p38, p-p38, AKT and p-AKT were obtained from Cell Signaling (Danvers, MA, USA). After attaching the secondary antibodies coupled to horseradish peroxidase, the blots of the membrane were visualized with an EZ-Western Lumi Plus solution (ATTO Corporation, Tokyo, Japan), pico EPD solution (ELPIS-Biotech., Inc, Daejeon, Korea) and Ez-Capture MG (ATTO Corporation).
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