Cells were harvested, washed with ice‐cold PBS and lysed in RIPA buffer supplemented with protease and phosphatase inhibitors for 10 minutes. Protein concentrations were determined by using a BCA Protein Assay Kit (Beyotime, Wuhan, China). 25 μg total protein was subjected to electrophoresis in denaturing 10% SDS‐PAGE, and then transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were then blocked with 5% non‐fat milk powder dissolved in Tris‐buffered saline with Tween‐20 for 1 hour and incubated with primary antibodies overnight at 4°C. The primary antibodies used include anti‐PLA2G16 (1:4000, # MA5‐26290; Thermo Fisher Scientific), anti‐p53 (1:250, #MA5‐14516; Thermo Fisher Scientific), anti‐KLF5 (1:1000, 21017‐1‐AP; Thermo Fisher Scientific). Then, the membranes were incubated with HRP‐conjugated secondary antibodies for 1 hour at room temperature. The blots were then visualized with BeyoECL Star reagent (Beyotime) and an ImageQuant LAS‐4000 imaging system (GE Healthcare, Piscataway, NJ, USA).
Beyoecl star reagent
Manufactured by Beyotime
Sourced in United States, China
BeyoECL Star reagent is a chemiluminescent substrate solution used for the detection of horseradish peroxidase (HRP) in western blotting applications. The reagent produces a luminescent signal when it reacts with HRP, allowing for the visualization of target proteins on a membrane.
Automatically generated - may contain errors
Lab products found in correlation
Imagequant las 4000 imaging system, by GE Healthcare (3 mentions)
Bca protein assay kit, by Beyotime (3 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Protease inhibitor, by Beyotime (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Anti p53, by Thermo Fisher Scientific (1 mentions)
Anti pla2g16, by Thermo Fisher Scientific (1 mentions)
Primary rabbit anti hla antibody, by Merck Group (1 mentions)
Hrp labeled goat anti rabbit igg, by Merck Group (1 mentions)
Np 40 buffer, by Beyotime (1 mentions)
Nuclear and cytoplasmic protein extraction kit, by Beyotime (1 mentions)
Bca protein analysis kit, by Beyotime (1 mentions)
Cbl b, by Proteintech (1 mentions)
Sds polyacrylamide gel electrophoresis, by Beyotime (1 mentions)
Ab80194, by Abcam (1 mentions)
Anti phospho nf κb p65 ser536 p p65, by Cell Signaling Technology (1 mentions)
Anti nf κb p65, by Cell Signaling Technology (1 mentions)
Anti iκbα, by Cell Signaling Technology (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
11 protocols using beyoecl star reagent
1
Western Blot Analysis of Protein Targets
2
Western Blot Analysis of Subcellular Fractions
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The cytoplasmic fraction and nuclear fraction of NMFH-1 cells were prepared following the method introduced in one previous study [17 (link)]. For western blot analysis, samples were then separated on 10% SDS-PAGE gels, and electrotransferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA) and subjected to immunoblotting with different antibodies. The blots were then visualized with BeyoECL Star reagent (Beyotime) and an ImageQuant LAS-4000 imaging system (GE Healthcare, Piscataway, NJ, USA). Protein band intensity was quantified using ImageJ software (NIH, USA). Primary antibodies used are provided in Table S2 .
3
Echinatin Inhibits α-Hemolysin in S. aureus
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Staphylococcus aureus cultures were treated with various echinatin concentrations (0 to 32 μg/mL) and incubated with shaking until the late logarithmic growth period was reached. The supernatant was then collected and mixed with protein loading buffer for 10% SDS–PAGE. Proteins were then transferred onto PVDF membranes by a semidry transfer device. The membranes were blocked with 5% bovine serum albumin (BSA) for 2 h at room temperature and then washed three times with PBST. The membrane was then incubated with the primary rabbit anti-Hla antibody (1:3000, Sigma–Aldrich) followed by incubation with the HRP-labeled goat anti-rabbit IgG (1:5000, Sigma–Aldrich) secondary antibody. After washing the membrane three times, the bands were observed by a chemiluminescence detection device after adding BeyoECL Star reagent (Beyotime, Beijing, China).
4
Protein Extraction and Analysis
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Total protein from the tissues and cells was collected and lyzed in NP-40 buffer (P0013F, Beyotime, Shanghai, China) with 1 mM PMSF. The nuclear and cytosolic proteins were extracted with a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. The protein concentration was measured using a BCA protein assay kit (P0009, Beyotime, Shanghai, China). Equal amounts (30 μg) of protein were separated by 10–15% SDS-PAGE and then transferred to a PVDF membrane. Subsequently, the PVDF membrane was blocked with 5% (w/v) BSA in Tris-buffered saline containing 0.1% Tween-20 (TBST) buffer for 1 h at room temperature and incubated with the corresponding primary antibodies overnight at 4°C. Subsequently, the membrane was washed three times with TBST and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. Finally, the membrane was washed and visualized with the BeyoECL Star reagent (P0018AM, Beyotime, Shanghai, China). The relative optical densities of the bands were quantified using ImageJ gel analysis software.
5
Protein Quantification and Immunoblotting Protocol
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Cells or tissues were lysed with RIPA buffer (Beyotime, Hangzhou, China) containing a mixture of protease inhibitors. The total protein was quantified using BCA protein analysis kit (Beyotime). SDS-polyacrylamide gel electrophoresis (Beyotime) was used to separate the protein cleavage products. The main monovalent antibodies used were as follows: Cbl-b (1:500, Proteintech, Chicago, IL, USA), Atrogin-1 (1:1000, Bioss Antibodies, Beijing, China), MuRE-1 (1:1000, Proteintech). Protein was detected with BeyoECL Star reagent (Beyotime).
6
Protein Expression Analysis by Western Blotting
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Western blotting was performed as previously described [17 (link)]. The following antibodies and dilutions were applied: anti-CXCR2 (1:1000, 20634-1-AP, Proteintech, Wuhan, China), anti-Phospho-IκBα (Ser32) (p-IκBα) (1:1000, #2859, Cell Signaling Technology, Danvers, MA, USA), anti-IκBα (1:1000, #4814, Cell Signaling Technology), anti-NF-κB p65 (1:1000, #8242, Cell Signaling Technology), anti-phospho-NF-κB p65 (Ser536) (p-p65) (1:1000, #3033, Cell Signaling Technology), anti-CD31 (1:2000, 11265-1-AP, Proteintech), anti-VE-cadherin (VE-cad, 1:1000, 27956-1-AP, Proteintech), anti-CD34 (1:1000, 14486-1-AP, Proteintech), anti-S100A4 (1:5000, 16105-1-AP, Proteintech), anti-α-SMA (1:2000, 14395-1-AP, Proteintech), anti-N-cadherin (N-cad, 1:2000, 22018-1-AP, Proteintech), anti-SLC7A11 (1:1000, 26864-1-AP, Proteintech), anti-GPX4 (1:2000, 67763-1-Ig, Proteintech), anti-HO-1 (1:1000, 10701-1-AP, Proteintech), anti-NRF2 (1:1000, 80593-1-RR, Proteintech), anti-TFR2 (1:1000, ab80194, Abcam, Cambridge, UK), or anti-β-tubulin (1:5000, 10094-1-AP, Proteintech), or anti-β-actin (1:1000, 20536-1-AP, Proteintech). The protein bands were visualized using BeyoECL Star reagent (Beyotime, Shanghai, China) and the Tanon 4600SF Chemiluminescent Imaging System (Tanon, Shanghai, China). The endogenous protein expression control used was β-actin.
7
Protein Expression Analysis of MCF-7 and BT-474 Cells
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Total proteins were extracted with cell lysis buffer with protease inhibitors (Beyotime, Wuhan, China) from MCF-7 and BT-474 cells 48h after indicating treatment and were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Membranes were blocked with 5% nonfat milk in Tris(hydroxymethyl)aminomethane-buffered saline Tween-20 and incubated with the primary antibody (anti-Cyclin D1, SAB4502603, 1:500, Sigma-Aldrich; anti-C-Myc, ab32072, 1:1000, Abcam, Cambridge, MA, USA). Then, the membranes were incubated with HRP-conjugated secondary antibodies for 1h at room temperature. The blots were then visualized with BeyoECL Star reagent (Beyotime) and an ImageQuant LAS-4000 imaging system (GE Healthcare, Piscataway, NJ, USA).
8
Protein Extraction and Western Blot Analysis
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Cell samples were harvested, washed, and lysed using a lysis buffer containing protease inhibitors. After centrifugation, the supernatant was collected to determine protein concentration using a BCA kit (Beyotime). 30 μg protein samples were loaded into each lane. The following antibodies and dilutions were used, including anti-KIF20A (1:1000, 15911-1-AP, Proteintech, Wuhan, China), anti-BTRC (1:1000, 37-3400, ThermoFisher Scientific, Waltham, MA, USA), anti-Flag tag (1:5000, 66008-4-Ig, Proteintech), anti-CDC25A (1:1000, 55031-1-AP, Proteintech), anti-HA tag (1:5000, 51064-2-AP, Proteintech), anti-Myc tag (1:3000, 16286-1-AP, Proteintech), and anti-β-actin (sc-47778, Santa Cruz Biotechnology, Santa Cruz, CA, USA). HRP-conjugated secondary antibodies were used. Then, the signals were developed using the BeyoECL Star reagent (Beyotime) and a LAS-4000 imaging system (GE Healthcare, Piscataway, NJ, USA).
9
Western Blot Analysis of Cytoskeletal Proteins
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Total protein was collected from cells using RIPA buffer (Solarbio Life Science, Beijing, China) containing protease inhibitors (Beyotime Institute of Biotechnology, Jiangsu, China). The concentration of the extracted protein samples was then quantified using a BCA Protein Assay Kit (Beyotime Institute of Biotechnology). The proteins (30 µg) were subjected to Western blotting with the Bio–Rad Bis-Tris gel system. The polyvinylidene difluoride (PVDF) membranes (Millipore, Burlington, MA, USA) were incubated with primary antibodies against RhoA (BM4479; Boster Bio, Pleasanton, CA, USA), ROCK1 (BM4203; Boster Bio), ROCK2 (BM5257; Boster Bio), pMLC (no. AF8010; Affinity Biosciences, Changzhou, China), MLC (DF7911; Affinity Biosciences) and Cx43 (ER1802-88; HuaBio, Hangzhou, China) diluted 1:1000 at 4°C overnight. Later, the PVDF membranes were probed with secondary antibody conjugated to horseradish peroxidase (Boster) for one hour. After rinsing, the PVDF membranes were placed in an Amersham Imager 600 system (Amersham, Piscataway, NJ, USA) and covered with BeyoECL Star reagent (Beyotime Institute of Biotechnology). The signals were captured with the fluorescence imaging system (Amersham Imager 600 RGB).
10
Western Blot Analysis of ATP6V0A4 Protein
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Protein samples from cells were collected using RIPA lysis buffer and protease inhibitor (Beyotime Institute of Biotechnology). Protein concentrations were determined using a BCA protein assay kit (Takara Bio, Inc.). A total of 20 µg protein/lane was loaded on 10% SDS-gels, resolved using SDS-PAGE and transferred to PVDF membranes, and then the membranes were blocked using 5% skimmed milk for 2 h at room temperature. Subsequently, the membranes were incubated with the primary antibodies overnight at 4°C, followed by subsequent incubation with the relevant secondary antibody for 1 h at room temperature. The primary antibodies used were an anti-ATP6V0A4 antibody (1:3,000; cat. no. ab97440; Abcam) and an anti-GAPDH antibody (1:10,000; cat. no. AC002; ABclonal Biotech Co., Ltd.). The secondary antibodies used were an anti-rabbit (1:2,000; cat. no. 7074; Cell Signaling Technology, Inc.) and an anti-mouse IgG HRP-linked antibody (1:2,000; cat. no. 7076; Cell Signaling Technology, Inc.). Signals were visualized using a chemiluminescence imaging system (Tanon-5200Multi; Tanon Science and Technology Co., Ltd.) and BeyoECL Star reagent (Beyotime Institute of Biotechnology). ImageJ v1.53 (National Institutes of Health) was used for densitometry.
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