For the expression analysis of PTEN and P27 proteins, ovaries of PD1, PD5, PD23, and PD35 C57BL/6 mice were isolated and collected. For the deletion analysis of Pten and p27 genes, granulosa cells of DKO females at PD23 and GCT cells of DKO females at 3-4 months were isolated and collected, respectively. For expression analysis of CD47 protein after RRX-001 treatment, tumor cells of 4 months GCT were collected and primarily cultured. Primary cells were collected following RRX-001 and DMSO treatment. Total proteins were extracted by using WIP lysis solution (1:10000) and Cell lysis solution (BioChip, 110000) according to the manufacturer’s protocol. Western blot experiments were conducted following standard procedure. The results were captured by using the SuperSignal chemiluminescent detection system (Thermo Scientific, 32109).
Supersignal chemiluminescent detection system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The SuperSignal chemiluminescent detection system is a laboratory equipment product that provides a method for detecting and quantifying target proteins or molecules in various assays, such as Western blotting and immunoassays. The system utilizes a chemiluminescent substrate that emits light upon interaction with the target analyte, which can then be measured and analyzed.
Automatically generated - may contain errors
Lab products found in correlation
Zb 2305, by ZSGB-BIO (3 mentions)
Zb 2301, by ZSGB-BIO (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Gapdh, by Thermo Fisher Scientific (1 mentions)
Phospho akt, by Cell Signaling Technology (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Protran nitrocellulose membrane, by Cytiva (1 mentions)
Ipvh00010, by Merck Group (1 mentions)
Ab52495, by Abcam (1 mentions)
Iseq00005, by Merck Group (1 mentions)
Mab3726, by Cell Signaling Technology (1 mentions)
Af5012, by Beyotime (1 mentions)
His tag, by Cell Signaling Technology (1 mentions)
Parp1, by Cell Signaling Technology (1 mentions)
β tubulin, by Santa Cruz Biotechnology (1 mentions)
Parp antibody, by Cell Signaling Technology (1 mentions)
8 protocols using supersignal chemiluminescent detection system
1
Expression and Deletion Analysis of PTEN and P27
2
Protein Expression Analysis in H9c2 Cells
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The protein expression of Akt, Bax, and Bad was evaluated as described previously with a slightly modification (Phosri et al., 2017 (link)). After treatment, H9c2 cells were solubilized in Triton X-100 lysis buffer (20 mM Tris pH 7.4, 0.8% Triton X-100, 150 mM NaCl, 2 mM EDTA, 10% glycerol, 100 μM phenylmethylsulfonyl fluoride, and protease inhibitor cocktail). After centrifugation, amount of protein in cell lysates was measured by a protein assay kit (Bio-Rad) and used bovine serum albumin as a standard. Samples were mixed with 4× SDS loading buffer and denatured by heating at 95°C for 5 min. After that, samples were subjected to SDS-PAGE gels and transferred to PVDF membrane (Bio-Rad), and separately immunoblotted with several antibodies such as Akt (Cell Signaling), phospho-Akt (Cell Signaling), Bax (Cell Signaling), Bad (Cell Signaling), and GAPDH (SantaCruz). Immunoblots were visualized with horseradish peroxidase-conjugated secondary antibodies and a SuperSignal chemiluminescent detection system (Thermo Scientific), using GAPDH as a loading control. The density of the band was calculated using ImageJ software.
3
Western Blot Analysis of Ovarian Proteins
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Ovaries were lysed in WIP (Beijing Cell Chip Biotechnology Co., Beijing, China) according to the manufacturer's protocol. Equal amounts of protein from lysates were subjected to 10% SDS–PAGE and then transferred to PVDF membranes (Millipore). PVDF membranes were blocked for 1 h with 5% (v/v) non-fat milk in phosphate buffered saline (PBS) with 0.1% (v/v) Tween 20 at room temperature. The blots were incubated with primary antibodies overnight at 4°C (antibody information is listed in Table S1 ). The blots were incubated with horseradish peroxidase-linked secondary antibodies (ZB-2301, ZB-2305 from ZSGB-BIO, Beijing, China) for 1 h at room temperature. Protein were detected by a Super Signal Chemiluminescent Detection System (34080; Thermo Scientific). The blots were acquired by Chemiluminescence image system (Tanon 5200, Beijing, China). The level of β-ACTIN and GAPDH were used as an internal control.
4
Membrane Protein Extraction and Western Blot
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Membrane proteins were extracted with ProteinExtTM Mammalian Membrane Protein Extraction Kit (Transgen Biotech, Beijing, China). Aliquots of proteins were resolved by 10% SDS–PAGE and transferred to pieces of Protran nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany). The membranes were blocked with 5% nonfat dry milk for 1 h and incubated overnight at 4 °C with primary antibodies. After five washes in TBST, the membranes were incubated with HRP-conjugated secondary antibody (1:5000, Zhong Shan) for 1 h at room temperature. The proteins on the membranes were visualized using the SuperSignal chemiluminescent detection system (Thermo Fisher Scientific). The density of blot was measured by AlphaEaseFC Software if needed.
5
Western Blot Analysis of HDAC3 Protein
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Total protein was extracted in WIP (CellChip, BJ Biotechnology Co., Ltd., Beijing, China) as recommended by the manufacturer. In brief, total protein from each sample was separated by 10% SDS-polyacrylamide gel electrophoresis and then electrophoretically transferred onto polyvinylidene fluoride membranes (IPVH00010, Millipore, MA, USA). The membranes were then incubated for 1 h at room temperature with 5% BSA, followed by an overnight incubation at 4 °C with an anti-HDAC3 antibody. After being washed in TBST, the membranes were incubated with secondary antibody (1:5000, ZB-2301 and ZB-2305, ZSGB-BIO, Beijing, China) in TBST, followed by detection with the SuperSignal chemiluminescent detection system (34080, Thermo Scientific, CA, USA). Glyceraldehyde 3-phosphate dehydrogenase was used as an internal control.
6
Western Blot Analysis of Protein Expression
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To detect protein expression in mouse materials, total proteins were extracted using WIP Tissue and Cell lysis solution (BioChip, 110000) according to the manufacturer’s protocol. Electrophoresis was performed with 50 μg total proteins separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, ISEQ00005). The membranes were incubated overnight at 4 °C with the appropriate primary antibodies listed below: anti-RDX (78 kDa, 1:500, ab52495, Abcam), anti-p-ERM (78 kDa, 1:500, mAb#3726, Cell Signaling Technologies). The appropriate secondary antibody (ZB-2301, ZB-2305 from ZSGB-BIO) was diluted 1:5000 in TBST (TBS plus 0.5% Tween 20). Tubulin (1:5000, Beyotime, AF5012) was used as an internal control. The membranes were visualized using the SuperSignal chemiluminescent detection system (Thermo Scientific, 32109). Original blots can be found in the Source data file.
7
Western Blot Analysis of Protein Targets
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Samples were resolved by electrophoresis through 10% SDS-polyacrylamide gels. Proteins were transferred to nitrocellulose membranes (Bio-Rad) and probed with corresponding antibodies. Antibodies used in this work are as follows: iNOS (Santa Cruz Biotechnology, SC-7271), PARP-1 (Cell Signaling Inc. #9532), β-tubulin (Santa Cruz Biotechnology, SC-55529), and His-tag (Cell Signaling Inc. #2365). The membranes were developed using the SuperSignal chemiluminescent detection system (Thermo Scientific Pierce). Quantification of immunoblotting results was performed using software ImageJ. Statistical summaries were obtained from at least 3 independent samples.
8
Quantifying S-nitrosated Proteome by Biotin-Switch
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